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learning and mastering procedural skills are major challenges in anesthesia practice and are essential in the process of achieving clinical competence .
anesthesiologists carry out many complex clinical tasks in their routine work which the trainee is expected to learn and master during training .
an increased public awareness of healthcare related issues has led to greater accountability of healthcare professionals .
this has very rightly led to an increasing focus on patient safety in clinical practice .
the supervisors have to undertake the important responsibility of deciding when a trainee can be allowed to perform the various procedures without direct supervision while ensuring patient safety .
supervisors and trainers must accept that not all trainees can be equally quick in learning and equally competent in performing practical procedures and reliable , and objective assessment is , therefore , mandatory .
airway management is an inherent part of the routine day - to - day work of anesthesiologists .
they are required to perform this procedure not only in the operation theater , but , also in the intensive care unit , the wards and the emergency department .
failure to perform the technique promptly and correctly can lead to serious consequences including death .
it is important to ensure that an anesthesia trainee is capable of performing tracheal intubation independently before he or she could be included in a cardiac arrest team , where direct supervision by a senior colleague is not always possible .
this requires robust and reliable assessment techniques such as direct observation by senior anesthesiologists using procedure - specific tools while the trainee is performing the procedure on actual patients . when constructing an assessment tool , it is important to explore the literature to see whether there is an already existing instrument that is appropriate and has established reliability and validity .
we were successful in retrieving tools for assessment of procedures performed by anesthesiologists , including rapid sequence induction of anesthesia and management of difficult airways .
however , we could not identify a structured tool for assessment of routine airway management with established reliability and validity .
the objectives of this study were to evaluate the inter - rater and test - retest reliability and construct validity of a tool designed to assess competence in bag - mask ventilation and tracheal intubation .
reliability of a tool is its ability to assess skills consistently by different assessors at different times while construct validity is the ability of the tool to differentiate among varying levels of expertise .
approval was granted by the university ethics review committee ( 1398-ane - erc-09 ) and written informed consent was obtained from all participants .
junior trainees were described as those having had more than two and < 4 months of anesthesia training , while senior residents recruited were those in the fourth year of training and already performing airway management independently .
the study protocol was presented in the departmental faculty meeting so as to share it with all faculty members .
the purpose of the study was explained to the participating residents at the time of informed consent .
the participants ' bag - mask ventilation and tracheal intubation skills were assessed by the use of a structured procedure - specific assessment tool .
all three authors participated in the construction of the tool and advice was taken from two other senior anesthesia consultants .
the tool comprised of five major categories with further sub - categories in each , in order to evaluate the performance of the trainee in all the essential steps involved in the procedure [ table 1 ] .
a simple 3-point scale was used to assess each step , where : steps of bag - mask ventilation and tracheal intubation assessed by direct observation in anesthesia trainees
1 ( one ) meant step not performed2 ( two ) meant performance below expectations3 ( three ) meant performance meets expectationsa column was added for steps not applicable during the performance .
1 ( one ) meant step not performed 2 ( two ) meant performance below expectations
3 ( three ) meant performance meets expectations a column was added for steps not applicable during the performance . performance below expectation was defined in the tool as unsuccessful attempt or incorrectly performed step , while meets expectation was defined as step performed adequately and successfully .
the procedural steps used for assessment of bag - mask ventilation and tracheal intubation skills are provided in table 1 . before finalizing the tool for the study
, we conducted a pilot study to identify any missing steps and to assess the practicality of using the tool in the operation theater .
the pilot study provided a chance for a final check on the content validity and served as a means of training the investigators in rating trainees ' performance by direct observation .
the authors also attended a half - day workshop on direct observation of procedural skills .
the residents were assessed while working in their assigned operation theater under the supervision of the assigned consultant anesthesiologist .
trainee 's assessment was not done if the patient being anesthetized was pregnant or had oral , faciomaxillary or neck pathology or anatomic anomaly , obesity ( body mass index > 30 ) , rheumatoid arthritis , ankylosing spondylitis , a history of difficult airway in the past or was found to have limited mouth opening , buck teeth , short thick neck with limited mobility , and mallampati grade iii or iv .
the assessment was done simultaneously by two of the investigators who are senior consultant anesthesiologists and registered supervisors for anesthesia training .
the trainee was observed while managing the airway with bag - mask ventilation and intubating the trachea with a tracheal tube .
the assessment time began once the patient was transferred to the operating table for induction of anesthesia and monitors were attached and ended when the endotracheal tube position was confirmed , and the tube was fixed . any decision to take over the procedure , in case
the trainee was unable to intubate the patient 's trachea , was left to the discretion of the supervising consultant .
it was planned to allow two attempts at laryngoscopy and intubation , and if the trainee was unsuccessful after two attempts , it was to be considered a failed attempt .
each resident was observed performing the same procedure again after 3 - 4 weeks by the same assessors to evaluate the test - retest reliability of the tool .
sample size was calculated using pass version 11 ( ncss llc , kaysville , utah ) . in a test for agreement between raters using the kappa statistic ,
a sample size of 20 subjects achieves 80% power to detect a true kappa value of 0.90 in a test of h0 : kappa = 0.50 versus h1 : kappa 0.50 using a two - tailed level of significance of 0.05 .
statistical analysis was performed using statistical packages for social sciences version 19 ( spss inc . ,
inter - rater and test - retest reliability were computed by percent agreement and kappa statistic .
kappa statistic was used to evaluate the level of agreement between assessors ' ratings and between the same assessor 's ratings at two points in time for each item of the structured assessment form .
kappa is positive when the agreement exceeds what is expected by chance ; kappa is negative when the observed agreement is less than the chance agreement . for the interpretation of kappa values
the rating indicators are : 0.0 - 0.2 slight agreement , 0.21 - 0.40 fair agreement , 0.41 - 0.60 moderate agreement , 0.61 - 0.80 substantial agreement , and 0.81 - 1.0 almost perfect or perfect agreement .
average agreement and the average kappa value was also calculated . for construct validity , the score of sub - categories of the main criteria were added for each rater in order to perform the analysis by using independent sample t - test and mann - whitney u - test ( as per rule of normality of the data ) to compare the scores between junior and senior residents .
statistical analysis was performed using statistical packages for social sciences version 19 ( spss inc . ,
inter - rater and test - retest reliability were computed by percent agreement and kappa statistic .
kappa statistic was used to evaluate the level of agreement between assessors ' ratings and between the same assessor 's ratings at two points in time for each item of the structured assessment form .
kappa is positive when the agreement exceeds what is expected by chance ; kappa is negative when the observed agreement is less than the chance agreement . for the interpretation of kappa values
the rating indicators are : 0.0 - 0.2 slight agreement , 0.21 - 0.40 fair agreement , 0.41 - 0.60 moderate agreement , 0.61 - 0.80 substantial agreement , and 0.81 - 1.0 almost perfect or perfect agreement .
average agreement and the average kappa value was also calculated . for construct validity , the score of sub - categories of the main criteria were added for each rater in order to perform the analysis by using independent sample t - test and mann - whitney u - test ( as per rule of normality of the data ) to compare the scores between junior and senior residents .
the inter - rater agreement between scores at the two assessments is presented in table 2 .
percent agreement and kappa values were found to be high for patient positioning , bag - mask ventilation , chin lift / jaw thrust , and leak around the facemask among the two assessors , and the options of absence of co2 trace , and difficulty in bag - mask ventilation exhibited 100% agreement .
the average kappa value for inter - rater reliability for the first assessment session was 0.91 and for the second assessment 0.99 , with an average agreement of 95% [ table 2 ] .
inter - rater reliability of the tool for assessment of bag - mask ventilation and tracheal intubation ( percentage agreement and kappa values ) kappa values and percent agreement for test - retest reliability are presented in table 3 .
the average agreement for test - retest reliability was 82% with a kappa value of 0.39 .
determination of construct validity [ table 4 ] showed that senior trainees obtained higher scores compared to the junior trainees in all areas of assessment .
this difference was statistically significant for the sums of scores for patient positioning , preoxygenation , and laryngoscopy technique .
test - retest reliability of the tool for assessment of bag - mask ventilation and tracheal intubation ( percentage agreement and kappa values ) construct validity of the assessment tool for bag - mask ventilation and tracheal intubation
assessment of competence in cognitive knowledge , judgment , communication , including history taking , physical examination , etc . , is routinely done by written , oral , and objective structured clinical examinations .
however , procedural skills have historically been assessed with subjective evaluations done by senior colleagues and supervisors without well - defined criteria or through procedure logs maintained by trainees .
work has been done on defining a minimum number of procedures required to attain competency in anesthetic procedures .
however the relationship between experience , as judged by number of procedures performed , and competence is difficult to define and differs markedly in trainees .
end - of - rotation global rating forms are often filled out by supervising faculty members who have not directly observed trainees performing the procedure on patients .
this form of assessment can not reliably assess procedural skills in their entirety and can not be justified for use in decisions about allowing trainees to perform procedures without direct supervision .
direct observation of the trainee , while performing a procedure on an actual patient , is recommended for a more reliable assessment of competence in procedural skills to enhance the quality of clinical training and ensure patient safety . the construction of procedure - specific assessment tools is therefore required for all complex procedural skills .
it is essential to ensure that the trainee masters the principal components of airway management before he / she is allowed to perform this procedure without direct supervision .
the tool employed in this study was designed specifically for novices in anesthesia and hence the technique was broken down into each of its basic steps forming a checklist with a simple rating scale of 1 - 3 so that the procedure could be assessed in its entirety as recommended for assessment of procedural skills .
the inter - rater reliability for the tool was high . during their training , the anesthesia trainees work at multiple sites with multiple consultants who are responsible for their assessment and provision of feedback .
good inter - rater reliability is , therefore , a basic requirement for this assessment tool .
this would allow the tool to be used by different assessors in different locations depending upon the initial rotations of the trainee .
many other researchers studying the inter - rater reliability of procedure - specific assessment tools for medical trainees have obtained good to excellent results for inter - rater reliability .
the test - retest reliability for the assessment tool does not show as high agreement or kappa values as for inter - rater reliability .
the most probable reason for this seems to be the learning effect involved due to the 3 - 4 weeks interval between the two assessment sessions .
the anesthesia trainees get frequent opportunities to perform bag - mask ventilation and tracheal intubation on a daily basis and thus get the adequate practice to learn and master the skills in the early months of their training .
therefore , their performance might have improved in the 3 - 4 weeks between the two assessments in this study .
we found that the senior trainees obtained higher scores for all steps of bag - mask ventilation and intubation , the difference being significant in many of the steps [ table 4 ] .
this indicates that this procedure - specific structured assessment tool has the ability to discriminate between junior and senior trainees , thus depicting good construct validity .
obtained similar results when testing validity and reliability of an assessment tool for brachial plexus regional anesthesia performance and have recommended their tool for routine use during anesthesia training .
the main use of the tool employed in the current study will be for assessment of junior anesthesia trainees in their first 6 months of training .
bag - mask ventilation and tracheal intubation are among the first few procedural skills that anesthesia trainees learn at the beginning of training and then use it for the rest of their professional career .
the authors hope to use the instrument for formative assessment in novices and for judgment of competence to perform the procedure without direct supervision .
the average assessment score obtained by the group of senior trainees could be used to ascertain the score that the junior trainees must reach before they are trained and assessed for more advanced airway management skills required during difficult intubations and rapid sequence induction . both percent agreement and kappa statistics
were used to analyze the reliability of the tool to increase the strength of the analysis .
the percent agreement does not take account of the possibility that raters may guess on some scores due to uncertainty .
it is therefore advised to calculate both percent agreement and kappa for analysis of inter - rater reliability .
a limitation of our study is that the assessments were done in real time , and , therefore , the assessors were not blinded to the trainees being assessed .
similar studies on assessment tools have been performed by assessing videotaped performance of procedural skills after masking the identity of the trainees or by employing assessors not known to the trainees and vice versa .
efforts were made to reduce this bias by the inclusion of residents who were not rotating with either of the two assessors at the time of assessment .
another limitation of this study is that a relatively long interval was allowed between the two assessment sessions .
this could have affected the value of test - retest reliability due to learning effect , which is the main shortcoming of test - retest reliability studies .
we recommend that the second assessment should be done after shorter intervals to ascertain the test - retest reliability of tools used for assessment of frequently performed procedure such as endotracheal intubation .
the absence of criteria for passing or failing the assessment may be considered as a limitation of the tool .
this has been overcome by adding a sentence : demonstrates ability to perform all aspects of the procedure independently with a yes / no option at the end of the procedural steps .
this section must be carefully filled by the assessors as it identifies whether or not the candidate was able to perform the entire procedure successfully and thus indicates that he / she has passed or not passed in performing the skill .
simulation - based skill assessment is now being described for assessment of residents ' ability to perform anesthetic skills .
however , financial constraints are a limiting factor in developing countries , where reliable and valid assessment tools like ours would be feasible and practical for routine assessment of trainees . as stated by cuschieri et al .
development of objective procedure - specific assessment tools for evaluation of procedural skills and their integration into training programs are the needs of the day . we believe that objective assessment with direct observation using well - defined criteria and rating scales has the potential to greatly improve assessment of procedural skills .
future research should focus on assessing improvement in procedural skills and quality of patient care with implementation of procedure - specific tools for assessment of skills in anesthesia training programs .
our results show that the tool designed by us to assess bag - mask ventilation and tracheal intubation skills in anesthesia trainees demonstrates good construct validity , excellent inter - rater reliability , and fair test - retest reliability . | background and aims : gaining expertise in procedural skills is essential for achieving clinical competence during anesthesia training .
supervisors have the important responsibility of deciding when the trainee can be allowed to perform various procedures without direct supervision while ensuring patient safety .
this requires robust and reliable assessment techniques .
airway management with bag - mask ventilation and tracheal intubation are routinely performed by anesthesia trainees at induction of anesthesia and to save lives during a cardiorespiratory arrest .
the purpose of this study was to evaluate the construct validity , and inter - rater and test - retest reliability of a tool designed to assess competence in bag - mask ventilation followed by tracheal intubation in anesthesia trainees.material and methods : informed consent was obtained from all participants .
tracheal intubation and bag - mask ventilation skills in 10 junior and 10 senior anesthesia trainees were assessed by two investigators on two occasions at a 3 - 4 weeks interval , using a procedure - specific assessment tool.results:average kappa value for inter - rater reliability was 0.91 and 0.99 for the first and second assessments , respectively , with an average agreement of 95% .
the average agreement for test - retest reliability was 82% with a kappa value of 0.39 .
senior trainees obtained higher scores compared to junior trainees in all areas of assessment , with a significant difference for patient positioning , preoxygenation , and laryngoscopy technique , depicting good construct validity.conclusion:the tool designed to assess bag - mask ventilation and tracheal intubation skills in anesthesia trainees demonstrated excellent inter - rater reliability , fair test - retest reliability , and good construct validity .
the authors recommend its use for formative and summative assessment of junior anesthesia trainees . | Introduction
Material and Methods
Data analysis
Results
Discussion
Conclusion
Financial support and sponsorship
Conflicts of interest |
materials rna oligonucleotides described in this work were
obtained from dharmacon and dna oligonucleotides were obtained from
invitrogen .
we cloned ns3 + and ns3 - 4a+-expressing
cdna from the hepatitis c viral genotype 1a ( version h77 , kindly donated by
dr .
the dna oligonucleotides
used for pcr of ns3 and ns3 - 4a were ns3 1a-1 and ns3 1a-2 or ns4a(1a)
bamhi .
an internal xhoi site in the ns3(1a)+ gene was removed through
a silent base pair change using a quikchange kit ( stratagene ) .
the dna
oligonucleotides used for the quikchange reaction were ns3 1a-3 and ns3
1a-4 .
the ns3 + and ns3/4a+ genes from hepatitis c viral
genotype 1b ( version n , kindly provided by dr .
stan lemon
( 24 ) ) were amplified and
cloned into pet15b according to the methods of beran et al .
the upstream dna
oligonucleotide used for pcr amplification was ns3 xhoi and the
downstream dna oligonucleotide was ns3 bamhi or ns4a. ns3 + and ns34a+ of both the 1a and 1b genotypes
were cloned into pet - sumo ( invitrogen ) using extaq pcr ( takara )
followed by ligation with linear pet - sumo according to the
manufacturer 's protocol .
the dna oligonucleotides used were the same as those
described above for pet15b cloning , except that the 5 pcr
oligonucleotide in the genotype 1a case was ns3(1a ) sumo start and in the
genotype 1b case was ns3(1b ) 5 sumo start . to produce pet - sumo - ns4a(1a)+ , the ns4a(1a)+ gene was pcr
amplified using the dna oligonucleotides ns4a 1a sumo start and ns4a 1a sumo
end and then cloned into pet - sumo . to produce the isolated ns3 protease
domain , a stop codon
was inserted in the ns3(1a)+ gene after amino
acid 188 in the pet - sumo - ns3(1a)+ plasmid
( 13 ) .
mutagenesis was
conducted via quikchange ( stratagene ) , using dna oligonucleotides
ns3prot(1a)-1 and ns3prot(1a)-2 . to create the ns3/4a polyprotein with an
ala
/ ala cleavage site instead of a thr / ser cleavage site , the wild - type
ns34a(1b)+ construct was mutated by quikchange ( stratagene ) ,
using oligonucleotides aa ns34a-1 and aa ns34a-2 .
all constructs
were verified initially through pcr screening as necessary and subsequently
sequenced for accuracy ( keck facility , yale university ) .
because there are a multitude of
reports describing the purification of various autocleaved and reconstituted
forms of the ns3 - 4a complex
( 17 ,
22 ,
23 ,
26 ,
27 ) and because all of these
factors greatly influence ns3 enzymatic function
( 13 ) , we believe it is
necessary to describe our constructs , purification strategies , and
reconstitutions in detail .
all proteins were purified according to the methods
described in beran et al . ,
( 13 ,
25 ) .
briefly , 4 liters of
escherichia coli culture in lb ( supplemented with 35 g / ml
kanamycin ) were grown at 37 c with shaking . upon reaching the exponential
growth phase ,
the temperature was shifted to 15 c , the cultures were
supplemented with 1 mm isopropyl
1-thio--d - galactopyranoside ( final concentration ) , and
subsequently incubated overnight at 15 c with shaking .
after lysing the
cells using an emusiflex c5 cell disruptor ( avestin ) , the cellular extract was
centrifuged at 10,000 rpm ( 14,500 g ) at 4 c for 10 min
before being passed through a nickel column .
the his6 and
his6-sumo fusion proteins were purified through a nickel column
( qiagen ) , treated with 10 units of sumo protease ( invitrogen ) overnight at 4
c , and subsequently passed through a gel filtration column ( hiload
superdex 200 16/60 ) equilibrated with a buffer containing 25 mm
hepes ( ph 8.0 ) , 0.3 m nacl , 10% glycerol , 1 mm
dithiothreitol , and 0.2% triton x-100 .
ninety - six 1.2-ml gel filtration
fractions were collected at a rate of 0.4 ml / min .
the ns3 - 4a complex eluted between fractions 45 and 55 , presumably in a
detergent micelle ( 17 ) .
fractions containing ns3 - 4a were identified using the ret - s1 protease assay
( the ret - s1 protease assay details are described later in this section ) as
well as through sds - page analysis . moreover , anti - ns3 and anti - ns4a
( monoclonal antibodies in both cases ) western blotting were used to confirm
the identity of the purified protein complex , which was then used for
experimentation ( see fig .
2 , b and
c , and the western blotting procedure described later in
this section ) .
free ns3 protein eluted from the gel filtration column between fractions 60
and 70 .
fractions containing ns3 were identified using rna unwinding assays
( 25 ) and sds - page analysis . in
addition
, anti - ns3 western blotting was used to confirm the identity of the
purified protein , which was then used for experimentation ( see
fig . 2b and
the
western blotting procedure described later in this section ) . because there is a 13-kda size difference between his - sumo tagged and
untagged protein ,
the untagged proteins were initially analyzed side by side
using sds - page with uncleaved his - sumo ns34a or uncleaved his - sumo - ns3
to verify that the purified proteins were , in fact , untagged species ( data not
shown ) . after purification , preparations were divided into 10-l aliquots and
stored at -80 c .
the ns3 and ns3 - 4a proteins were examined for purity and
their sizes compared using sds - page ( fig .
protein samples ( 40 pmol ) were subjected to
electrophoresis on a nupage 412% bistris gel ( invitrogen ) in mes - sds
buffer for 2 h at 200 v. the gel was subsequently stained with coomassie
blue .
for reconstitution of the protease complex using purified ns3 and purified
ns4a proteins ( ns3 + ns4a ) , we expressed his - sumo - ns3 as well as
his - sumo - ns4a in e. coli using the pet - sumo vector system
( invitrogen ) .
after partial purification using a nickel column , his - sumo - ns3
and a 4-fold excess of his - sumo - ns4a were mixed and incubated together at 4
c overnight in the presence of 10 units of sumo protease ( invitrogen ) .
reconstituted , untagged ns3 + 4a was subsequently isolated from untagged ns3
and untagged ns4a ( based upon size differences ) by passing the protein mixture
through a gel filtration column ( hiload superdex 200 16/60 ) using the same
methods as described above for autocleaved ns3 - 4a .
the same procedure was
followed to reconstitute and purify untagged ns3 protease domain with untagged
ns4a ( ns3 protease domain + ns4a ) . in this case ,
ns3 protease
domain alone eluted between gel filtration fractions 75 and 80 and ns3
protease domain + ns4a eluted between fractions 70 and 75 .
the ret - s1 serine
protease assay as well as sds - page analysis was used to identify the fractions
containing reconstituted ns3 protease domain + ns4a . in all cases ,
sds - page was performed using a bio - rad
miniprotean ii apparatus according to the manufacturer 's protocols .
the
western blot transfer was performed using a bio - rad transfer apparatus
according to the manufacturer 's instructions .
anti - ns3 and anti - ns4a
monoclonal antibodies were purchased from virogen ( watertown , ma ) and diluted
according to the manufacturer 's instructions . in these experiments ( as shown
in fig .
2 , b and
c ) , 25 pmol of each protein was subjected to
electrophoresis on a 12% sds - polyacrylamide gel for 1 h at 200 v.
subsequently , the proteins were transferred to a nitrocellulose membrane by
applying 100 v for 1 h at 4 c .
finally , the blots were incubated with
either anti - ns3 or anti - ns4a monoclonal antibody and subsequently developed
using a pierce supersignal western blot analysis kit .
protease assays protease assays were performed at 37 c
using the resonance energy transfer - s1 substrate ( ret - s1 ) ( anaspec ) designed
by taliani et al .
ret - s1 is an ns4a / ns4b junction mimic that fluoresces upon cleavage . all
assays , unless otherwise indicated , were performed in 60-l reaction
volumes containing 40 nm ns3 - 4a and 5 m ret - s1 .
the
data shown in fig . 6 and
table 1 were collected using
120 nm protein with the indicated amounts of ret - s1 substrate . in
table 1 , the
kcat values were calculated based upon the observation
that 75% of the protein was active in each case
( fig . 5 ) .
the buffer conditions
were the same as those normally used for helicase assays
( 13 ) : 25 mm
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\begin{equation*}{\mathrm{mops - nh}}_{4}^{+}\end{equation*}\end{document } ( ph 6.5 ) , 3 mm
mgcl2 , 1% glycerol , 2 mm dithiothreitol , 30
mm nacl , 0.2% ( v / v )
data
were collected using a cary eclipse spectrophotometer ( varian ) with a
temperature - controlled cuvette holder .
the initial background value ( the
value at time 0 , just before substrate addition ) was subtracted from all
subsequent time points .
table 1the ns3 helicase domain enhances ns3 - 4a protease activity the
proteolysis data were determined by monitoring ret - s1 cleavage on a
fluorescence spectrophotometer ( see experimental procedures ) .
the data shown were determined using proteins of the 1a genotype and is the
average of three experiments . the error values represent standard
deviation.proteinkmvmaxkcatkcat/km mpmol ret - s1 cleaved / spmol ret - s1 cleaved / s / pmol enzymepmol ret - s1 cleaved / s / pmol enzyme/m substrate ns3/4a
10.0 3.0
17 2
3.15 0.30
0.315
ns3 + ns4a
3.0 1.0
0.50 0.05
0.09 0.01
0.030
ns3 protease domain + ns4a
4.0 1.2
0.11 0.01
0.020 0.002
0.005
the ns3 helicase domain enhances ns3 - 4a protease activity the
proteolysis data were determined by monitoring ret - s1 cleavage on a
fluorescence spectrophotometer ( see experimental procedures ) .
the data shown were determined using proteins of the 1a genotype and is the
average of three experiments . the error values represent standard
deviation . the fraction of active ns3 - 4a protein was determined for both tagged and
untagged preparations by incubating a small amount of enzyme ( 40
nm ) with a large amount of ret - s1 substrate ( 5 m ) .
a
line was fit to the slope of the proteolysis data during the steady - state
phase of reaction .
this line was extrapolated to the intersection with the
y axis ( sigma plot , systat software ) , which corresponds to the
concentration of active enzyme present in the reaction
( 28 ) . in all cases throughout
this work ,
importantly ,
similar values for the fraction of active enzyme ( 75% ) were observed when
the ns3 - 4a concentration was raised to 120 nm or when the ret - s1
concentration was raised well above the km ( 10 3
m ) ( table 1 ) to
20 m ( data not shown ) . similarly , when reconstituted ns3
protease domain + ns4a and ns3 + ns4a were assayed with ret - s1 concentrations
well above the km values ( 810 m
ret - s1 , whereas km = 4.0 1.2 and 3.01.0
m , respectively ) ( table
1 ) , a similar enzymatic active fraction was observed for both
complexes ( 75% ) ( data not shown ) . when protease activities of ns3 - 4a(1a )
and ns3 - 4a(1b ) were compared using the ret - s1 analyses , results were similar
for both genotypes
the ns3 - 4a complex can be purified in a highly active
form to study ns3 - 4a protease function , we needed to purify ns3 in
complex with its ns4a co - factor ( i.e. ns3 - 4a ) . in previous studies
,
ns3 - 4a has been overexpressed in insect cells
( 27 ) or in e. coli
( 12 ,
17 ) . in all of these cases ,
additional amino acids were fused to the ns3 n terminus for the purpose of
protein purification ( 12 ,
17 ) and lysine residues were
added to the ns4a c terminus to increase solubility
( 17 ) . to our knowledge , a
recombinant ns3 - 4a complex that lacks tags or additional modifying amino acids
has never been produced .
we therefore designed an approach for generating
native , full - length ns3 - 4a complex ( e.g. lacking modifications to the
terminal sequences of ns3 or ns4a ) using the pet - sumo expression system in
e. coli ( fig .
the protein of interest is tagged at the n terminus with the sumo
polypeptide , which enhances solubility of the protein and is then readily
removed upon incubation with the sumo protease , thereby resulting in native
protein that lacks additional amino acids
( 29 ) .
we expressed ns3 - 4a as a his - sumo - ns3/4a polyprotein in e. coli ,
designing the construct so that ns3 would be expected to spontaneously cleave
ns4a from its c terminus ( fig .
1b ) ( 17 ,
27 ) , thereby forming an active
protease complex ( 4 ) .
after
purification on a nickel column , the his - sumo fusion protein was cleaved by
sumo protease from ns3 , generating a native n terminus
( fig .
the protein
mixture was subsequently passed through a gel filtration column to separate
native protein complex from uncleaved fusion protein ( see experimental
procedures ) .
the resultant cleaved , purified ns3 - 4a complex was
95% pure ( fig .
2a ) . to determine whether the purified ns3 - 4a protein had autocatalytically
cleaved properly , forming two separate proteins ( ns3 and ns4a ) , we subjected
the complex to denaturing electrophoresis side by side with ns3 and an
uncleavable ns3/4a polyprotein control .
the uncleavable ns3/4a polyprotein was
produced by mutating the conserved junction amino acid sequence between ns3
and ns4a from thr / ser to ala / ala .
sds - page and western blot analysis show that
the ns3 component of the wild - type ns3 - 4a preparation has the same
electrophoretic mobility as ns3 ( in fig .
2a , compare lanes 2 and 3 of the
coomassie - stained gel and in fig .
2b , compare lanes 1 and 2 of the
anti - ns3 western blot ) .
however , the uncleaved ns3/4a polyprotein migrates
more slowly than ns3 , as expected due to its larger size ( in
fig .
2a , compare
lane 4 to lanes 2 and 3 and in
fig .
coomassie staining does
not reveal any ns3/4a polyprotein in our ns3 - 4a preparation
( fig . 2a , lane
3 ) . however , western blots are more sensitive than coomassie staining ,
and consistent with this , the anti - ns3 western blots reveal a small fraction
of uncleaved ns3/4a polyprotein in the wild - type ns34a preparation
( fig . 2b , note the
faint upper band in lane 2 that migrates similarly to the ns3/4a
polyprotein in lane 3 ) . taken together , these results demonstrate
that the preparation of wild - type ns3 - 4a complex is composed primarily of
fully cleaved ns3 and ns4a molecules . the catalytic function and
conformational homogeneity of this preparation
figure 1.composition and purification of ns3 - 4a . the ns3 - 4a complex
organization and
a , pro refers to the serine protease domain
and the roman numerals indicate the respective ns3 helicase
subdomains .
the regions where atp , rna , and the ns4a co - factor bind are
indicated as well .
the protein construct expressed in e. coli is
depicted in panel b. the numbers below the map refer to the
hcv polyprotein numbering of the amino acids of ns3 - 4a . in panel c ,
his - sumo - ns3 and his - sumo - ns4a fusion construct designs are presented .
these
proteins were purified separately and their his - sumo tags were removed using
the same methods as for ns3 - 4a .
subsequently , the native ns3 or native ns3
protease domain was reconstituted with native ns4a ( see experimental
procedures ) .
the ns3 - 4a complex
organization and construct design are illustrated schematically ( a
and b ) . in panel
a , pro refers to the serine protease domain
and the roman numerals indicate the respective ns3 helicase
subdomains . the regions where atp , rna , and the ns4a co - factor bind are
indicated as well .
the protein construct expressed in e. coli is
depicted in panel b. the numbers below the map refer to the
hcv polyprotein numbering of the amino acids of ns3 - 4a . in panel c ,
his - sumo - ns3 and his - sumo - ns4a fusion construct designs are presented .
these
proteins were purified separately and their his - sumo tags were removed using
the same methods as for ns3 - 4a .
subsequently , the native ns3 or native ns3
protease domain was reconstituted with native ns4a ( see experimental
procedures ) .
the presence of ns4a in the ns3 - 4a preparation was assessed using
denaturing electrophoresis and western blot analysis with antibodies against
ns4a .
the anti - ns4a western blots indicate that the wild - type ns3 - 4a
preparation consists of both free ns4a ( which is difficult to detect because
it is so small and diffuses rapidly from the gel ) and a fraction of uncleaved
ns3/4a polyprotein ( in fig .
the free
ns4a migrates at the correct position , 6 kda , near the bottom of the gel
( fig .
2c , lane
2 ) . taken together , the electrophoretic and immunoaffinity methods
provide physical evidence that the ns3 - 4a preparation consists of cleaved ns3
and ns4a components . nonetheless , it was still essential to demonstrate that
the complex was catalytically active and to assess the fraction of active
molecules in the preparation .
a , purified
proteins were subjected to denaturing electrophoresis on a 412%
gradient gel .
purified ns3 ( lane 2 ) , ns3 - 4a ( lane 3 ) , and
ns3/4a polyprotein ( lane 4 ) were subjected to electrophoresis side by
side for comparison of mobilities .
the band shown in lane 3
represents the ns3 component of a native , fully cleaved ns3 - 4a preparation .
the band shown in lane 4 represents an ns3/4a polyprotein preparation
produced by mutating the thr / ser cleavage site between ns3 and ns4a to a
non - cleavable sequence ( aa ) . in panels b and c ,
anti - ns3 and
anti - ns4a western blot analysis confirm the identity of our purified proteins
( see experimental procedures ) .
b , lane 1 contains purified ns3 ,
lane 2 contains purified , full - length ns3 - 4a , and lane 3
contains purified ns3/4a polyprotein .
truncated forms of ns3 are visible below
the full - length protein in each lane in the anti - ns3 blot .
these truncated
forms of ns3 are likely produced during bacterial expression as well as during
the multiday purification performed in the absence of protease inhibitors .
these truncated forms could represent either n- or c - terminal truncations of
ns3 as the monoclonal anti - ns3 antibody binds to the central region of the
helicase domain .
lane 1 contains purified ns3 , lane 2 contains
purified , full - length ns3 - 4a , and lane 3 contains purified ns3/4a
polyprotein .
truncated forms of the ns3/4a polyprotein are visible
below the full - length polyprotein in the anti - ns4a blot .
these
truncated forms of ns3/4a polyprotein likely represent n - terminal degraded
ns3/4a as the monoclonal anti - ns4a antibody binds the final 11 c - terminal
residues of ns4a .
a , purified
proteins were subjected to denaturing electrophoresis on a 412%
gradient gel .
purified ns3 ( lane 2 ) , ns3 - 4a ( lane 3 ) , and
ns3/4a polyprotein ( lane 4 ) were subjected to electrophoresis side by
side for comparison of mobilities .
the band shown in lane 3
represents the ns3 component of a native , fully cleaved ns3 - 4a preparation .
the band shown in lane 4 represents an ns3/4a polyprotein preparation
produced by mutating the thr / ser cleavage site between ns3 and ns4a to a
non - cleavable sequence ( aa ) . in panels
b and c , anti - ns3 and
anti - ns4a western blot analysis confirm the identity of our purified proteins
( see experimental procedures ) .
b , lane 1 contains purified ns3 ,
lane 2 contains purified , full - length ns3 - 4a , and lane 3
contains purified ns3/4a polyprotein .
truncated forms of ns3 are visible below
the full - length protein in each lane in the anti - ns3 blot .
these truncated
forms of ns3 are likely produced during bacterial expression as well as during
the multiday purification performed in the absence of protease inhibitors .
these truncated forms could represent either n- or c - terminal truncations of
ns3 as the monoclonal anti - ns3 antibody binds to the central region of the
helicase domain .
lane 1 contains purified ns3 , lane 2 contains
purified , full - length ns3 - 4a , and lane 3 contains purified ns3/4a
polyprotein .
truncated forms of the ns3/4a polyprotein are visible
below the full - length polyprotein in the anti - ns4a blot .
these
truncated forms of ns3/4a polyprotein likely represent n - terminal degraded
ns3/4a as the monoclonal anti - ns4a antibody binds the final 11 c - terminal
residues of ns4a . to quantify the fraction of fully processed , active ns3 - 4a in the
preparation , we analyzed the proteolysis of a depsipeptide substrate known as
ret - s1 , which was originally derived from the ns4a
both free ns3
and the uncleavable mutant ns3/4a polyprotein are proteolytically inactive , as
they fail to react with ret - s1 ( see below ) .
however , the wild - type ns3 - 4a
preparation displays efficient protease activity against ret - s1 , with a rate
constant of 0.020 0.001 m product / s ) .
this value is
significantly faster than values obtained by others for ns3 - 4a with a
non - native n terminus incubated with a 5ab peptide substrate
( 27 ) . importantly ,
burst
kinetics experiments reveal that the ns3 - 4a preparation contains 75
14% active enzyme ( fig .
3 ) ,
indicating that the majority of the population is properly folded , assembled ,
and catalytically active .
that this value falls short of 100% is consistent
with the electrophoretic analyses , which indicated that 20% of the
wild - type ns3 - 4a preparation remains uncleaved
( fig .
the
physical and functional information provided by both the electrophoretic and
proteolysis assays indicate that we have successfully overexpressed untagged ,
unmodified ns3 - 4a in a form that is conformationally homogeneous ,
appropriately cleaved , and highly reactive .
this approach now makes it
possible to quantitatively assess the proteolysis and helicase activities of
the ns3 - 4a complex , and to compare these activities with the behavior of other
ns3 constructs .
figure 3.steady-state proteolysis of ret - s1 by ns3 - 4a and his - ns3 - 4a .
the
y intercept of the line in each case corresponds to the active
fraction of protein ( see experimental procedures ) . for ns3 - 4a
( solid circles ) ,
for
his - ns3 - 4a ( hatched squares ) , the active fraction was 25
12% .
ns3/4a polyprotein did not display any measurable serine protease
activity ( solid squares ) .
similar results were observed using ns3 - 4a(1a )
and his - ns3 - 4a(1a ) .
this data are the average of three experiments and the
error values represent standard deviation .
steady - state proteolysis of ret - s1 by ns3 - 4a and his - ns3 - 4a .
the
y intercept of the line in each case corresponds to the active
fraction of protein ( see experimental procedures ) . for ns3 - 4a
( solid circles ) , the active fraction was 75 14% . for
his - ns3 - 4a ( hatched squares ) ,
ns3/4a polyprotein did not display any measurable serine protease
activity ( solid squares ) .
similar results were observed using ns3 - 4a(1a )
and his - ns3 - 4a(1a ) .
this data are the average of three experiments and the
error values represent standard deviation .
most previous work on ns3 has utilized n - terminal his - tagged variants of
the protein ( 12 ,
13 ,
30 ,
31 ) . for comparison purposes
,
we also constructed an ns3 - 4a complex with a his tag on the n terminus of ns3 .
interestingly , kinetic analysis indicates that the his - tagged ns3 - 4a
preparation reacts 2-fold more slowly than the untagged variant ( 0.010
0.001 m product / s ) , and contains only a 25
12% active population ( fig . 3
and experimental procedures ) .
a his tag at the ns3 n terminus is
therefore detrimental for folding and/or assembly of the protease domain , and
it also diminishes the ultimate levels of proteolytic activity by the tagged
ns3 - 4a complex .
these findings are consistent with the fact that n - terminal
ns3 his tags are in close proximity to the protease active site .
uncleaved ns3/4a polyprotein lacks protease and helicase
activities the uncleavable ns3/4a polyprotein construct ( containing
2 alanines where the threonine / serine cleavage sequence n - terminal to ns4a
would have been ) was synthesized originally as a tool that would provide a
size standard for determining the presence of any uncleaved ns3/4a polyprotein
in wild - type preparations of the complex .
however , the mutant ns3/4a construct
has also provided valuable additional information about the activities that we
should expect for hcv polyproteins . to our knowledge , catalytic activities of
uncleaved ns3/4a constructs have never been tested .
here we observed , using
the mutant polyprotein , that uncleaved ns3/4a molecules are catalytically
inactive ( i.e. they lack both serine protease and helicase activity ) .
they fail to display protease activity for substrates provided in
trans ( i.e. ret - s1 ) ( fig .
thus , proper
assembly of the cleaved , ns3 - 4a complex is required for all the enzymatic
activities of this protein .
it is therefore likely that many sections of the
polyprotein are not appropriately folded or functional until the individual
proteins are cleaved apart .
figure 4.steady-state velocity curves for ns3 - 4a proteolysis of ret - s1 under a
range of ph and salt conditions .
the steady - state rates of proteolysis
were 0.006 0.001 m product / s at ph 6.5 , 30
mm nacl ( solid circles ) , 0.019
0.001m product / s at ph 8 , 30 mm nacl ( pierced
circles ) , 0.019 0.001 m product / s at ph 6.5 , 75
mm nacl ( solid diamonds ) , 0.020 0.001
m product / s at ph 8 , 75 mm nacl ( pierced
diamonds ) , 0.010 0.001 m product / second at ph
6.5 , 200 mm nacl ( solid squares ) , and 0.010 0.001
m product / s at ph 8 , 200 mm nacl ( pierced
squares ) .
the active fraction in each case , as determined by intersection
with the y intercept , was 68 9% for ph 6.5 , 30 mm
nacl , 84 16% for ph 8.0 , 30 mm nacl , 84 15% for
ph 6.5 , 75 mm nacl , 70 12% for ph 8.0 , 75 mm
nacl , 95 5% for ph 6.5 , 200 mm nacl , and 95 5%
for ph 8.0 , 200 mm nacl .
the data shown were determined using
ns3 - 4a of the 1b genotype and represent the steady - state data points fit to a
line .
this data are the
average of three experiments and the error values represent standard
deviation .
steady - state velocity curves for ns3 - 4a proteolysis of ret - s1 under a
range of ph and salt conditions .
the steady - state rates of proteolysis
were 0.006 0.001 m product / s at ph 6.5 , 30
mm nacl ( solid circles ) , 0.019
0.001m product / s at ph 8 , 30 mm nacl ( pierced
circles ) , 0.019 0.001 m product / s at ph 6.5 , 75
mm nacl ( solid diamonds ) , 0.020 0.001
m product / s at ph 8 , 75 mm nacl ( pierced
diamonds ) , 0.010 0.001 m product / second at ph
6.5 , 200 mm nacl ( solid squares ) , and 0.010 0.001
m product / s at ph 8 , 200 mm nacl ( pierced
squares ) . the active fraction in each case , as determined by intersection
with the y intercept , was 68 9% for ph 6.5 , 30 mm
nacl , 84 16% for ph 8.0 , 30 mm nacl , 84 15% for
ph 6.5 , 75 mm nacl , 70 12% for ph 8.0 , 75 mm
nacl , 95 5% for ph 6.5 , 200 mm nacl , and 95 5%
for ph 8.0 , 200 mm nacl .
the data shown were determined using
ns3 - 4a of the 1b genotype and represent the steady - state data points fit to a
line .
this data are the
average of three experiments and the error values represent standard
deviation .
ns3 - 4a exhibits robust serine protease activity under a variety of salt
and ph conditions it has previously been reported that ns3 - 4a only
functions as a robust serine protease under conditions of high salt ( 150
mm nacl ) and high ph ( 7.58.0 )
( 17 ,
27 ) .
these same conditions are
incompatible with those required for ns3 helicase activity
( 13 ,
17 ,
32 ) , and it therefore has been
suggested that the two activities ( proteolysis and unwinding ) might be
mutually exclusive ( 17 ) .
we
therefore sought to determine whether protease activity of the wild - type
ns3 - 4a complex is restricted to the narrow range of previously reported
conditions . to this end , we measured ns3 - 4a serine protease activity under a
variety of ph ( 6.5 and 8.0 ) and salt conditions ( 30 , 75 , and 200 mm
nacl ) ( fig .
4 ) . we did not
observe significant differences in the active fraction of protein
( i.e. the y intercept of the velocity plots ) under these
varying ph and salt conditions , particularly when accounting for error
( fig .
the
steady - state proteolysis velocities in 30 mm nacl at ph 6.5 and 8.0
were observed to be 0.006 0.001 and 0.019 0.001
m / s , respectively . in 200 mm nacl at ph 6.5 and 8.0 ,
the steady - state proteolysis velocities were observed to be 0.010
0.001 m / s in both cases .
therefore , native , full - length ns3 - 4a
is a robust serine protease under a broad range of salt and ph conditions ,
including those that are compatible with helicase activity .
figure 5.steady-state proteolysis of ret - s1 by reconstituted ns3 + ns4a and ns3
protease domain + ns4a .
the steady - state velocity for ns3 + ns4a is 0.005
001 m / s ( pierced circles ) and for ns3
protease ns4a is 0.001 0.001 m / s ( solid
squares ) .
the y intercept of the fitted lines show ns3
+ ns4a and ns3 protease domain + ns4a to have 75 10% active fraction
and 75 12% active fraction , respectively .
the data shown are the
average of three experiments and the error values represent standard
deviation .
steady - state proteolysis of ret - s1 by reconstituted ns3 + ns4a and ns3
protease domain + ns4a .
the steady - state velocity for ns3 + ns4a is 0.005
001 m / s ( pierced circles ) and for ns3
protease ns4a is 0.001 0.001 m / s ( solid
squares ) .
the y intercept of the fitted lines show ns3
+ ns4a and ns3 protease domain + ns4a to have 75 10% active fraction
and 75 12% active fraction , respectively .
the data shown are the
average of three experiments and the error values represent standard
deviation .
ns3hel enhances ns3 - 4a serine protease activity given that
efficient ns3 helicase activity requires the protease domain
( 13 ) , we asked whether ns3
protease activity is enhanced by the helicase domain . to answer this question ,
it was necessary to build new types of protein expression constructs because
polyprotein precursors containing the isolated protease domain ( i.e.
a his - sumo - ns3 protease domain / ns4a polyprotein construct ) did not undergo
autocatalytic cleavage to form an ns3 protease-4a complex ( data not shown ) .
we
therefore expressed the ns3 protease domain and full - length ns4a as separate
his - sumo fusion proteins ( fig .
the partially purified his - sumo - ns3 protease domain and his - sumo - ns4a proteins
were then combined in the presence of sumo protease and incubated to form an
active complex of the native ns3 protease domain and native ns4a ( ns3 protease
+ ns4a , see experimental procedures ) .
similarly , we
reconstituted full - length ns3 + ns4a as a control for comparative purposes
( fig .
similar to the preparation of co - expressed ns3 - 4a , 75 10% of the
reconstituted ns3 + ns4a preparation and 75 12% of the reconstituted
ns3 protease + ns4a preparations were proteolytically active
( fig .
comparison of the
kinetic parameters for ns3 - 4a , ns3 protease domain + ns4a , and ns3 + ns4a
proteolysis of ret - s1 revealed that the km values for
cleavage of substrate did not differ significantly ( km =
10.0 3.0 m for ns3 - 4a , 4.0 1.2
m for ns3 protease + ns4a , and 3.0 1.0
m for ns3 + ns4a ) ( fig .
6 and table 1 ) ,
indicating that ret - s1 binds similarly to all the constructs .
figure 6.steady-state proteolysis rates of ret - s1 by ns3 - 4a , ns3 + ns4a , and ns3
protease domain + ns4a in the presence of varying substrate
concentrations .
the data were fit to the michaelis - menten equation to
determine km , vmax , and
kcat values .
steady - state proteolysis rates of ret - s1 by ns3 - 4a , ns3 + ns4a , and ns3
protease domain + ns4a in the presence of varying substrate
concentrations .
the data were fit to the michaelis - menten equation to
determine km , vmax , and
kcat values .
however , the maximal velocities of proteolysis by the three enzyme
constructs were substantially different .
the rates of cleavage for ns3 - 4a , ns3
+ 4a , and ns3 protease + 4a were 17.0 2.0 , 0.50 0.05 , and
0.11 0.01 pmol of ret - s1 cleaved / s , respectively
( fig .
the large rate
differences between the co - expressed ns3 - 4a and the reconstituted ns3 + ns4a
constructs suggest that the ns4a cofactor does not associate and promote
protease folding as effectively when presented in trans
( i.e. 25 mm hepes ( ph 8.0 ) , 0.3 m nacl , 10%
glycerol , 1 mm dithiothreitol , 0.2% triton x-100 ) .
many conditions were explored to optimize reconstitution with ns4a in
trans . despite varying detergent concentrations ( 0.11% , using
triton x-100 or chaps ) ,
varying glycerol concentrations from 10 to 50% , and
varying nacl from 30 to 200 mm , increases in proteolytic activity
were not observed ( data not shown ) . taken together ,
these data suggest that the protease domain active site is
optimally formed only upon co - expression with ns4a .
nonetheless , the
reconstituted complexes retain significant levels of activity and provide
valuable tools for structure / function studies
( 5 ,
22 ) . given the inherently higher reactivity of co - expressed ns3 - 4a , it is clear
that proteolytic activity of the isolated protease domain ( ns3 protease + 4a )
is directly comparable only with the reconstituted complex containing
full - length ns3 ( ns3 + 4a ) . when these two constructs are compared , the
isolated protease domain is much less reactive than full - length ns3
( fig .
indeed , ns3
protease + ns4a is 5- and 6-fold less efficient than ns3 + ns4a when
vmax and kcat / km
values are compared , respectively ( table
1 ) .
it is notable that the major effects are on
kcat , which suggests that the helicase domain directly
influences catalytic function of the protease active site .
therefore , ns3
protease activity is enhanced by the presence of the ns3hel domain , indicating
that the two domains have evolved to become completely interdependent .
by creating new types of ns3 constructs and monitoring their enzymatic
activities , we have evaluated the structural integrity and catalytic potential
of the serine protease domain .
we have established that ns3 protease activity
is strongly influenced by its structural context , and that positive and
negative effects on catalysis are induced by the presence of adjacent domains
and uncleaved polyprotein moieties , respectively . taken together
, our findings
suggest that ns3 protease activity is tuned and regulated by other viral
components , which have coevolved to maximize the role of the protease in the
hcv viral lifecycle .
the helicase and the protease are interdependent enzymes within a
single protein like many viral proteins , ns3 is multifunctional and
it contains different enzymatic activities within a single protein chain .
it
has long been known that the protease and atpase / helicase activities of ns3
are located on separate domains of ns3 , and given the disparate nature of
these activities , and their presumably different roles in function of the
virus , it was generally assumed that the protease and helicase activities had
no relationship to one another .
however , structural analysis revealed that the
protease domain is located in close proximity to the atpase and rna binding
sites of ns3hel ( 33 ,
34 ) .
we therefore wondered
whether the two enzymes in ns3 might actually be interdependent , having
evolved to function optimally as a unit .
allosteric coactivation would have
important ramifications for the function of the hcv replication complex and
for the testing of hcv inhibitors , which tends to be studied with the isolated
domains . in our first study of this problem
, we showed that rna binding , atpase , and
rna unwinding activities of the helicase are all stimulated by the presence of
the covalently attached ns3 protease domain
( 13 ) . here
we demonstrate that
the ns3 - 4a serine protease activity is stimulated by the presence of ns3hel as
well .
importantly , the ns3hel domain must be covalently attached to the ns3
protease domain , as adding ns3hel in trans had no effect on ns3
protease domain + ns4a protease activity ( data not shown ) .
thus , the different
enzymatic domains of ns3 are fully interdependent , which suggests that they
have coevolved and that their presence on a single protein may have provided a
selective advantage to the virus .
perhaps most importantly , the results
suggest that these seemingly disparate enzymatic activities may somehow be
coupled during some aspect of viral function .
the ns3/4a polyprotein fails to catalyze proteolysis or
unwinding just as the helicase domain activates serine protease
activity , the presence of an uncleaved ns3/4a junction inhibits serine
protease activity .
the data presented herein show that polyprotein cleavage
must occur before ns3 becomes proteolytically active .
even ns3 helicase
activity requires cleavage of the ns3/4a junction sequence before any rna
unwinding activity is observed ( data not shown ) .
taken together , these data
indicate that polyprotein processing must necessarily be a very early event ,
as it is required for catalytic functions of viral constituents .
proteolysis and helicase activities are not mutually
exclusive previous work has suggested that ns3 - 4a protease activity
occurs under conditions that are incompatible with helicase activity .
this
finding implies that helicase and protease activities might be mutually
exclusive , or that their viral functions are completely unrelated .
importantly , it had been reported that ns3 - 4a protease activity is very
sensitive to salt concentrations when the protein is studied at low
concentrations ( < 1 nm ) and less sensitive to salt concentrations
at higher protein concentrations ( 1 nm )
( 22 ) .
we therefore conducted
protease assays with relatively high ns3 - 4a concentrations ( 40120 nm )
that fall within the range typically employed for rna helicase assays
( 12 ,
13 ,
25 ,
31 ) .
our ability to observe
helicase and protease activity under the same conditions may also stem from
the fact that we utilized untagged ns3 - 4a , which is more reactive than
his - tagged ns3 - 4a .
an n - terminal his tag reduces both the rate of serine
protease activity and the active fraction of protein
( fig .
proteolysis
by untagged ns3 - 4a is not highly salt dependent and the reaction is efficient
under diverse conditions ( fig .
4 ) .
earlier work has demonstrated that ns3 functions as a robust rna helicase
at nacl concentrations as high as 100 mm and it can unwind rna at
even higher nacl concentrations , although less efficiently
( 13 ) . the fact that ns3 can
cleave protein or unwind rna in the same range of salt and ph conditions
( including ionic conditions that approach physiological ( 150 mm ) )
indicates that ns3 - 4a is capable of transitioning smoothly between proteolysis
and rna unwinding during various stages of hcv replication . immediately after
ns3 - 4a autocleaves from the hcv polyprotein
, it may unwind hcv rna or use
ns3hel as a motor to translocate along the hcv polyprotein and scan for
subsequent peptide cleavage sites .
protein translocation by ns3hel has never
been demonstrated , but its potential importance is underscored by the
translocase activities of related proteins such as clpx
( 3537 ) . reconstituted forms of ns3 - 4a are significantly less reactive than
autocleaved ns3 - 4a to perform this study , it was necessary to
create reconstituted forms of the ns3 - 4a complex , in which the 4a protein was
added in trans to the serine protease domain or to the full - length
ns3 .
this provided an opportunity to compare the reactivities of ns3 - 4a
molecules that , despite identical sequences , were produced in different ways
( through autocleavage of polyprotein or reconstitution from separate
proteins ) . here
we observe that autoproteolyzed ns3 - 4a is a much more active
serine protease than complexes that were reconstituted from separate ns4a
molecules ( ns3 + ns4a ) .
consistent with this , the ns3 - 4a complex that is
disrupted by dilution into low salt buffers
( 17 ) can not be restored to
normal levels of the protease activity by the addition of salt ( data not
shown ) .
specifically , we observe that ns3 - 4a has a vmax
for ret - s1 proteolysis that is 34 times faster than ns3 + ns4a
( table 1 ) .
intriguingly , the
km values for ns3 - 4a and ns3 + ns4a proteolysis of ret - s1
do not vary significantly ( table
1 ) . taken together
, the kinetic analysis of proteolysis by ns3 - 4a ,
ns3 + ns4a , and ns3 protease + ns4a suggests that all of these constructs bind
substrate with a similar affinity , but they have large differences in
efficiency of chemical catalysis .
one possible explanation is that the ns4a co - factor may not properly
intercalate with the ns3 protease domain when ns3 and ns4a are simply mixed ,
potentially resulting in misalignment of active site residues within the ns3
protease domain .
autocleavage between ns3 and ns4a and the concurrent
intercalation of ns4a into the ns3 protease domain requires a specific
cleavage site sequence that slows the proteolysis reaction ( thr / ser as opposed
to cys / ser at other hcv polyprotein sites )
( 38 ) .
presumably , this slow
autocleavage between ns3 and ns4a facilitates the correct intercalation of
ns4a into the ns3 protease domain as ns4a is being cleaved from the ns3 c
terminus ( 38 ) .
therefore ,
whereas our findings suggest that the helicase domain allosterically
influences the protease active site , an alternative interpretation of the
results is that a coexpressed helicase domain is required for proper folding
of the protease domain and/or appropriate docking of the 4a cofactor . in
addition
, the expression strategy employed in this work may not optimally
recapitulate folding of the individual proteins . but
regardless of the
mechanistic basis for the apparent codependence of the protease and the
helicase , the data clearly show that reconstituted ns3 and 4a constructs are
significantly impaired , which has major implications for the design of protein
constructs used in drug screening and for structure / function efforts on
hcv .
many investigators have aimed to disrupt ns3 - 4a protease activity as an
approach toward antiviral therapeutics and various forms of the complex have
been employed in these studies
( 1923 ,
39 ) .
although it has been
suggested that ns3hel might enhance ns3 - 4a protease activity
( 23 ) , this synergy was not
demonstrated until now .
our findings suggest that screens for ns3 - 4a protease
inhibitors should involve full - length ns3 constructs rather than truncated
protease domains .
moreover , these screens should utilize constructs in which
ns3 - 4a is produced through autoproteolysis rather than reconstitution .
indeed ,
a recent study comparing the effects of ns3 - 4a serine protease inhibitors on
full - length ns3 - 4a produced through autocleavage , ns3 + ns4a peptide , and ns3
protease domain + ns4a peptide demonstrated that biln 2061 and vx-950 are much
better inhibitors of autocleaved ns3 - 4a serine protease activity than of
reconstituted ns3 + ns4a peptide or ns3 protease domain + ns4a
( 22 ) .
the greater protease
activity of autocleaved ns3 - 4a creates the potential for increased sensitivity
when measuring protease activity in the presence of new drugs ( i.e.
more easily measured inhibition of protease activity in screening assays of
compound libraries ) , which will lead to a greater diversity of promising lead
compounds . given the synergies observed among components of the ns3
helicase - protease , it will be interesting to monitor the influence of ns4a on
rna binding , atpase , and rna unwinding activities of ns3 in future
studies . | non - structural protein 3 ( ns3 ) is a multifunctional enzyme possessing
serine protease , ntpase , and rna unwinding activities that are required for
hepatitis c viral ( hcv ) replication .
hcv non - structural protein 4a ( ns4a )
binds to the n - terminal ns3 protease domain to stimulate ns3 serine protease
activity .
in addition , the ns3 protease domain enhances the rna binding ,
atpase , and rna unwinding activities of the c - terminal ns3 helicase domain
( ns3hel ) . to determine whether ns3hel enhances the ns3 serine protease
activity , we purified truncated and full - length ns3 - 4a complexes and examined
their serine protease activities under a variety of salt and ph conditions .
our results indicate that the helicase domain enhances serine protease
activity , just as the protease domain enhances helicase activity .
thus , the
two enzymatic domains of ns3 - 4a are highly interdependent .
this is the first
time that such a complete interdependence has been demonstrated for a
multifunctional , single chain enzyme .
ns3 - 4a domain interdependence has
important implications for function during the viral lifecycle as well as for
the design of inhibitor screens that target the ns3 - 4a protease . | EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material |
many efforts have been undertaken to unravel the role of the immune system in the pathogenesis of nonalcoholic steatohepatitis ( nash ) . the balance between proinflammatory th17 and the regulatory t cells ( tregs ) , which plays a major role in the control of inflammation , appears to be disturbed in various diseases , such as autoimmune , infectious , inflammatory , and metabolic diseases .
th17 are a subset of effector t cells that express the nuclear receptor retinoid - related orphan receptor t ( rort ) and produce il17 .
an upregulation of the th17 pathway has been described in the liver in fatty liver disease and in liver fibrosis , which is the hallmark of progressive liver disease .
opposite to these findings , a protective role of il17 was described in obesity , since il17 acted as a negative regulator of adipogenesis and glucose metabolism in mice and delayed the development of obesity .
accordingly , a reduction of th17 in abdominal adipose tissue ( aat ) was described in mice fed a high fat diet ( hfd ) .
tregs derive from cd4 + th0 cells and constitutively express cd25 ( the il2 receptor chain ) .
in addition , they express foxp3 ( forkhead / winged helix transcription factor ) that is crucial for their function . in a hfd mouse model , a liver - specific and reversible depletion of tregs was observed , while human studies showed their increase in liver biopsies of nash patients with a more severe disease .
moreover , tregs were specifically reduced in the aat of preclinical insulin - resistant models of obesity [ 6 , 10 ] , while in obese patients some authors described an increase of foxp3 rna in the subcutaneous adipose tissue ( sat ) and others found a downregulation of foxp3 rna only in obese patients without insulin resistance , while no difference was found when comparing insulin - resistant obese patients and lean controls .
other key factors in the pathogenesis of nash are the adipokines , which are mainly produced in the white adipose tissue .
high to normal serum leptin levels can be found in nash patients independently from the body mass index ( bmi ) .
leptin has also important proinflammatory effects , acting both on the innate and on the adaptive immunity [ 14 , 15 ] .
recent lines of evidence have shown that leptin is implicated in the upregulation of the th17 pathway [ 16 , 17 ] and in the suppression of treg proliferation . in order to further elucidate the differential role of th17 and tregs at different anatomical sites in the pathogenesis of nash , we analyze in the present study the treg and th17 populations in mice fed hfd and their location - specific modifications with particular interest in liver and aat and sat .
moreover we analyze the possible correlation between the tissue - specific variations of these cells and the diet - induced disturbances of metabolic homeostasis .
c57bl6/j six - to - eight - week - old male mice were purchased ( charles river laboratories , brussels , belgium ) and kept at the animal facility of the university of antwerp in temperature- and light - controlled conditions .
mice were fed for 36 weeks with either hfd ( with 60% of kcals derived from fat , 20% from proteins , and 20% from carbohydrate ) ( research diets ) or normal chow , in line with what was previously described .
mice were weighed weekly and just prior to sacrifice . at sacrifice blood samples , sat , epididymal fat tissue and liver were obtained . in mice
epididymal fat is able to influence the major components of the metabolic syndrome , as shown by the improvement of insulin sensitivity and the decrease of leptin secretion by sat after its removal .
the protocols were approved by the antwerp university ethical committee on animal experiments ( permit number : 2012 - 47 ) .
the animals received human care and were treated according to the helsinki declaration , the national guidelines for animal protection , and the guide for the care and use of laboratory animals
blood samples on ethylenediaminetetraacetic acid ( edta ) were collected from anesthetized mice by cardiac puncture . sat and aat and liver were excised after flushing organs through the right ventricle with 5 ml of cold phosphate buffered saline ( pbs ) ( sigma ) and cut into small pieces of 1 - 2 mm .
liver tissue was smashed through a sieve and intrahepatic immune cells were harvested after centrifugation with 33% percoll ( sigma ) and heparin .
fat tissue fragments were incubated for 60 minutes with collagenase type ii ( sigma ) , filtered , and incubated with dnase i ( sigma ) for 20 min .
the cells were incubated with the following fluorochrome conjugated antibodies : cd45-ef450 ( bd ) , cd3-percpcy5.5 ( bio - legend ) , cd4-fitc ( e - bioscience ) , and cd25-alexafluor700 ( e - bioscience ) .
intracellular stains for foxp3-apc ( e - bioscience ) and rort - pe ( e - bioscience ) were performed using the foxp3 staining buffer set ( e - bioscience ) according to the manufacturer 's instructions .
samples were measured on a fascanto ii flow cytometer with diva software ( bd ) and analyzed with kaluza software ( beckman coulter ) .
t lymphocytes were selected as cd45+cd3 + . among this population , tregs were defined as cd4+cd25+foxp3 + and th17 cells as cd4+rort++ .
the different populations were defined as the level of the positive population above the 99th percentile of a fluorescence - minus - one ( fmo ) sample .
after fixation and sectioning , the liver tissue samples were stained with haematoxylin - eosin and trichrome - masson .
steatosis , lobular inflammation , and ballooning were scored in a blinded way by one single experienced hepatologist according to the nash clinical research network scoring system .
the nash activity score ( nas ) was calculated as the sum of the scores for steatosis , ballooning , and lobular inflammation .
nash was diagnosed if some degree of steatosis , lobular inflammation , and ballooning were simultaneously present [ 24 , 25 ] .
total rna was extracted from stored frozen liver samples using the column - based technique ( rneasy minikit , qiagen , kj venlo , netherlands ) , according to the manufacturer 's instructions .
the total rna extraction for the sat and aat samples was based on the trizol ( invitrogen , life technology , belgium ) procedure from w. m. keck foundation biotechnology microarray resource laboratory at yale university .
purified total rnas were treated with dnase to obtain dna - free rna ( turbo dnase free , ambion , life technology , belgium ) .
cdna was synthesized using a transcriptor first strand cdna synthesis kit ( roche applied science , indianapolis , in ) .
taqman gene expression assay for il10 ( life technologies , belgium , gene i d 16153 , reference mm00439614_m1 ) , il17a ( life technologies , belgium , gene i d 16171 , reference mm00439618_m1 ) , and leptin ( gene i d 16846 , reference mm00434759_m1 ) was performed on an abi prism 7300 sequence detector system ( applied biosystems , life technology , belgium ) in 25 l reaction volumes containing 2 l cdna and 23 l master mix .
the master mix was prepared using 12.5 l taqman universal pcr master mix ( life technologies , belgium ) , 1.25 l primer , and 9.25 l rnasednase free water ( gibco , life technologies , belgium ) .
the parameter for pcr amplification was followed by 50 cycles of 95c for 15 s ( denaturation ) and 60c for 1 minute ( annealing and extension ) .
glyceraldehyde 3-phosphate dehydrogenase ( gadph ) was used as a housekeeping gene to normalize the results ( life technologies , belgium , gene i d 14433 , reference mm99999915_g1 ) .
the ct value corresponds to the number of cycles to reach a defined threshold and therefore it increases with a decreasing amount of the template .
alanine aminotransferase ( alt ) was measured by means of enzyme - linked immunosorbent assay ( elisa ) .
the mann - whitney u test and student 's t - test were used to compare independent variables , as appropriate .
liver histology showed steatosis , lobular inflammation , and ballooning in the mice fed hfd and therefore confirmed the presence of nash .
moreover the blood tests showed higher fasting glucose levels and alt levels in the hfd group in comparison with the nd group .
these data confirmed the validity of the experimental model to obtain hfd - induced nash together with an impairment of glucose metabolism and obesity - like disturbances .
no modifications in the percentage of leukocytes ( cd45 + cells ) , t - lymphocytes ( cd45+cd3 + cells ) , and cd4 + t - lymphocytes were found in mice fed a hfd in comparison with the lean counterparts , except for a decrease of the cd4 + in the aat after hfd .
tregs were significantly increased in the sat of mice fed hfd versus mice fed nd ( p = 0.02 ) , while no difference was found in aat or liver ( figure 2(a ) ) . moreover ,
an increase of the percentage of the highly polarized th17 ( cd4+rort++ ) was detected in the liver ( p = 0.02 ) and the aat ( p = 0.01 ) of mice fed hfd , in comparison with mice fed nd ( figure 2(a ) ) .
rt - pcr showed a positive cytokine response to hfd : both il10 and il17a were expressed in liver ( ct values , resp .
, 15.5 ( 15.416.5 ) and 21.7 ( 21.222.3 ) ) and both the aat ( resp . , 10.8 ( 5.110.9 ) and 8 ( 6.98.2 ) ) and the sat ( resp . ,
11.3 ( 811.7 ) and 6.4 ( 5.56.9 ) ) of mice fed hfd , while they were nondetectable in mice fed nd .
moreover leptin gene expression was increased in the adipose tissue of mice fed hfd compared to mice fed nd .
this increase was more pronounced in the sat district : in the abdominal district leptin increased 2-fold versus 986-fold in the sat ( figure 2(b ) ) .
in addition the significant correlations ( table 3 ) between the t - cell modifications and metabolic parameters and histology were investigated .
the aat - derived cd4+rort++ cells positively correlated with the weight gain and with liver histology : a positive correlation was detected with the nas as well as with its subscores steatosis , ballooning , and lobular inflammation .
moreover the sat - derived tregs positively correlated with the weight gain , the glucose levels , and liver histology ( nas , ballooning , and inflammation ) ( table 3 ) .
there is a growing interest in the role of the immune system as a key contributor to the pathogenesis of nash and the metabolic syndrome .
given the discordant data on the role of tregs and th17 in the development of nash , we performed a study to further elucidate the differential role of these subsets of cells at the different anatomical sites involved in the pathogenesis of nash and we analyzed the correlation between the tissue specific variations of these cells and the diet - induced metabolic disturbances .
the histologic features fulfilled the histologic criteria for nash ( defined as the simultaneous presence of steatosis , inflammation , and ballooning ) .
moreover the mice fed a hfd were obese and showed an impaired glucose tolerance and a leptin upregulation .
our model hence mimics all features of human nash [ 27 , 28 ] . when considering the different organs investigated ( figure 3 ) , the liver showed an increase of cd4+rort++ cells in mice fed a hfd in comparison with mice fed a nd , while there was no difference in tregs .
thus , the balance between th17 and tregs in the liver was shifted towards th17 .
importantly , the increase in cd4+rort++ cells showed a correlation with liver inflammation ( which is an important determinant of liver damage in nash ) , but not with metabolic disturbances . in the aat ,
similar to the liver , we observed an increase of the cd4+rort++ in the mice fed a hfd , while tregs did not show statistically significant variations .
also in this organ hfd shifted the balance between th17 and th10 towards the th17 . in line with the active role of the aat in the onset of nash and of the metabolic syndrome , the aat cd4+rort++ correlated with liver histology as well as with some features of metabolic impairment , including weight gain . in the sat
the tregs played a predominant role as we observed no statistically significant variations of the cd4+rort++ while the tregs were significantly increased in the mice fed a hfd .
this increase correlated with liver histology impairment and with metabolic alterations , namely , glucose impairment and weight gain .
as far as the th17 are concerned , our results are in agreement with tang et al . , who demonstrated an increased number of hepatic th17 and an increased hepatic gene expression in a hfd mouse model , as well as an attenuation of the lps - induced liver injury after neutralization of il17 .
others , however , found a decrease of th17 in the aat of mice fed hfd .
moreover , in an il17-deficient mouse model il17 appeared to act as a negative regulator of adipogenesis and glucose metabolism and to delay the development of diet - induced obesity .
this discrepancy could be explained by the different animal models used . in our model , which was able to represent the main characteristics of human nash , not only in terms of liver histology but also in terms of metabolic impairment , the th17 were increased in the liver and in the aat which are key organs in the onset of nash and positively correlated with liver histology and obesity .
this observation , although it can not mechanistically explain the role of the th17 in nash , suggests that th17 could be key players in nash and the associated metabolic disease . regarding the tregs , previous preclinical studies
reported that aat is a preferential site of accumulation of tregs in mice fed a nd and that they decrease after hfd [ 10 , 29 ] .
others showed an increase of tregs in the sat in obesity [ 29 , 30 ] . in agreement with the latter ,
our data confirm an increase of the tregs specifically in the sat in histologically proven nash accompanied by metabolic alterations .
these data suggest that also the sat plays an active role in liver disease - related and metabolic - related inflammation .
it could also be speculated that in this hfd model the treg increase in the sat is an attempt to restore the proinflammatory prone th17/treg balance .
finally we found a leptin upregulation in mice fed a hfd , mainly in the sat , which , through its systemic effect , may have contributed to the stimulation of immune cells , and particularly the th17 , at distance .
it is known that leptin is able to stimulate the th17 pathway : in experimental conditions of leptin deficiency , lower levels of il17 and an impairment of the th17 pathway are observed while the administration of increasing doses of leptin is able to restore and increase the th17 pathway .
this process could explain , at least in part , the increase of the cd4+rort++ cells in the aat and the liver as a consequence of increased levels of circulating leptin , mainly originating from the sat . in addition leptin has shown proinflammatory , profibrogenic , and prodiabetogenic effects .
this is in line with our observation of a significant correlation between leptin and the alt levels and hepatocellular ballooning , highlighting the link between leptin disturbances and hepatocellular damage . in line with the ability of leptin to stimulate the inflammatory immune response
, we found an upregulation of mrna of il17a in the liver and adipose tissue of mice fed hfd .
these results nicely fit with the aforementioned observed increase of cd4+rort++ in the liver and the abdominal adipose tissue .
in contrast to what could be expected , however , this increase in the proinflammatory il17a was not counterbalanced by a decrease of the il10 mrna in the same sites .
moreover we observed a positive correlation between adipose tissue - derived il10 and hepatic inflammation at histology .
its role , however , in diet - induced steatohepatitis and insulin resistance is controversial .
although il10 has been reported to be protective against diet - induced insulin resistance , it has also been reported that il10 does not improve hepatic or systemic insulin sensitivity in high fat feeding and therefore it does not protect against insulin resistance .
in contrast to the flow - cytometric data , the gene expression analysis showed a cytokine upregulation in the liver and in both the aat and sat . this discrepancy can be explained by the contribution to cytokine secretion by other cell types , which can not be discriminated by this method .
il10 increase could be explained , at least in part , due to a tissue - recruitment of m2 macrophages in response to hfd , as previously described .
il17a on the other hand can be produced by cell types other than th17 , such as nkt and t lymphocytes . given the active role of these cells in the onset of nash and the correlated metabolic disease [ 39 , 40 ] , it could be speculated that also these cells constitute a source of il17 . in conclusion
our results show a stimulation of both the th17 and the treg axis in nash , also in correlation with its metabolic complications .
these pathways seem to act differently at different sites , the th17 axis being upregulated at the level of the aat and the liver , and the treg axis being upregulated in the sat .
it has recently been shown that sat expresses genes that are implicated in inflammation that correlate with liver damage and is therefore also potentially implicated in nash pathogenesis .
the treg upregulation in sat observed in our study could hence be as a tentative to increase the anti - inflammatory mechanisms in response to inflammation and , therefore , it could represent a compensatory mechanism to counterbalance the th17 enhancement .
furthermore , obesity - related overproduction of leptin in sat could be one of the drivers of the upregulation of the th17 pathway , further highlighting the potential contribution of the sat , besides that of the aat .
as this is , however , a cross - sectional study , it is not possible to draw clear cause - effect conclusions . |
background and aims .
inflammatory mediators that cross - talk in different metabolically active organs are thought to play a crucial role in the pathogenesis of nonalcoholic steatohepatitis ( nash ) .
this study was aimed at investigating the cd4+rort+ t - helper cells and their counterpart , the cd4+cd25+foxp3 + regulatory t cells in the liver , subcutaneous adipose tissue ( sat ) , and abdominal adipose tissue ( aat ) in a high fat diet ( hfd ) mouse model .
methods .
c57bl6 mice were fed a hfd or a normal diet ( nd ) .
liver enzymes , metabolic parameters , and liver histology were assessed .
the expression of cd4+rort+ cells and regulatory t cells in different organs ( blood , liver , aat , and sat ) were analyzed by flow cytometry .
cytokine and adipokine tissue expression were studied by rt - pcr .
results .
mice fed a hfd developed nash and metabolic alterations compared to normal diet .
cd4+rort++ cells were significantly increased in the liver and the aat while an increase of regulatory t cells was observed in the sat of mice fed hfd compared to nd .
inflammatory cytokines were also upregulated .
conclusions .
cd4+rort++ cells and regulatory t cells are altered in nash with a site - specific pattern and correlate with the severity of the disease .
these site - specific differences are associated with increased cytokine expression . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
two proven vector control strategies are currently advocated to reduce transmission of malarial disease in africa , namely indoor residual spraying ( irs ) [ 1 - 5 ] and insecticide - treated bednets ( itns ) [ 6 - 9 ] . both methods are based on the use of residual insecticides in the intra - domiciliary domain and target mosquito vectors either before ( itns ) or after host - feeding ( irs ) .
impressive reductions in childhood morbidity and mortality have been demonstrated in a variety of epidemiological settings , and it can be expected that irs / itns will remain in the forefront of malaria vector control for at least the remainder of this decade . in spite of their proven effectiveness [ 5 - 9 ] ,
both methods have some drawbacks and limitations , such as insecticide resistance [ 10 - 13 ] , environmental or human health concerns and socio - economic or cultural acceptance by communities .
it is also clear that these powerful tools are not sufficient on their own to eliminate or drastically reduce the malaria burden from the most intensely endemic regions of the tropics , notably sub - saharan africa .
an expansion of this limited arsenal of vector - control tools , with new strategies to reduce human exposure , the size of mosquito populations , or transmissibility of disease , is therefore needed , and preferably appropriate for use in an integrated fashion with irs / itns [ 19 - 21 ] .
new innovative strategies , involving the release of genetically - engineered mosquitoes , aimed at rendering vector populations less susceptible to infection by human pathogens have seen enormous developments over the past few years [ 22 - 27 ] . if transposable genetic elements can be used to drive genes coding for refractoriness into fixation in wild vector populations , a substantial reduction in disease transmission may result .
however , advances to date have been confined to laboratory settings and many questions relating to the fitness , behaviour , ecology and phenotypic characteristics of transformed insects remain unanswered [ 27 - 29 ] .
the spread of desired traits , such as refractoriness to plasmodium infection , will depend on the reproductive fitness , evolutionary cost of the introduced trait , and manifestation of life - history behaviours , such as dispersal and mating , by engineered specimens .
for instance , given the likelihood of assortative mating , transgenic males and females may face strong competition upon release , which necessitates an increased understanding of the behavioural and ecological determinants of gene flow in mosquito populations . the characteristics of genetically - engineered mosquitoes should preferably be similar to those of their wild con - specifics but may be compromised by genetic modification , selection for specific traits or routine laboratory maintenance and difficult to assess realistically under standard laboratory conditions .
many of the ecological and population biology issues thus remain serious challenges to the application of genetically - engineered mosquitoes .
moreover , until such time that the probability of potential public health benefits can be maximised , it will be unlikely that approval for release can and will be granted .
the use of large contained field - based research settings is now widely advocated to face the shortfalls in our understanding of the behaviour and ecology of genetically - engineered vectors , prior to their release in the wild .
contained semi - field systems have been used for a variety of studies on mosquitoes , albeit outside africa [ 37 - 40 ] . in kenya , we have recently rejuvenated this approach and developed semi - field systems to study the behavioural ecology of malaria vectors [ 32,41 - 44 ] . by doing so
we present the first attempts to complete the life - cycle of this important vector in such systems , as a first step towards studies involving genetically - engineered specimens .
we transformed an existing greenhouse ( cambridge glass house co. ltd . , uk ) , measuring 11.4 7.1 m ( fig .
1a,1b,1c,1d ) into a ' malariasphere ' ( an enclosed environment with all components of a natural anopheles ecosystem ) by replacing all glass parts with dark - green shade netting ( density 90% ) permitting airflow ( wind ) and precipitation to enter the system . consequently ,
a sliding door provides entrance to the sphere , after passing a double layer of similar shade netting to prevent escape of released mosquitoes and entry of wild ones .
the sphere is located on the shores of lake victoria , west kenya at the mbita point research & training centre ( 0025 's , 3413'e ) of the international centre of insect physiology and ecology ( icipe ) .
the area experiences two rainy seasons ; the long rains from mid - march through june and short rains from october through december .
relatively high temperatures prevail throughout the year , ranging from 16c to 34c . during the rainy season , there are ample breeding sites in the mbita area for an .
suba district is inhabited by some 156,000 people , mostly belonging to the luo ethnic group , who practice mainly fishing and subsistence farming .
; data - loggers are shown as grey cubes ) and photographs of the hut ( b , note the white arrow showing the breeding site in front of the hut ( c , d ) .
further details see text . inside the sphere , a traditional luo house ( 3.2 2.8 1.7 m , fig .
a mixture of wood ash and clay was used for plastering and smoothing the wall surfaces .
the roof ( 2.8 m at its apex ) was made of grass thatch mounted on a wooden frame .
the house has a single door and no windows , which is typical for a local ' simba ' house .
eaves ( height 15 cm ) all around provide ventilation and serve as the predominant entry point for mosquitoes .
we maintained the depth of the site at 15 cm by replenishing it with water collected from lake victoria .
in each site we suspended a hobooptic stowaway tidbit data logger just below the water surface , and recorded the temperature at 30 min intervals . in order to monitor climatic conditions inside the system , we fitted six more hoboh8 data loggers , three inside the hut ( at 0.5 , 1.5 and 2.5 m from ground level ) and three outside the hut on a pole at similar heights ( fig .
climatic data were collected in february ( peak summer ) and june ( onset of the cold season ) .
we allowed plants to emerge from seeds present in the soil brought into the sphere , and in addition to this we planted a variety of food crops normally found around local homesteads ( table 1 ) . prior to the release of mosquitoes and during construction of the house and planting of crops , a wide variety of other organisms entered the sphere , including some known mosquito predators such as ants ( formicidae ) , spiders ( e.g. salticidae ) and geckos ( geckonidae ) .
mosquitoes used in the experiments originates from njage village ( 70 km from ifakara ) , south - east tanzania , and has been maintained under laboratory conditions since april 1996 .
adult insects were kept in standard 30 30 30 cm netting cages and offered 6% glucose as a carbohydrate source .
the cages were kept under ambient climatic conditions and females given the opportunity to feed on an arm of a volunteer for 10 min three times per week .
eggs were collected on wet filter paper disks ( 9 cm diameter ) and transferred to plastic containers containing water from lake victoria .
larvae were fed daily on tetraminfish food . upon pupation , insects were transferred to cages for emergence . in order to assess
whether all major life - history behaviours ( i.e. mating , sugar feeding , host seeking and oviposition ) occurred successfully in the sphere , we attempted to complete the life - cycle during three separate experiments by introducing i ) a group of 100 blood - fed females , ii ) groups of 500 virgin females and 1500 males or iii ) batches of 500 eggs in both breeding sites : i ) in the first experiment we introduced 100 three - day - old females ( f0 ) , which had been held in cages with males since the time of emergence .
they were blood fed ( for the first time ) on the forearm of a volunteer for 15 min and subsequently released ( at 21.30 hrs ) from a paper cup placed on the bed inside the hut .
we then monitored the presence and development of eggs , larvae and pupae by inspecting the breeding sites at daily intervals .
following emergence of the first adults , we deliberately waited for six days before entering the greenhouse at night , in order to assess whether mosquitoes would successfully mate and survive / feed on the plants in the system . on day 17
, 19 , 20 and 21 following the introduction of females , a volunteer slept inside the hut from 21.30 hrs until 07.00 the following day , which allowed the f1 population , and any of their parents that had survived , to feed on human blood .
ii ) as some of the parental ( f0 ) females could have survived until day 17 , it needed to be ascertained that virgin ( newly emerged ) insects survived , mated and blood - fed successfully too .
we therefore introduced 500 , 3 to 5 day - old virgin females , which had emerged from the pupa individually in glass vials , together with 1500 , 5 to 7 day - old males at 21.00 hrs .
starting three days afterwards , a volunteer slept in the hut for 5 consecutive nights .
we observed daily whether eggs were laid in the breeding sites to ensure that insemination , blood feeding and oviposition had taken place for two weeks following the release .
all pupae were collected from the breeding sites as they appeared so that assessments of survival by the f0 generation would not be confounded by the emergence of an overlapping f1 generation .
iii ) a third experiment was started by introducing 500 eggs at night ( 22.30 hrs ) in each of the two breeding sites .
concurrently we reared one thousand eggs from the same batch under standard laboratory conditions described above .
this enabled us to determine the sex ratio and thus the number of males and females released .
a volunteer occupied the hut for four consecutive nights , starting on day 22 after the start of the experiment .
thereafter , the breeding sites were monitored daily for the presence of eggs / larvae until day 32 .
a research protocol for the above experiments was submitted to the kenya national ethical review committee , based at the kenya medical research institute ( kemri ) , in which the discomfort and potential risks of ( non - infectious ) mosquito bites to volunteers was explained .
bnn , bgjk and gfk were involved in the experiments , and do not object to their names being revealed for publication .
a parasite - free environment was ensured through a ) regular screening of the volunteers ' peripheral blood for plasmodium parasites and b ) non - occupancy of the sphere beyond 5 days after the mosquitoes were given the first opportunity to obtain a blood meal .
as malaria infections in mosquitoes take at least 10 days to reach the sporozoite - infective stage , this procedure minimised risks of volunteers being infected within the experimental set up .
we transformed an existing greenhouse ( cambridge glass house co. ltd . , uk ) , measuring 11.4 7.1 m ( fig .
1a,1b,1c,1d ) into a ' malariasphere ' ( an enclosed environment with all components of a natural anopheles ecosystem ) by replacing all glass parts with dark - green shade netting ( density 90% ) permitting airflow ( wind ) and precipitation to enter the system . consequently ,
a sliding door provides entrance to the sphere , after passing a double layer of similar shade netting to prevent escape of released mosquitoes and entry of wild ones .
the sphere is located on the shores of lake victoria , west kenya at the mbita point research & training centre ( 0025 's , 3413'e ) of the international centre of insect physiology and ecology ( icipe ) .
the area experiences two rainy seasons ; the long rains from mid - march through june and short rains from october through december .
relatively high temperatures prevail throughout the year , ranging from 16c to 34c . during the rainy season , there are ample breeding sites in the mbita area for an .
suba district is inhabited by some 156,000 people , mostly belonging to the luo ethnic group , who practice mainly fishing and subsistence farming .
; data - loggers are shown as grey cubes ) and photographs of the hut ( b , note the white arrow showing the breeding site in front of the hut ( c , d ) .
further details see text . inside the sphere , a traditional luo house ( 3.2 2.8 1.7 m , fig .
a mixture of wood ash and clay was used for plastering and smoothing the wall surfaces .
the roof ( 2.8 m at its apex ) was made of grass thatch mounted on a wooden frame .
the house has a single door and no windows , which is typical for a local ' simba ' house .
eaves ( height 15 cm ) all around provide ventilation and serve as the predominant entry point for mosquitoes .
we maintained the depth of the site at 15 cm by replenishing it with water collected from lake victoria .
in each site we suspended a hobooptic stowaway tidbit data logger just below the water surface , and recorded the temperature at 30 min intervals . in order to monitor climatic conditions inside the system , we fitted six more hoboh8 data loggers , three inside the hut ( at 0.5 , 1.5 and 2.5 m from ground level ) and three outside the hut on a pole at similar heights ( fig .
climatic data were collected in february ( peak summer ) and june ( onset of the cold season ) .
we allowed plants to emerge from seeds present in the soil brought into the sphere , and in addition to this we planted a variety of food crops normally found around local homesteads ( table 1 ) . prior to the release of mosquitoes and during construction of the house and planting of crops , a wide variety of other organisms entered the sphere , including some known mosquito predators such as ants ( formicidae ) , spiders ( e.g. salticidae ) and geckos ( geckonidae ) .
mosquitoes used in the experiments originates from njage village ( 70 km from ifakara ) , south - east tanzania , and has been maintained under laboratory conditions since april 1996 .
adult insects were kept in standard 30 30 30 cm netting cages and offered 6% glucose as a carbohydrate source .
the cages were kept under ambient climatic conditions and females given the opportunity to feed on an arm of a volunteer for 10 min three times per week .
eggs were collected on wet filter paper disks ( 9 cm diameter ) and transferred to plastic containers containing water from lake victoria .
in order to assess whether all major life - history behaviours ( i.e. mating , sugar feeding , host seeking and oviposition ) occurred successfully in the sphere , we attempted to complete the life - cycle during three separate experiments by introducing i ) a group of 100 blood - fed females , ii ) groups of 500 virgin females and 1500 males or iii ) batches of 500 eggs in both breeding sites : i ) in the first experiment we introduced 100 three - day - old females ( f0 ) , which had been held in cages with males since the time of emergence .
they were blood fed ( for the first time ) on the forearm of a volunteer for 15 min and subsequently released ( at 21.30 hrs ) from a paper cup placed on the bed inside the hut .
we then monitored the presence and development of eggs , larvae and pupae by inspecting the breeding sites at daily intervals .
following emergence of the first adults , we deliberately waited for six days before entering the greenhouse at night , in order to assess whether mosquitoes would successfully mate and survive / feed on the plants in the system . on day 17 , 19 , 20 and 21 following the introduction of females , a volunteer slept inside the hut from 21.30 hrs until 07.00 the following day , which allowed the f1 population , and any of their parents that had survived , to feed on human blood .
ii ) as some of the parental ( f0 ) females could have survived until day 17 , it needed to be ascertained that virgin ( newly emerged ) insects survived , mated and blood - fed successfully too .
we therefore introduced 500 , 3 to 5 day - old virgin females , which had emerged from the pupa individually in glass vials , together with 1500 , 5 to 7 day - old males at 21.00 hrs .
starting three days afterwards , a volunteer slept in the hut for 5 consecutive nights .
we observed daily whether eggs were laid in the breeding sites to ensure that insemination , blood feeding and oviposition had taken place for two weeks following the release .
all pupae were collected from the breeding sites as they appeared so that assessments of survival by the f0 generation would not be confounded by the emergence of an overlapping f1 generation .
iii ) a third experiment was started by introducing 500 eggs at night ( 22.30 hrs ) in each of the two breeding sites .
concurrently we reared one thousand eggs from the same batch under standard laboratory conditions described above .
this enabled us to determine the sex ratio and thus the number of males and females released .
a volunteer occupied the hut for four consecutive nights , starting on day 22 after the start of the experiment .
thereafter , the breeding sites were monitored daily for the presence of eggs / larvae until day 32 .
a research protocol for the above experiments was submitted to the kenya national ethical review committee , based at the kenya medical research institute ( kemri ) , in which the discomfort and potential risks of ( non - infectious ) mosquito bites to volunteers was explained .
bnn , bgjk and gfk were involved in the experiments , and do not object to their names being revealed for publication .
a parasite - free environment was ensured through a ) regular screening of the volunteers ' peripheral blood for plasmodium parasites and b ) non - occupancy of the sphere beyond 5 days after the mosquitoes were given the first opportunity to obtain a blood meal . as malaria infections in mosquitoes
take at least 10 days to reach the sporozoite - infective stage , this procedure minimised risks of volunteers being infected within the experimental set up .
figure 2 shows the climatic conditions recorded in the greenhouse over a 3-week period in june 2000 , coinciding with the time of the third experiment ( onset cold dry season ) .
similar data sets for february 2000 ( peak of the main hot and dry season ) showed an average temperature at the water surface of 24.0c ( range 20.029.8c ) .
other studies have recorded slightly lower average temperatures and larger ranges over which these fluctuate , both in artificially constructed and natural sites .
haddow recorded a range of 19.034.5c in pans of similar size that were supplemented with soil and had water of similar depth near kisumu ( 80 km from mbita point ) .
gimnig et al . recorded an average of 26.4 0.7c from natural sites between march and august 1998 in that same area , as did koenraadt et al .
( pers . comm . ) , with 25.8 4.2c , in two subsequent years .
the lower temperatures recorded from sites in the sphere were probably caused by reduced infiltration of sunlight through the roof 's shade netting .
temperature ( a ) and relative humidity ( b ) data recorded in one of the two the breeding sites and different heights ( 0.5 , 1.5 and 2.5 m ) inside the hut over a 3-week period in june 2000 . arrows on y - axis show maximum and minimum recorded and accompanying figures show the same data for data - loggers outside the hut at those same heights .
2a ) inside the hut at various heights fluctuated much more considerably but nevertheless remained relatively consistent throughout the observational periods .
average temperatures increased both inside ( 23.1 , 23.8 and 23.9c for 0.5 , 1.5 and 2.5 m respectively ) and outside the hut with height as did the range over which these fluctuated daily . corresponding data for february inside the hut showed higher averages ( 24.8 , 25.1 and 25.2c for 0.5 , 1.5 and 2.5 m respectively ) .
between the seasons , maximum variation between temperatures was found outside the hut at 2.5 m , from 37.0c ( february - maximum ) to 16.4c ( june - minimum ) .
the smallest variation was observed inside the hut at 0.5 m , from 28.7c ( february - maximum ) to 19.7c ( june - minimum ) . as such
, the range over which temperatures fluctuated between seasons was 2.3 times larger outside ( 2.5 m ) than inside ( 0.5 m ) the hut .
haddow recorded mean temperatures between october and december in local huts ( at 1 m height ) in the kisumu area and found an average temperature of 24.2c ( range 21.027.0c ) .
our own measurements inside the hut in the sphere between 18 october and 15 november ( 2000 ) at 1.5 m height gave values of 24.0 1.76c ( range 19.829.1c ) whereas measurements inside 4 local houses in mbita of similar design during that same period gave higher average values ( 0.50.8c ) and absolute maxima ( 0.8c ) .
ambient conditions inside local houses are more constant than outdoor climatic conditions ( this study and ) and it would seem that the sphere itself exerts a similar , albeit small , insulating effect : slightly lower temperatures and smaller ranges over which these fluctuate , both inside and outside the hut . relative humidity ( rh ) data ( fig .
relative humidity is fairly constant and averages inside the hut during june ranged from 63.5% ( 1.0 m ) to 69.3% ( 2.5 m ) .
minimum values were always higher inside the hut than outside , providing more suitable microclimatic conditions for resting mosquitoes .
measurements in october / november , both in the sphere and a local house of similar design in mbita point , showed slightly higher average rh values outdoors in both settings than indoors with minimal differences between the sphere and the village hut .
overall , as with temperatures , the range over which the rh fluctuated was smaller inside than outside the hut and minima inside the sphere were always 3 to 4 % higher than those measured in village huts .
although small , these climatic differences may affect development of immature stages and survival of adults , and research findings from experiments inside the sphere should be compared with field conditions at slightly higher altitudes .
the introduction of blood - fed females into the greenhouse resulted in the presence of eggs in the breeding sites on day 3 ( 2.5 days after release ) , and eggs continued to be observed in the sites until day 7 ( fig .
3 ) . larvae ( from l1 to l4 stage ) were seen feeding at the water surface until day 23 , when the last l4 larva pupated .
the first five pupae were seen in the breeding sites in the evening of day 10 , meaning that the variation in maturation time from egg to pupa was 720 days . in spite of having conditions with higher larval density and smaller water surface area , gimnig et al . recorded much reduced periods to pupation , from 5 to 12 days . in total , 57 pupae were counted in the breeding site 3.8 m in front of the hut and 130 in the site 1.1 m behind it , and on average they harboured only 0.08 and 0.18 larva / cm , respectively .
these densities are lower than those observed in natural habitats , and given the fact that we observed algal growth , considered important for larval growth , it seems that the prolonged time to pupation in this trial may be attributed to the relatively small range over which temperatures fluctuate .
alternatively , re - introducing mosquitoes that had been maintained under laboratory conditions for several years in a more natural setting may have caused these effects , and poor adaptation to these conditions may have stunted their development .
completion of the anopheles gambiae life - cycle in the greenhouse over a 27-day period .
blood - fed females were released on day 1 , and arrows show time periods during which the various developmental stages were observed .
the first adults were seen inside the hut on day 11 , and continued to be present until the end of the experiment ( day 27 ) .
starting in the morning of day 22 , we observed new eggs in the breeding sites and subsequent larval development . from the above it
can be deduced that specific behaviours of the adult insects occurred during certain time periods ( fig .
oviposition activity took place twice during this trial , meaning that females survived until eggs were mature , that they successfully located a breeding site , accepted it for oviposition , and laid eggs .
other potential breeding sites , like the leaf axils of the banana trees in the sphere , were examined but were not found to harbour eggs , larvae or pupae .
the period for reaching sexual maturity for males may be at least one day and for females up to 60 hrs , so mating of the f1 generation may not have taken place until dusk on day 14 . in spite of regular observations during dusk , we did not see any swarming activity typically associated with mating in an .
this is a particularly interesting point , because in contrast with other settings ( e.g. ) , where mating swarms were frequently observed we failed to do so over a 3-year period in the mbita area as did charlwood ( pers.comm . ) who observed only one anopheline swarm during 4 years in the kilombero valley of tanzania , raising the question as to whether swarming is an obligate component of the an .
alternatively , maintenance of mosquitoes in laboratory cages for several years forced this strain to become stenogamous ( i.e. the ability to mate in small cages ) , and this adaptation may have interfered with its ability to swarm when introduced into the sphere . as newly emerged adults rarely survive for more than 48 hrs without the availability of an energy source , mosquitoes must have supplemented their energy reserves with carbohydrates from the plants in the sphere ( table 1 ) for up to 6 days before they were allowed access to a blood source , in the form of a human volunteer sleeping in the hut .
some plants like castorbean ( ricinus communis l. ) were flowering at the time of the experiment , and may have provided nectar sources . in cage experiments ( impoinvil et al . , in prep . )
we have observed a mean survival time of 7.0 0.2 days on this plant , which is comparable to mosquitoes given 6% glucose ( 8.7 0.2 days ) .
gambiae mosquitoes emerge from the pupal stage with a deficit not only in carbohydrates , but also lipid and protein , which usually is compensated for by consuming a ( small ) blood meal within the first few days of adult life , it is likely that mortality during the 6-day post - emergence period in the current trial was too high to have a good number of the 8090 females that emerged survive long enough to obtain their first blood meal . within 15 min of entering the hut at night ,
the volunteer noticed the sound of mosquitoes and subsequently felt mosquito bites on his exposed lower limbs .
this implies that females were receptive to host cues , entered the hut , probably through the eaves , and then successfully located and fed on the human host . at sunrise ,
several engorged females were seen resting on the walls , indicating successful blood feeding and endophily ( indoor resting ) , which is typical for this species . following maturation of eggs ,
the second oviposition began during the night of day 21 , thus completing the life - cycle .
we continued to observe both immature and adult insects ( presumably mostly from the f1 generation ) until day 27 , when the experiment was terminated ( by refraining from entering the sphere for about 1 month ) .
the second experiment , in which we released 500 virgin females together with 1500 males demonstrated that mating does occur in a relatively small , semi - field system .
after the third night that a volunteer had slept in the hut , we observed eggs in the breeding sites .
the production of offspring , though , was low , and we only collected 40 pupae by the end of the trial period .
this may have been caused by heavy rainfall during three consecutive nights ( day 24 ) , which may have affected the survival of the adults and/or larvae or washed away the larvae from the breeding sites due to overflow .
since we observed few mosquitoes , we decided to conduct a human landing catch during two nights inside the hut , starting two nights after sleeping in the hut had ended .
nevertheless , the life - cycle was completed , as manifested by the harvested pupae , which were removed from the breeding sites to prevent emergence of the f1 generation which would have compromised interpretation of survival of the f0 generation .
the third experiment started by introducing 500 eggs into each of the breeding sites , whilst 1000 eggs ( from the same original batch ) were reared under laboratory conditions . in the laboratory , larvae developed at the same rate and
most reached maturity by day 10 , when the first pupae were observed ( fig .
this was similar for the breeding sites in the sphere , except that development was highly asynchronous , i.e. some larvae pupated by day 10 , whereas others took up to day 24 before pupation .
these times to pupation are similar to those observed in the first trial , but are again in contrast with other studies .
on day 7 we counted all larvae and observed 887 in the insectary , as opposed to 652 in the sphere .
overall , the laboratory batch yielded 804 pupae , versus 495 from the breeding sites .
on the basis of these data , the average daily survival was 0.90 for the laboratory larvae , and 0.83 for the larvae in the sphere . with nearly half the larvae surviving to the adult stage , these results contrast sharply with much higher mortalities ( up to 90% ) observed in the kisumu area and so tome ( charlwood , pers .
the sex ratio of emerging adults in the laboratory was 2:3 , which translated into a female population in the sphere of 297 , on the assumption that no insects died during emergence . on day 28 , after the release of eggs in the breeding sites , we observed that eggs had been laid by the f1 generation , but with only 6 and 3 eggs in the two breeding sites respectively .
cumulative percentage of pupation of eggs introduced in the two breeding sites inside the greenhouse ( ) or under insectary conditions ( ) .
our results have shown that by starting either at the post - blood feeding , pre - mating , or egg stage , a new generation of insects can be reared under these semi - field conditions , and that all life - history behaviours were successfully completed to a lesser or greater extent .
this therefore represents the first and promising step towards continuous maintenance of parasite - free an .
gambiae populations under semi - natural conditions that can be experimentally manipulated in studies of malaria vector ecology and transmission control .
russell and rao used a large outdoor cage , based on a design by hackett and bates , to study swarming and oviposition behaviour of an .
culicifacies giles in india , and showed that such systems can be used to unravel aspects of the behavioural ecology of anophelines .
our study shows that such systems can now also be developed for studies on african malaria vectors in order to start filling the gaps in our understanding of the behavioural ecology of these insects .
first and foremost , it provides a suitable intermediate between laboratory - based studies addressing mosquito behaviour and ecology , and the field situation .
too often , conclusions are drawn from results obtained under laboratory conditions that necessitate speculation as to what may or may not happen in the field .
fixed climatic conditions , cage - experiments , olfactometers and windtunnels , in which the mosquito strains used have been laboratory - reared for sometimes decades , may readily distort behavioural and ecological phenomena .
here we have shown that , beyond introducing f1 generation malaria - free mosquitoes from wild populations , it may be possible to rear vectors in situ within a semi - natural system that may minimize such artefacts .
conversely , the advantage of using insectary - reared mosquitoes is in the level of control that may be exerted that would not otherwise be possible : experiments can be conducted all - year - round , with fixed numbers of insects , of known age and physiological status , in a malaria parasite - free environment under ambient climatic conditions . this enables more direct inferences to be drawn from data analysis as compared to longitudinal field studies , because of constant conditions and simplified experimental design .
we have recently evaluated the efficacy of several plants traditionally used by the luo community as repellents in a similar semi - field set - up , and simple logistic regression , on data collected during four nights per plant , yielded significant results . within a year of nearly continuous experimentation
, the repellency of 8 plant species and 3 combinations thereof was evaluated through thermal expulsion or direct burning , and 9 species and 2 combinations thereof were tested in potted form .
such studies would have taken several years under field conditions , and would be limited to times when mosquito densities would have been sufficiently high to permit meaningful experiments .
gambiae maintained on a variety of diets ( blood or sugar alone , or a combination of both ) and revealed similar results to those obtained under laboratory conditions .
within one year of starting studies on the behaviour of mosquitoes around bednets in a semi - field setup , we transformed a regular conical bednet into a trap that may catch up to 70% of the females released .
recent field evaluation of this trap shows it to be a promising replacement for the human landing catch ( mathenge et al .
with the trap now being considered for commercial manufacturing ( it took a mere two years to reach this stage ) , this would have been impossible without the availability of a semi - field set - up in which continuous experimentation ensured rapid progress towards product development .
even though our system resembles more closely the field situation , it remains to be ascertained to what extent .
our current study was mainly qualitative in design and focused on life - cycle completion .
for instance , observation of eggs in the breeding sites in the morning of day 22 during the first trial implies that these originated from females that fed once on day 19 , as those that fed on day 17 should have laid before .
however , it is likely that these females fed twice , on day 17 and day 19 , and should be classified as pre - gravids , before fully maturing a batch of eggs
. furthermore , in the absence of a human host , mosquitoes survived up to six days after emergence , confirming field results that feeding on plant sugars does occur and may be an important feature of the life - cycle in this species ( foster and knols , unpublished data ) .
there may be other , yet unknown , factors that affect the behavioural ecology of the insects in such systems . or , as bates wrote , following his outdoor cage studies in albania : " one still can not be sure that the reactions of the mosquito are ' natural ' because there is always the barrier of wire liable to be encountered on extended flights ; and when the flight of a mosquito has been interrupted by this wire barrier its further activity may be definitely unnatural " .
rightly so , and even though we did not observe any obvious distorted behaviours , it is imperative that findings from semi - field studies be verified under natural outdoor conditions
. additional studies in which the release and performance of field - collected , blood - fed mosquitoes in the sphere is compared with that of laboratory specimens in terms of egg - recovery , developmental periods and important behavioural characteristics ( like swarming ) will provide further insight to what extent such systems mimic the natural anopheles environment and colony adaptation impairs natural behaviours . nevertheless , since bates ' days , advances in science merit a renewed impetus towards semi - field studies in contained near - natural environments , particularly with respect to transgenic mosquitoes .
fitness evaluations of engineered strains of vectors are mandatory for transformation technology to become an established disease control tool in africa . perhaps this alone , is ample justification for more intensive sphere studies , hopefully not only in kenya , but also in other african countries likely to be involved in this endeavour .
studies on gene flow , mating behaviour and reproductive fitness , combined with studies on the effects of laboratory maintenance on the genetic make - up of transformed strains to be released , can be conducted in semi - field systems .
such systems , particularly when used to study genetically - engineered mosquitoes will require more advanced containment levels than the system described here . guidelines for facility location , physical and biological containment , safety practices and calamity control need to be developed and adapted from existing arthropod containment guidelines .
there are several good reasons to further such studies in disease - endemic settings . under such conditions it will be possible to transform offspring from wild mosquitoes , conduct experiments under local ambient climatic conditions and evaluate transgene spread and fixation in offspring from field - collected gravid females that emerge in a semi - field setup .
last , but not least , it will enable scientists from developing countries to become more directly involved in evaluating the potential use and application of transgenic mosquitoes for future malarial disease control .
figure 2 shows the climatic conditions recorded in the greenhouse over a 3-week period in june 2000 , coinciding with the time of the third experiment ( onset cold dry season ) .
similar data sets for february 2000 ( peak of the main hot and dry season ) showed an average temperature at the water surface of 24.0c ( range 20.029.8c ) .
other studies have recorded slightly lower average temperatures and larger ranges over which these fluctuate , both in artificially constructed and natural sites .
haddow recorded a range of 19.034.5c in pans of similar size that were supplemented with soil and had water of similar depth near kisumu ( 80 km from mbita point ) .
gimnig et al . recorded an average of 26.4 0.7c from natural sites between march and august 1998 in that same area , as did koenraadt et al .
( pers . comm . ) , with 25.8 4.2c , in two subsequent years .
the lower temperatures recorded from sites in the sphere were probably caused by reduced infiltration of sunlight through the roof 's shade netting .
temperature ( a ) and relative humidity ( b ) data recorded in one of the two the breeding sites and different heights ( 0.5 , 1.5 and 2.5 m ) inside the hut over a 3-week period in june 2000 . arrows on y - axis show maximum and minimum recorded and accompanying figures show the same data for data - loggers outside the hut at those same heights .
2a ) inside the hut at various heights fluctuated much more considerably but nevertheless remained relatively consistent throughout the observational periods .
average temperatures increased both inside ( 23.1 , 23.8 and 23.9c for 0.5 , 1.5 and 2.5 m respectively ) and outside the hut with height as did the range over which these fluctuated daily . corresponding data for february inside the hut showed higher averages ( 24.8 , 25.1 and 25.2c for 0.5 , 1.5 and 2.5 m respectively ) .
between the seasons , maximum variation between temperatures was found outside the hut at 2.5 m , from 37.0c ( february - maximum ) to 16.4c ( june - minimum ) .
the smallest variation was observed inside the hut at 0.5 m , from 28.7c ( february - maximum ) to 19.7c ( june - minimum ) . as such
, the range over which temperatures fluctuated between seasons was 2.3 times larger outside ( 2.5 m ) than inside ( 0.5 m ) the hut .
haddow recorded mean temperatures between october and december in local huts ( at 1 m height ) in the kisumu area and found an average temperature of 24.2c ( range 21.027.0c ) .
our own measurements inside the hut in the sphere between 18 october and 15 november ( 2000 ) at 1.5 m height gave values of 24.0 1.76c ( range 19.829.1c ) whereas measurements inside 4 local houses in mbita of similar design during that same period gave higher average values ( 0.50.8c ) and absolute maxima ( 0.8c ) .
ambient conditions inside local houses are more constant than outdoor climatic conditions ( this study and ) and it would seem that the sphere itself exerts a similar , albeit small , insulating effect : slightly lower temperatures and smaller ranges over which these fluctuate , both inside and outside the hut . relative humidity ( rh ) data ( fig .
relative humidity is fairly constant and averages inside the hut during june ranged from 63.5% ( 1.0 m ) to 69.3% ( 2.5 m ) .
minimum values were always higher inside the hut than outside , providing more suitable microclimatic conditions for resting mosquitoes .
measurements in october / november , both in the sphere and a local house of similar design in mbita point , showed slightly higher average rh values outdoors in both settings than indoors with minimal differences between the sphere and the village hut .
overall , as with temperatures , the range over which the rh fluctuated was smaller inside than outside the hut and minima inside the sphere were always 3 to 4 % higher than those measured in village huts .
although small , these climatic differences may affect development of immature stages and survival of adults , and research findings from experiments inside the sphere should be compared with field conditions at slightly higher altitudes .
the introduction of blood - fed females into the greenhouse resulted in the presence of eggs in the breeding sites on day 3 ( 2.5 days after release ) , and eggs continued to be observed in the sites until day 7 ( fig .
3 ) . larvae ( from l1 to l4 stage ) were seen feeding at the water surface until day 23 , when the last l4 larva pupated .
the first five pupae were seen in the breeding sites in the evening of day 10 , meaning that the variation in maturation time from egg to pupa was 720 days . in spite of having conditions with higher larval density and smaller water surface area , gimnig et al . recorded much reduced periods to pupation , from 5 to 12 days . in total , 57 pupae were counted in the breeding site 3.8 m in front of the hut and 130 in the site 1.1 m behind it , and on average they harboured only 0.08 and 0.18 larva / cm , respectively .
these densities are lower than those observed in natural habitats , and given the fact that we observed algal growth , considered important for larval growth , it seems that the prolonged time to pupation in this trial may be attributed to the relatively small range over which temperatures fluctuate .
alternatively , re - introducing mosquitoes that had been maintained under laboratory conditions for several years in a more natural setting may have caused these effects , and poor adaptation to these conditions may have stunted their development .
completion of the anopheles gambiae life - cycle in the greenhouse over a 27-day period .
blood - fed females were released on day 1 , and arrows show time periods during which the various developmental stages were observed .
the first adults were seen inside the hut on day 11 , and continued to be present until the end of the experiment ( day 27 ) .
starting in the morning of day 22 , we observed new eggs in the breeding sites and subsequent larval development . from the above it
can be deduced that specific behaviours of the adult insects occurred during certain time periods ( fig .
oviposition activity took place twice during this trial , meaning that females survived until eggs were mature , that they successfully located a breeding site , accepted it for oviposition , and laid eggs .
other potential breeding sites , like the leaf axils of the banana trees in the sphere , were examined but were not found to harbour eggs , larvae or pupae . the period for reaching sexual maturity for
males may be at least one day and for females up to 60 hrs , so mating of the f1 generation may not have taken place until dusk on day 14 . in spite of regular observations during dusk , we did not see any swarming activity typically associated with mating in an .
this is a particularly interesting point , because in contrast with other settings ( e.g. ) , where mating swarms were frequently observed we failed to do so over a 3-year period in the mbita area as did charlwood ( pers.comm . ) who observed only one anopheline swarm during 4 years in the kilombero valley of tanzania , raising the question as to whether swarming is an obligate component of the an .
alternatively , maintenance of mosquitoes in laboratory cages for several years forced this strain to become stenogamous ( i.e. the ability to mate in small cages ) , and this adaptation may have interfered with its ability to swarm when introduced into the sphere . as newly emerged adults rarely survive for more than 48 hrs without the availability of an energy source , mosquitoes must have supplemented their energy reserves with carbohydrates from the plants in the sphere ( table 1 ) for up to 6 days before they were allowed access to a blood source , in the form of a human volunteer sleeping in the hut . some plants like castorbean ( ricinus communis l. ) were flowering at the time of the experiment , and may have provided nectar sources . in cage experiments ( impoinvil et al . , in prep . )
we have observed a mean survival time of 7.0 0.2 days on this plant , which is comparable to mosquitoes given 6% glucose ( 8.7 0.2 days ) .
gambiae mosquitoes emerge from the pupal stage with a deficit not only in carbohydrates , but also lipid and protein , which usually is compensated for by consuming a ( small ) blood meal within the first few days of adult life , it is likely that mortality during the 6-day post - emergence period in the current trial was too high to have a good number of the 8090 females that emerged survive long enough to obtain their first blood meal . within 15 min of entering the hut at night ,
the volunteer noticed the sound of mosquitoes and subsequently felt mosquito bites on his exposed lower limbs .
this implies that females were receptive to host cues , entered the hut , probably through the eaves , and then successfully located and fed on the human host . at sunrise
, several engorged females were seen resting on the walls , indicating successful blood feeding and endophily ( indoor resting ) , which is typical for this species . following maturation of eggs ,
the second oviposition began during the night of day 21 , thus completing the life - cycle .
we continued to observe both immature and adult insects ( presumably mostly from the f1 generation ) until day 27 , when the experiment was terminated ( by refraining from entering the sphere for about 1 month ) .
the second experiment , in which we released 500 virgin females together with 1500 males demonstrated that mating does occur in a relatively small , semi - field system .
after the third night that a volunteer had slept in the hut , we observed eggs in the breeding sites
. the production of offspring , though , was low , and we only collected 40 pupae by the end of the trial period .
this may have been caused by heavy rainfall during three consecutive nights ( day 24 ) , which may have affected the survival of the adults and/or larvae or washed away the larvae from the breeding sites due to overflow .
since we observed few mosquitoes , we decided to conduct a human landing catch during two nights inside the hut , starting two nights after sleeping in the hut had ended .
nevertheless , the life - cycle was completed , as manifested by the harvested pupae , which were removed from the breeding sites to prevent emergence of the f1 generation which would have compromised interpretation of survival of the f0 generation .
the third experiment started by introducing 500 eggs into each of the breeding sites , whilst 1000 eggs ( from the same original batch ) were reared under laboratory conditions . in the laboratory , larvae developed at the same rate and
most reached maturity by day 10 , when the first pupae were observed ( fig .
this was similar for the breeding sites in the sphere , except that development was highly asynchronous , i.e. some larvae pupated by day 10 , whereas others took up to day 24 before pupation .
these times to pupation are similar to those observed in the first trial , but are again in contrast with other studies . on day 7
we counted all larvae and observed 887 in the insectary , as opposed to 652 in the sphere .
overall , the laboratory batch yielded 804 pupae , versus 495 from the breeding sites .
on the basis of these data , the average daily survival was 0.90 for the laboratory larvae , and 0.83 for the larvae in the sphere . with nearly half the larvae surviving to the adult stage , these results
contrast sharply with much higher mortalities ( up to 90% ) observed in the kisumu area and so tome ( charlwood , pers .
. the sex ratio of emerging adults in the laboratory was 2:3 , which translated into a female population in the sphere of 297 , on the assumption that no insects died during emergence .
on day 28 , after the release of eggs in the breeding sites , we observed that eggs had been laid by the f1 generation , but with only 6 and 3 eggs in the two breeding sites respectively .
cumulative percentage of pupation of eggs introduced in the two breeding sites inside the greenhouse ( ) or under insectary conditions ( ) .
our results have shown that by starting either at the post - blood feeding , pre - mating , or egg stage , a new generation of insects can be reared under these semi - field conditions , and that all life - history behaviours were successfully completed to a lesser or greater extent .
this therefore represents the first and promising step towards continuous maintenance of parasite - free an .
gambiae populations under semi - natural conditions that can be experimentally manipulated in studies of malaria vector ecology and transmission control .
russell and rao used a large outdoor cage , based on a design by hackett and bates , to study swarming and oviposition behaviour of an .
culicifacies giles in india , and showed that such systems can be used to unravel aspects of the behavioural ecology of anophelines .
our study shows that such systems can now also be developed for studies on african malaria vectors in order to start filling the gaps in our understanding of the behavioural ecology of these insects .
first and foremost , it provides a suitable intermediate between laboratory - based studies addressing mosquito behaviour and ecology , and the field situation .
too often , conclusions are drawn from results obtained under laboratory conditions that necessitate speculation as to what may or may not happen in the field .
fixed climatic conditions , cage - experiments , olfactometers and windtunnels , in which the mosquito strains used have been laboratory - reared for sometimes decades , may readily distort behavioural and ecological phenomena .
here we have shown that , beyond introducing f1 generation malaria - free mosquitoes from wild populations , it may be possible to rear vectors in situ within a semi - natural system that may minimize such artefacts .
conversely , the advantage of using insectary - reared mosquitoes is in the level of control that may be exerted that would not otherwise be possible : experiments can be conducted all - year - round , with fixed numbers of insects , of known age and physiological status , in a malaria parasite - free environment under ambient climatic conditions .
this enables more direct inferences to be drawn from data analysis as compared to longitudinal field studies , because of constant conditions and simplified experimental design .
we have recently evaluated the efficacy of several plants traditionally used by the luo community as repellents in a similar semi - field set - up , and simple logistic regression , on data collected during four nights per plant , yielded significant results . within a year of nearly continuous experimentation
, the repellency of 8 plant species and 3 combinations thereof was evaluated through thermal expulsion or direct burning , and 9 species and 2 combinations thereof were tested in potted form .
such studies would have taken several years under field conditions , and would be limited to times when mosquito densities would have been sufficiently high to permit meaningful experiments .
gambiae maintained on a variety of diets ( blood or sugar alone , or a combination of both ) and revealed similar results to those obtained under laboratory conditions .
within one year of starting studies on the behaviour of mosquitoes around bednets in a semi - field setup , we transformed a regular conical bednet into a trap that may catch up to 70% of the females released .
recent field evaluation of this trap shows it to be a promising replacement for the human landing catch ( mathenge et al .
, in preparation . ) . with the trap now being considered for commercial manufacturing ( it took a mere two years to reach this stage ) ,
this would have been impossible without the availability of a semi - field set - up in which continuous experimentation ensured rapid progress towards product development .
even though our system resembles more closely the field situation , it remains to be ascertained to what extent .
our current study was mainly qualitative in design and focused on life - cycle completion .
for instance , observation of eggs in the breeding sites in the morning of day 22 during the first trial implies that these originated from females that fed once on day 19 , as those that fed on day 17 should have laid before .
however , it is likely that these females fed twice , on day 17 and day 19 , and should be classified as pre - gravids , before fully maturing a batch of eggs .
furthermore , in the absence of a human host , mosquitoes survived up to six days after emergence , confirming field results that feeding on plant sugars does occur and may be an important feature of the life - cycle in this species ( foster and knols , unpublished data ) .
obviously , there are limitations associated with these studies . some phenomena , like dispersal ,
there may be other , yet unknown , factors that affect the behavioural ecology of the insects in such systems . or , as bates wrote , following his outdoor cage studies in albania : " one still can not be sure that the reactions of the mosquito are ' natural ' because there is always the barrier of wire liable to be encountered on extended flights ; and when the flight of a mosquito has been interrupted by this wire barrier its further activity may be definitely unnatural " .
rightly so , and even though we did not observe any obvious distorted behaviours , it is imperative that findings from semi - field studies be verified under natural outdoor conditions .
additional studies in which the release and performance of field - collected , blood - fed mosquitoes in the sphere is compared with that of laboratory specimens in terms of egg - recovery , developmental periods and important behavioural characteristics ( like swarming ) will provide further insight to what extent such systems mimic the natural anopheles environment and colony adaptation impairs natural behaviours . nevertheless , since bates ' days , advances in science merit a renewed impetus towards semi - field studies in contained near - natural environments , particularly with respect to transgenic mosquitoes .
fitness evaluations of engineered strains of vectors are mandatory for transformation technology to become an established disease control tool in africa . perhaps this alone , is ample justification for more intensive sphere studies , hopefully not only in kenya , but also in other african countries likely to be involved in this endeavour .
studies on gene flow , mating behaviour and reproductive fitness , combined with studies on the effects of laboratory maintenance on the genetic make - up of transformed strains to be released , can be conducted in semi - field systems .
such systems , particularly when used to study genetically - engineered mosquitoes will require more advanced containment levels than the system described here . guidelines for facility location , physical and biological containment , safety practices and calamity control need to be developed and adapted from existing arthropod containment guidelines .
there are several good reasons to further such studies in disease - endemic settings . under such conditions it will be possible to transform offspring from wild mosquitoes , conduct experiments under local ambient climatic conditions and evaluate transgene spread and fixation in offspring from field - collected gravid females that emerge in a semi - field setup .
last , but not least , it will enable scientists from developing countries to become more directly involved in evaluating the potential use and application of transgenic mosquitoes for future malarial disease control .
the introduction of blood - fed females into the greenhouse resulted in the presence of eggs in the breeding sites on day 3 ( 2.5 days after release ) , and eggs continued to be observed in the sites until day 7 ( fig .
3 ) . larvae ( from l1 to l4 stage ) were seen feeding at the water surface until day 23 , when the last l4 larva pupated .
the first five pupae were seen in the breeding sites in the evening of day 10 , meaning that the variation in maturation time from egg to pupa was 720 days . in spite of having conditions with higher larval density and smaller water surface area , gimnig et al . recorded much reduced periods to pupation , from 5 to 12 days . in total , 57 pupae were counted in the breeding site 3.8 m in front of the hut and 130 in the site 1.1 m behind it , and on average they harboured only 0.08 and 0.18 larva / cm , respectively .
these densities are lower than those observed in natural habitats , and given the fact that we observed algal growth , considered important for larval growth , it seems that the prolonged time to pupation in this trial may be attributed to the relatively small range over which temperatures fluctuate .
alternatively , re - introducing mosquitoes that had been maintained under laboratory conditions for several years in a more natural setting may have caused these effects , and poor adaptation to these conditions may have stunted their development .
completion of the anopheles gambiae life - cycle in the greenhouse over a 27-day period .
blood - fed females were released on day 1 , and arrows show time periods during which the various developmental stages were observed .
the first adults were seen inside the hut on day 11 , and continued to be present until the end of the experiment ( day 27 ) .
starting in the morning of day 22 , we observed new eggs in the breeding sites and subsequent larval development . from the above it
can be deduced that specific behaviours of the adult insects occurred during certain time periods ( fig .
oviposition activity took place twice during this trial , meaning that females survived until eggs were mature , that they successfully located a breeding site , accepted it for oviposition , and laid eggs .
other potential breeding sites , like the leaf axils of the banana trees in the sphere , were examined but were not found to harbour eggs , larvae or pupae . the period for reaching sexual maturity for
males may be at least one day and for females up to 60 hrs , so mating of the f1 generation may not have taken place until dusk on day 14 . in spite of regular observations during dusk , we did not see any swarming activity typically associated with mating in an .
this is a particularly interesting point , because in contrast with other settings ( e.g. ) , where mating swarms were frequently observed we failed to do so over a 3-year period in the mbita area as did charlwood ( pers.comm . ) who observed only one anopheline swarm during 4 years in the kilombero valley of tanzania , raising the question as to whether swarming is an obligate component of the an .
alternatively , maintenance of mosquitoes in laboratory cages for several years forced this strain to become stenogamous ( i.e. the ability to mate in small cages ) , and this adaptation may have interfered with its ability to swarm when introduced into the sphere . as newly emerged adults rarely survive for more than 48 hrs without the availability of an energy source , mosquitoes must have supplemented their energy reserves with carbohydrates from the plants in the sphere ( table 1 ) for up to 6 days before they were allowed access to a blood source , in the form of a human volunteer sleeping in the hut . some plants like castorbean ( ricinus communis l. ) were flowering at the time of the experiment , and may have provided nectar sources . in cage experiments ( impoinvil et al . , in prep . )
we have observed a mean survival time of 7.0 0.2 days on this plant , which is comparable to mosquitoes given 6% glucose ( 8.7 0.2 days ) .
gambiae mosquitoes emerge from the pupal stage with a deficit not only in carbohydrates , but also lipid and protein , which usually is compensated for by consuming a ( small ) blood meal within the first few days of adult life , it is likely that mortality during the 6-day post - emergence period in the current trial was too high to have a good number of the 8090 females that emerged survive long enough to obtain their first blood meal . within 15 min of entering the hut at night ,
the volunteer noticed the sound of mosquitoes and subsequently felt mosquito bites on his exposed lower limbs .
this implies that females were receptive to host cues , entered the hut , probably through the eaves , and then successfully located and fed on the human host . at sunrise
, several engorged females were seen resting on the walls , indicating successful blood feeding and endophily ( indoor resting ) , which is typical for this species . following maturation of eggs ,
the second oviposition began during the night of day 21 , thus completing the life - cycle .
we continued to observe both immature and adult insects ( presumably mostly from the f1 generation ) until day 27 , when the experiment was terminated ( by refraining from entering the sphere for about 1 month ) .
the second experiment , in which we released 500 virgin females together with 1500 males demonstrated that mating does occur in a relatively small , semi - field system .
after the third night that a volunteer had slept in the hut , we observed eggs in the breeding sites .
the production of offspring , though , was low , and we only collected 40 pupae by the end of the trial period .
this may have been caused by heavy rainfall during three consecutive nights ( day 24 ) , which may have affected the survival of the adults and/or larvae or washed away the larvae from the breeding sites due to overflow .
since we observed few mosquitoes , we decided to conduct a human landing catch during two nights inside the hut , starting two nights after sleeping in the hut had ended .
nevertheless , the life - cycle was completed , as manifested by the harvested pupae , which were removed from the breeding sites to prevent emergence of the f1 generation which would have compromised interpretation of survival of the f0 generation .
the third experiment started by introducing 500 eggs into each of the breeding sites , whilst 1000 eggs ( from the same original batch ) were reared under laboratory conditions . in the laboratory , larvae developed at the same rate and
most reached maturity by day 10 , when the first pupae were observed ( fig .
this was similar for the breeding sites in the sphere , except that development was highly asynchronous , i.e. some larvae pupated by day 10 , whereas others took up to day 24 before pupation .
these times to pupation are similar to those observed in the first trial , but are again in contrast with other studies . on day 7
we counted all larvae and observed 887 in the insectary , as opposed to 652 in the sphere .
overall , the laboratory batch yielded 804 pupae , versus 495 from the breeding sites .
on the basis of these data , the average daily survival was 0.90 for the laboratory larvae , and 0.83 for the larvae in the sphere . with nearly half the larvae surviving to the adult stage , these results contrast sharply with much higher mortalities ( up to 90% ) observed in the kisumu area and so tome ( charlwood , pers .
. the sex ratio of emerging adults in the laboratory was 2:3 , which translated into a female population in the sphere of 297 , on the assumption that no insects died during emergence . on day 28 , after the release of eggs in the breeding sites , we observed that eggs had been laid by the f1 generation , but with only 6 and 3 eggs in the two breeding sites respectively .
cumulative percentage of pupation of eggs introduced in the two breeding sites inside the greenhouse ( ) or under insectary conditions ( ) .
our results have shown that by starting either at the post - blood feeding , pre - mating , or egg stage , a new generation of insects can be reared under these semi - field conditions , and that all life - history behaviours were successfully completed to a lesser or greater extent .
this therefore represents the first and promising step towards continuous maintenance of parasite - free an .
gambiae populations under semi - natural conditions that can be experimentally manipulated in studies of malaria vector ecology and transmission control .
russell and rao used a large outdoor cage , based on a design by hackett and bates , to study swarming and oviposition behaviour of an .
culicifacies giles in india , and showed that such systems can be used to unravel aspects of the behavioural ecology of anophelines .
our study shows that such systems can now also be developed for studies on african malaria vectors in order to start filling the gaps in our understanding of the behavioural ecology of these insects .
first and foremost , it provides a suitable intermediate between laboratory - based studies addressing mosquito behaviour and ecology , and the field situation .
too often , conclusions are drawn from results obtained under laboratory conditions that necessitate speculation as to what may or may not happen in the field .
fixed climatic conditions , cage - experiments , olfactometers and windtunnels , in which the mosquito strains used have been laboratory - reared for sometimes decades , may readily distort behavioural and ecological phenomena .
here we have shown that , beyond introducing f1 generation malaria - free mosquitoes from wild populations , it may be possible to rear vectors in situ within a semi - natural system that may minimize such artefacts .
conversely , the advantage of using insectary - reared mosquitoes is in the level of control that may be exerted that would not otherwise be possible : experiments can be conducted all - year - round , with fixed numbers of insects , of known age and physiological status , in a malaria parasite - free environment under ambient climatic conditions .
this enables more direct inferences to be drawn from data analysis as compared to longitudinal field studies , because of constant conditions and simplified experimental design .
we have recently evaluated the efficacy of several plants traditionally used by the luo community as repellents in a similar semi - field set - up , and simple logistic regression , on data collected during four nights per plant , yielded significant results . within a year of nearly continuous experimentation , the repellency of 8 plant species and 3 combinations thereof was evaluated through thermal expulsion or direct burning , and 9 species and 2 combinations thereof were tested in potted form .
such studies would have taken several years under field conditions , and would be limited to times when mosquito densities would have been sufficiently high to permit meaningful experiments .
gambiae maintained on a variety of diets ( blood or sugar alone , or a combination of both ) and revealed similar results to those obtained under laboratory conditions .
within one year of starting studies on the behaviour of mosquitoes around bednets in a semi - field setup , we transformed a regular conical bednet into a trap that may catch up to 70% of the females released .
recent field evaluation of this trap shows it to be a promising replacement for the human landing catch ( mathenge et al .
with the trap now being considered for commercial manufacturing ( it took a mere two years to reach this stage ) , this would have been impossible without the availability of a semi - field set - up in which continuous experimentation ensured rapid progress towards product development .
even though our system resembles more closely the field situation , it remains to be ascertained to what extent .
our current study was mainly qualitative in design and focused on life - cycle completion .
for instance , observation of eggs in the breeding sites in the morning of day 22 during the first trial implies that these originated from females that fed once on day 19 , as those that fed on day 17 should have laid before .
however , it is likely that these females fed twice , on day 17 and day 19 , and should be classified as pre - gravids , before fully maturing a batch of eggs .
furthermore , in the absence of a human host , mosquitoes survived up to six days after emergence , confirming field results that feeding on plant sugars does occur and may be an important feature of the life - cycle in this species ( foster and knols , unpublished data ) . obviously , there are limitations associated with these studies
there may be other , yet unknown , factors that affect the behavioural ecology of the insects in such systems . or , as bates wrote , following his outdoor cage studies in albania
: " one still can not be sure that the reactions of the mosquito are ' natural ' because there is always the barrier of wire liable to be encountered on extended flights ; and when the flight of a mosquito has been interrupted by this wire barrier its further activity may be definitely unnatural " .
rightly so , and even though we did not observe any obvious distorted behaviours , it is imperative that findings from semi - field studies be verified under natural outdoor conditions .
additional studies in which the release and performance of field - collected , blood - fed mosquitoes in the sphere is compared with that of laboratory specimens in terms of egg - recovery , developmental periods and important behavioural characteristics ( like swarming ) will provide further insight to what extent such systems mimic the natural anopheles environment and colony adaptation impairs natural behaviours . nevertheless , since bates ' days , advances in science merit a renewed impetus towards semi - field studies in contained near - natural environments , particularly with respect to transgenic mosquitoes .
fitness evaluations of engineered strains of vectors are mandatory for transformation technology to become an established disease control tool in africa . perhaps this alone , is ample justification for more intensive sphere studies , hopefully not only in kenya , but also in other african countries likely to be involved in this endeavour .
studies on gene flow , mating behaviour and reproductive fitness , combined with studies on the effects of laboratory maintenance on the genetic make - up of transformed strains to be released , can be conducted in semi - field systems .
such systems , particularly when used to study genetically - engineered mosquitoes will require more advanced containment levels than the system described here . guidelines for facility location , physical and biological containment , safety practices and calamity control need to be developed and adapted from existing arthropod containment guidelines
. there are several good reasons to further such studies in disease - endemic settings . under such conditions it will be possible to transform offspring from wild mosquitoes , conduct experiments under local ambient climatic conditions and evaluate transgene spread and fixation in offspring from field - collected gravid females that emerge in a semi - field setup .
last , but not least , it will enable scientists from developing countries to become more directly involved in evaluating the potential use and application of transgenic mosquitoes for future malarial disease control .
bgjk conceived of the study , and developed the system and experiments together with bnn , emm and wrm .
jcb and gfk actively contributed to the interpretation of the findings and drafting of the final manuscript .
bgjk and emm are engaged in commercialising the mbita bednet trap , developed in semi - field systems similar in nature to that described in this article , in collaboration with the vestergaard frandsen group ( denmark ) .
bos of wageningen university and research centre ( the netherlands ) for assistance with plant nomenclature .
this research was supported by the national institutes of health , usa ( grant numbers u19 ai45511 , d43 tw01142 , d43 tw00920 ) .
emm and wrm receive financial support from the undp / world bank / who special programme for research and training in tropical diseases ( tdr ) under grants i d 980794 and 980692 respectively . | backgroundthe development and implementation of innovative vector control strategies for malaria control in africa requires in - depth ecological studies in contained semi - field environments .
this particularly applies to the development and release of genetically - engineered vectors that are refractory to plasmodium infection .
here we describe a modified greenhouse , designed to simulate a natural anopheles gambiae giles ecosystem , and the first successful trials to complete the life - cycle of this mosquito vector therein.methodswe constructed a local house , planted crops and created breeding sites to simulate the natural ecosystem of this vector in a screen - walled greenhouse , exposed to ambient climate conditions , in western kenya . using three different starting points for release ( blood - fed females , virgin females and males , or eggs ) , we allowed subsequent stages of the life - cycle to proceed under close observation until one cycle was completed.resultscompletion of the life - cycle was observed in all three trials , indicating that the major life - history behaviours ( mating , sugar feeding , oviposition and host seeking ) occurred successfully.conclusionthe system described can be used to study the behavioural ecology of laboratory - reared and wild mosquitoes , and lends itself to contained studies on the stability of transgenes , fitness effects and phenotypic characteristics of genetically - engineered disease vectors .
the extension of this approach , to enable continuous maintenance of successive and overlapping insect generations , should be prioritised .
semi - field systems represent a promising means to significantly enhance our understanding of the behavioural and evolutionary ecology of african malaria vectors and our ability to develop and evaluate innovative control strategies . with regard to genetically - modified mosquitoes , development of such systems
is an essential prerequisite to full field releases . | Background
Methods
Design
Mosquitoes
Life-cycle completion
Ethical considerations
Results and Discussion
Microclimate
Life-cycle completion
Blood-fed females
Males and virgin females
Eggs
Authors contributions
Competing interests
Acknowledgements |
causality assessment ( ca ) , is a method of evaluation used in pharmacovigilance to find out the relationship between drugs exposed and reported adverse drug reactions ( adr ) .
it includes , finding the temporal relationship between drugs and reported adr , dechallenge , rechallenge , clinical and pathological characteristics of the events .
it is difficult for the practitioner with careful monitoring to identify the drugs causing adr .
in such a condition the withdrawal of drugs one at a time and evaluating the reaction of dechallenge has become essential .
dechallenge is a response observed in a patient such as reduction or disappearance of adr after withdrawal of a drug .
there are two types of dechallenge namely positive dechallenge which resolves with the withdrawal of drug and the negative dechallenge which follows a course of its own .
decision on the withdrawal of drug has been considered from the point of adr underlying the disease .
a typical procedure has to be followed before attempting rechallenge , with an understanding of risk involved for the patient .
so the prescribers and the patients may not come forward for the procedures involved except on a few occasions .
the prescribers too prefer to adopt dechallenge rather than rechallenge . detecting adverse drug reactions and dechallenge
data mining is a kind of statistical approach for discovering useful patterns from enormous amount of data .
it contains algorithms to find out the pattern by means of various approaches like classification , prediction , clustering and association .
data - mining algorithm called multi - item gamma position shrinker is now used for signaling potential adrs by food and drug administration ( fda ) in us .
the naive bayes ( nb ) a statistical classifier predicts class membership probabilities widely used by researchers in data mining for classification . it is assumed that all the variables contributing toward classification are mutually independent .
medical dictionary for regulatory activities ( meddra ) , an international medical terminology developed under the auspices of the international conference of harmonization of technical requirements for registration of pharmaceuticals for human use ( ich ) is a controlled medical vocabulary for describing adverse events with five levels : the coarsest is system organ class ( soc ) , followed by high level group term ( hlgt ) , higher level term ( hlt ) , preferred term ( pt ) , and lowest level term ( llt ) , the finest grained description .
the duplicate reports were deleted in accordance to fda 's recommendation of adopting in recent case number as described in one of the files asc - nts.doc from the website of the fda .
fda had stored diseases category at preferred terms ( pt ) level . among the five levels of adverse events hierarchy of meddra
, soc level was used to classify the diseases category by referring to cancer therapy evaluation program simplified disease classification v4.0 ( meddra v 12.0 ) .
researchers suggested that it might be more advantageous to perform data mining , using a coarser grained adverse event representation soc than pt level .
the data were loaded from fda 's text file to oracle database using extract , transform and load ( etl ) tools .
records with soc as gastrointestinal , renal and urinary , metabolism and nutrition disorders were considered for dechallenge classification .
the attributes considered to evaluate dechallenge were the diseases categories denoted as system organ class ( soc ) in meddra , drug with valid trade and verbatim name represented by code 1 and 2 by fda , outcoming like life - threatening ( lt ) , death ( de ) , congenital anomaly ( ca ) , hospitalization - initial or prolonged ( ho ) , disability ( ds ) , required intervention ( ri ) to prevent permanent impairment had been considered for determining the occurrence of dechallenge .
the process of determining suitable algorithm for detecting dechallenge was accomplished by comparing nb and nb .
the performance of data mining algorithm was estimated by the parameters like percentage of accuracy , error , precision and receiver operating characteristic ( roc ) curve .
all the parameters were depicted by 2 2 confusion matrixes , containing the total number of true positive ( tp ) , true negative ( tn ) , false positive ( fp ) and false negative ( fn ) , where positive referred to identified set and negative to rejected set .
accuracy , precision and error were calculated by formula 1 , 2 and 3 as follows : roc curve was used to convey the graphical representation of perfect , liberal , random and conservative performance of an algorithm .
statistical data mining techniques had been implemented in the field of post - marketing surveillance .
safety signal detection problems in pharmacovigilance were examined by roy and jeffrey et al . , statistical data sources and data mining methods used in safety signal were studied by atsuko and manfred et al . , liang and rongzhan et al .
corani and zaffalon proposed an extension of nb named as naive credal classifier to issue reliable classifications for a domain with high uncertain information .
zhang et al . , proposed a novel model called hidden naive bayes to avoid computational complexity . for data mining model , each patient
the disease category was denoted in soc level , by mapping the pt of fda with meddra pt using extensible markup language ( xml ) mapping .
nb theorem given in formula 4 had been used to calculate the probability of an outcome .
the class label attribute dechallenge had two distinct values ( yes , no ) represented by hypothesis ( h ) .
p(h / x ) is the posterior probability where hypothesis ( h ) represents the presence of dechallenge with x as known disease category , drug code and outcome .
p ( x / h ) is the posterior probability of x on the subject of h. p ( h ) is the prior probability of h regardless of disease category , drug code and outcome .
p(x ) is the prior probability of x. for calculating the prior probability p ( x ) , dechallenge record sets with
then the posterior probability was calculated based on outcome , disease category and drug code .
the data set of 2011 and 2012 records contained the constraints mentioned for the failure of naive bayes classifier . to overcome this , nb algorithm proposed by balamurugan et al . , was applied in the present study as detailed below .
this algorithm starts with the influence factor as the first step to determine the dependability of an attribute value on the class attribute .
influence factor was calculated for the attributes drug code , disease category and outcome on the class label dechallenge .
where i(x / ci)=influence factor n(x\ci ) = number of records in which attribute value x had the class label ci and n(ci)=total number of records in which the class label were ci .
statistical data mining techniques had been implemented in the field of post - marketing surveillance .
safety signal detection problems in pharmacovigilance were examined by roy and jeffrey et al . , statistical data sources and data mining methods used in safety signal were studied by atsuko and manfred et al . , liang and rongzhan et al .
corani and zaffalon proposed an extension of nb named as naive credal classifier to issue reliable classifications for a domain with high uncertain information .
zhang et al . , proposed a novel model called hidden naive bayes to avoid computational complexity . for data mining model , each patient
the disease category was denoted in soc level , by mapping the pt of fda with meddra pt using extensible markup language ( xml ) mapping . the fundamental assumption to attribute independence
nb theorem given in formula 4 had been used to calculate the probability of an outcome .
the class label attribute dechallenge had two distinct values ( yes , no ) represented by hypothesis ( h ) .
p(h / x ) is the posterior probability where hypothesis ( h ) represents the presence of dechallenge with x as known disease category , drug code and outcome .
p ( x / h ) is the posterior probability of x on the subject of h. p ( h ) is the prior probability of h regardless of disease category , drug code and outcome .
p(x ) is the prior probability of x. for calculating the prior probability p ( x ) , dechallenge record sets with
then the posterior probability was calculated based on outcome , disease category and drug code .
the data set of 2011 and 2012 records contained the constraints mentioned for the failure of naive bayes classifier . to overcome this , nb algorithm proposed by balamurugan et al . , was applied in the present study as detailed below .
this algorithm starts with the influence factor as the first step to determine the dependability of an attribute value on the class attribute .
influence factor was calculated for the attributes drug code , disease category and outcome on the class label dechallenge .
/ ci)=influence factor n(x\ci ) = number of records in which attribute value x had the class label ci and n(ci)=total number of records in which the class label were ci .
statistical data mining techniques had been implemented in the field of post - marketing surveillance .
safety signal detection problems in pharmacovigilance were examined by roy and jeffrey et al . , statistical data sources and data mining methods used in safety signal were studied by atsuko and manfred et al . , liang and rongzhan et al .
corani and zaffalon proposed an extension of nb named as naive credal classifier to issue reliable classifications for a domain with high uncertain information .
zhang et al . , proposed a novel model called hidden naive bayes to avoid computational complexity .
the other data were outcome of drug and disease category . drug classified by fda as 1 for valid trade and 2 for verbatim name .
the disease category was denoted in soc level , by mapping the pt of fda with meddra pt using extensible markup language ( xml ) mapping .
nb theorem given in formula 4 had been used to calculate the probability of an outcome .
the class label attribute dechallenge had two distinct values ( yes , no ) represented by hypothesis ( h ) .
p(h / x ) is the posterior probability where hypothesis ( h ) represents the presence of dechallenge with x as known disease category , drug code and outcome .
p ( x / h ) is the posterior probability of x on the subject of h. p ( h ) is the prior probability of h regardless of disease category , drug code and outcome .
p(x ) is the prior probability of x. for calculating the prior probability p ( x ) , dechallenge record sets with
then the posterior probability was calculated based on outcome , disease category and drug code .
the data set of 2011 and 2012 records contained the constraints mentioned for the failure of naive bayes classifier . to overcome this , nb algorithm proposed by balamurugan et al . , was applied in the present study as detailed below .
this algorithm starts with the influence factor as the first step to determine the dependability of an attribute value on the class attribute .
influence factor was calculated for the attributes drug code , disease category and outcome on the class label dechallenge .
where i(x / ci)=influence factor n(x\ci ) = number of records in which attribute value x had the class label ci and n(ci)=total number of records in which the class label were ci .
it is observed from table 1 , influence factor is high for outcomes such as ho and lt , drug code with code 1 and disease category such as gastrointestinal disorder .
yes for combinations like gastrointestinal disorder with outcome as ho and lt and for drugs with code 1 . hence classifying the dechallenge for unknown records with same combinations of attributes can be predicted as
influence factor analysis of outcome , disease category , and drug code with dechallenge performance analysis of nb and nb provided in table 2 , where the average accuracy of nb is 90.11 and nb is 70.25 , average error of nb is 19.8 percent higher than nb , and precision of nb is 7.4 percent higher than nb . it is observed from experimental result , nb performs well in case of attributes with categorical values and zero probability issue . in the roc graph shown in figure 1 ,
although nb fits in the category of liberal performance with true positive rate as ( .7 , .8 , .9 , .9 ) , there is also substantial number of false positive rate ( .6 , .7 , .6 , .5 ) ; whereas nb fits in perfect performance with true positive rate as ( .9 , .9 , .9 , .9 ) , and minimal false positive rate as ( .2 , .8 , .0 , 0 ) .
comparison of performance analysis of error , precision and accuracy of nb and nb receiver operating characteristic curve for improved nave bayes and naive bayes
the fda uses data mining to screen the aers database using bayesian protocol for the presence of disproportionality in large adverse event - drug product pairs , but the data must be evaluated to determine causality reviews like dechallenge .
the performance of any data - mining algorithm depends on the type of attributes and its application .
a common means of identifying the association between drug and disease in pharmacovigilance is through disproportionality analysis .
this produces the results based on 2 2 tables as there are relevant drug and adr combination .
hence for large amount of data , this method will produce more number of tables which reduces the effectiveness of the approach .
several studies reported the need of data mining algorithms to review the data to make authoritative conclusion .
many studies reported the usage of mining algorithms like proportional reporting ratio , multi - item gamma - poisson shrinker .
further investigation of statistical methods to analyze large amount of data is essential to improve the effectiveness of pharmacovigilance activities .
hence in this study the data mining algorithms like nb and nb for determining the performance of algorithms in enormous data for detecting dechallenge have been investigated . among the 26 soc disease categories ,
the results presented here are based on 3 socs such as gastrointestinal , metabolism and nutrition , renal and urinary disorders . when nb is used to detect the dechallenge ,
the posterior probability is zero for records with outcome as ca and disease category as gastrointestinal disorder .
hence 72 records of the year 2011 fourth quarter are classified as unknown by nb algorithm .
the algorithm fails when the probability of a particular outcome or disease is uniformly distributed .
nb resolved zero probability issue by determining attributes with high influence factor and thus reducing the noise present in data for effective detection of dechallenge .
nb can be applied to any dataset which suffers from zero probability exertion . from the experimental analysis ,
it is clear that nb can be used for large data set in detecting causality reviews . for the fda
record sets , nb produced higher accuracy than nb and detected the dechallenge value as yes for drugs with code 1 and gastrointestinal disorder with outcome as ho and lt .
influence factor analysis in nb proved the usage of this algorithm in pharmacovigilance for predicting unknown samples .
most data available in fda and world health organisation ( who ) have neither brought in health science education , nor trained to utilise for patient care purpose .
post - marketing surveillance techniques like detecting dechallenge will help the practitioners and prescribers to gain knowledge about drugs with various reactions .
the outcome of the classification algorithms show that nb outperformed nb in traditional interesting measures like accuracy and minimal error in classifying dechallenge . | aim : dechallenge is a response observed for the reduction or disappearance of adverse drug reactions ( adr ) on withdrawal of a drug from a patient . currently available algorithms to detect dechallenge have limitations .
hence , there is a need to compare available new methods . to detect dechallenge in spontaneous reporting systems , data - mining algorithms like naive bayes and improved
naive bayes were applied for comparing the performance of the algorithms in terms of accuracy and error .
analyzing the factors of dechallenge like outcome and disease category will help medical practitioners and pharmaceutical industries to determine the reasons for dechallenge in order to take essential steps toward drug safety.materials and methods : adverse drug reactions of the year 2011 and 2012 were downloaded from the united states food and drug administration 's database.results:the outcome of classification algorithms showed that improved naive bayes algorithm outperformed naive bayes with accuracy of 90.11% and error of 9.8% in detecting the dechallenge.conclusion:detecting dechallenge for unknown samples are essential for proper prescription .
to overcome the issues exposed by naive bayes algorithm , improved naive bayes algorithm can be used to detect dechallenge in terms of higher accuracy and minimal error . | Introduction
Materials and Methods
None
Related Works
Data Mining Model
The Algorithms Used
Improved Naive Bayes
Results
Discussion
Conclusions |
metformin is a first - line drug in the pharmacotherapy of type 2 diabetes . apart from the low cost , good efficacy , and beneficial effects on body weight ,
several studies have reported association of vitamin b12 deficiency in type 2 diabetes patients treated with metformin .
the reported vitamin b12 deficiency in the general population of india varies from 12% to 67% .
the impact of metformin use on an already vitamin b12 deficient population is an interesting question .
the patients were recruited from the endocrinology outpatient department of a tertiary care hospital from august 2014 to december 2015 , after taking informed consent .
patients were defined as type 2 diabetes if they met the american diabetic association criteria with an age at diagnosis above 30 years .
pregnancy , pernicious anemia , malabsorption syndrome , gastrointestinal surgery , autoimmune thyroid disease , chronic hepatitis , chronic kidney disease stage 4 and above , chronic alcohol abuse , and the use of oral / parenteral vitamin b12 or multivitamin supplements were exclusion criteria .
metformin group consisted of patients with type 2 diabetes mellitus with ongoing treatment with metformin with duration of metformin use 3 months while no metformin group consisted of patients with type 2 diabetes mellitus who had never received metformin .
the patients who did not consume eggs or any form of meat were considered as vegetarian while those consuming eggs or meat were considered as nonvegetarian .
history about diabetes onset , dose , and duration of metformin usage as well as recent ( within 3 months ) glycosylated hemoglobin , hemoglobin , and mean corpuscular volume ( mcv ) were obtained from hospital records .
the hospital records , patient 's prescriptions , and medicines were also searched for prescription of any vitamin b12-containing supplements and patients were shown a list of commonly available multivitamins containing vitamin b12 and were asked about their use at any time in the past .
assessment for peripheral neuropathy was done using diabetic neuropathy symptom score ( dns ) and diabetic neuropathy examination score ( dne ) .
dns score 1 was considered suggestive of neuropathy while dne score 3 was considered suggestive of peripheral neuropathy .
vitamin b12 estimation was done by a solid phase , competitive chemiluminescent enzyme immunoassay on immulite 1000 analyzer using commercial kits from siemens healthcare diagnostics inc .
the interassay coefficients of variation ( cv ) were 7.4% ( 310 pg / ml ) , 6.1% ( 660 pg / ml ) , and 17% ( 1192 pg / ml ) while the intra - assay cv were 11.3% ( 159 pg / ml ) , 10.3% ( 204 pg / ml ) , 6.7% ( 401 pg / ml ) , 5.3% ( 736 pg / ml ) , and 5.9% ( 1308 pg / ml ) .
vitamin b12 deficiency was defined as levels below 150 pmol / l and borderline deficiency levels between 150 and 221 pmol / l .
statistical analysis was performed using statistical package for social sciences spss version 20 ( spss inc . ,
the comparison between the metformin group and no metformin group was done with student 's t - test for continuous variables and chi - square test for categorical variables .
linear regression analyses were carried out to study the effect of duration since the diagnosis of diabetes , use of metformin , and duration of metformin use on serum vitamin b12 levels .
table 1 shows the baseline characteristics of the metformin and no metformin groups . the two groups were comparable except for duration of diabetes which was significantly greater in the metformin group .
maximum daily dose of metformin was 834.1 754.2 mg ( range 5002550 mg ) .
the cumulative dose of metformin was 980.6 1576.1 g ( range 7510,950 g ) .
baseline characteristics of metformin and no metformin groups the mean unadjusted vitamin b12 was not significantly different in the metformin and no metformin groups : 267.7 194.4
pmol / l ( 95% confidence interval [ ci ] 233.6302.7 ) versus 275.1 197.2 pmol / l ( 95% ci 229.3334.1 ) , respectively , p = 0.78 . however , the mean duration of diabetes was longer in the metformin group as compared to the nonmetformin group ( 5.6 4.7 years vs. 2.0 3.9 years , p < 0.001 ) .
vitamin b12 deficiency was present in 35.5% of metformin group and in 33.8% of the no metformin group ( p = 0.93 ) while borderline deficiency was seen in 22.3% of metformin group and 21% of no metformin group .
on univariate linear regression analysis with vitamin b12 levels as the dependent variable and metformin use ( no and yes ) as the predictor variable , metformin use was associated with a 8.7 30.9 pmol / l ( 95% ci 69.752.3 , p = 0.78 ) lower vitamin b12 level .
on univariate linear regression analysis with vitamin b12 levels as the dependent variable and duration of diabetes as the predictor variable , vitamin b12 levels were 12.2 3.0 pmol / l ( 95% ci 6.418.0 , p < 0.001 ) higher for every 1 year increase in the duration of diabetes .
to further study the association of vitamin b12 levels and duration of diabetes , we performed a stratified analysis .
figure 1 shows the box plot of serum vitamin b12 with duration of diabetes categorized into newly diagnosed ( < 1 year ) , 15 years , and 5 years .
serum vitamin b12 levels were higher by 41.5 pmol / l in patients with diabetes duration of 15 years compared to those with recently diagnosed diabetes ( p = 0.31 ) .
pmol / l in patients with duration of diabetes 5 years compared to those with recently diagnosed diabetes ( p < 0.01 ) .
similarly , serum vitamin b12 levels were 77.7 pmol / l higher in 5 years diabetes duration group compared to 15 year duration of diabetes group ( p = 0.01 ) .
vitamin b12 levels and duration of diabetes ( new onset , 05 years and 5 years ) on univariate linear regression analysis with vitamin b12 levels as the dependent variable and duration of metformin use as the predictor variable , duration of metformin use predicted a 0.8 0.4 pmol / l ( 95% ci 0.0041.7 pmol / l , p = 0.05 ) lower vitamin b12 levels for every 1 month increase in the duration of metformin use .
on stratifying duration of metformin use into no metformin use , 02 years , 25 years , and more than 5 years , it was found that a 20.2 pmol / l ( p = 0.62 ) and 37.1 pmol / l lower serum vitamin b12 concentration was observed in individuals with a 02 years and 25 year duration of metformin use , respectively , compared with the group which had not received metformin .
in contrast , the serum concentration of vitamin b12 was higher by 45.2 pmol / l ( p = 0.29 ) in individuals who had received metformin for more than 5 years compared to those who had never received metformin [ figure 2 ] . to understand the interplay of duration of diabetes and metformin use on serum vitamin b12 levels ,
a stratified analysis was carried out . a multivariate linear regression analysis with serum vitamin b12 levels as the dependent variable and metformin use ( no / yes ) and duration of diabetes ( stratified as 15 years and 5 years ) as predictor variables
metformin use group was associated with a 87.7 37.7 pmol / l ( 95% ci , 162.13.3 , p = 0.02 ) lower serum vitamin b12 levels .
48.0 pmol / l ( 95% ci , 8.0197.4 , p = 0.03 ) higher in the 15 year duration of diabetes group while they were 192.1 51.9
pmol / l ( 95% ci , 89.8294.5 , p 0.001 ) higher in 5 year duration of diabetes group . effect of duration of metformin use on serum vitamin b12 levels the serum vitamin b12 levels in the metformin and no metformin
groups stratified according to duration of diabetes ( new onset , 15 years and 5 years ) are shown in the box plot given in figure 3 .
the serum vitamin b12 levels were 97.5 pmol / l lower in the metformin group as compared to the no metformin group in the new onset diabetes ( < 1-year duration since diagnosis of diabetes ) category .
in the 15 year duration of diabetes category , the serum vitamin b12 levels were 30.8 45.7 pmol / l ( p = 0.50 ) lower in the metformin group as compared to the no metformin group .
on analyzing the 5 years duration of diabetes category , the serum vitamin b12 levels were 203.9 75.8 pmol / l ( p < 0.001 ) lower in the metformin group as compared to the no metformin group .
vitamin b12 levels by metformin use and duration of diabetes on comparing patients with vitamin b12 deficiency to those with normal vitamin b12 levels , vitamin b12-deficient patients did not have significantly higher percentage of dne or dns positives .
mean hemoglobin and mcv were also not significantly different in the vitamin b12 deficiency and normal vitamin b12 groups [ table 2 ] .
the present study involving 183 patients with type 2 diabetes mellitus ( 121 metformin and 62 without metformin ) showed lower vitamin b12 levels with metformin use , when adjusted for duration since diagnosis of diabetes , which is consistent with other studies published earlier . without adjusting for duration of diabetes , there was neither a significant difference in serum vitamin b12 levels nor in the prevalence of vitamin b12 deficiency . however , the prevalence of vitamin b12 deficiency in patients on metformin in our study is higher than that reported in western literature . in different studies ,
vitamin b12 deficiency was found in 5.8% , 8.6% , 6.3% , of patients with type 2 diabetes mellitus on metformin .
the higher prevalence in our study ( 35.5% ) is not surprising considering that the prevalence of vitamin b12 in the apparently healthy populations in india have been reported to be as high as 33.3%67% .
a predominantly vegetarian diet could be one of the causes of higher prevalence of vitamin b12 deficiency in india .
metformin - induced vitamin b12 deficiency has been ascribed to the binding of the hydrophobic tail of biguanide to the hydrocarbon core of membranes .
the biguanide group being positively charged ( protonated ) gives a positive charge to the membrane and can displace divalent cations such as calcium .
the uptake of vitamin b12 into the ileal cells is calcium dependent and can thus be impaired by metformin .
however , in a cross - sectional study of two different ethnic groups with type 2 diabetes mellitus in india and the united kingdom , the prevalence of vitamin b12 deficiency of 12% was found in the indian population compared to 27% in a european population .
the authors cited the use of fermented foods by the indian population as the probable cause of lower vitamin b12 deficiency , but it underscores the dependence of vitamin b12 levels on dietary factors , which vary in different populations .
an interesting finding from our study is the statistically significant rise in vitamin b12 with increasing duration of diabetes . in a cross - sectional study from the us comprising 1621 patients with type 2 diabetes mellitus ( 575 on metformin and 1046 not on metformin ) and 6867 persons without diabetes , the diabetes without metformin group had the lowest prevalence of vitamin b12 deficiency ( 2.4% ) as compared to 5.8% in the diabetes on metformin group and the 3.3% in the group without diabetes .
interestingly , patients not on metformin group in that study had a statistically significant longer duration of diabetes . however , diabetes not on metformin group included patients who might have earlier received metformin and the authors speculated that these patients might have been diagnosed with vitamin b12 deficiency earlier and hence either treated for vitamin b12 deficiency or given dietary modifications to increase vitamin b12 levels in the diet .
we did make an active effort to exclude patients who had been given vitamin b12-containing supplements for any indication ( review of available medical records was done , and patients were asked about the use of vitamin b12-containing multivitamin supplements ) , but these preparations are available over the counter , and we can not be sure that patients had never taken these medications earlier .
in addition , the higher vitamin b12 levels with greater duration of diabetes were seen in the no metformin group also who were not at the risk of metformin - related vitamin b12 deficiency ( as they never received metformin ) and hence are less likely to have received vitamin b12 treatment .
the impact of vitamin b12 deficiency on hematological parameters such as mcv and hemoglobin as well as on peripheral neuropathy ( dns and dne scores ) was studied .
we did not find any increase in mcv or decrease in hemoglobin in the vitamin b12-deficient patients .
the dns and dne scores were positive in a greater percentage of patients who were not vitamin b12 deficient ; however , this group also had a longer duration since the diagnosis of diabetes .
in other words , vitamin b12 deficiency as defined by serum vitamin b12 levels did not impact hematological or neurological parameters assessed in our study .
however , serum total vitamin b12 levels may not accurately reflect vitamin b12 status of the body and therefore biochemical deficiency often does not result in clinical deficiency .
the limitation of our study is that we did not measure functional markers of vitamin b12 deficiency ( serum homocysteine and serum methylmalonic acid levels ) which may better reflect the status of vitamin b12 levels in the body as compared to serum vitamin b12 levels .
the strength of this study is that the no metformin group included only those patients who had never taken metformin .
the reason for choosing such a group was to exclude a possible long - term effect of metformin use on vitamin b12 status .
vitamin b12 is stored in the liver and several years may pass before the stores are depleted and detectable vitamin b12 deficiency manifests .
thus , including patients with a history of metformin use in the no metformin group is a potential confounder which we excluded in this study .
in our study , metformin use was associated with a significantly lower serum vitamin b12 levels when adjusted for duration of diabetes .
. further study of the impact of duration of diabetes on serum vitamin b12 levels and of functional markers of vitamin b12 deficiency on hematological and neurological parameters will be interesting . this study was supported by education grant from the indian council of medical research . | context : there are limited data about the effect of metformin use on serum vitamin b12 levels in type 2 diabetes patients from india.aims:we studied serum vitamin b12 levels in patients with type 2 diabetes mellitus who were receiving metformin and compared them to those never treated with metformin.subjects and methods : a total of 183 patients ( metformin group 121 , no metformin group 63 ) of type 2 diabetes from the endocrinology clinic of a tertiary care center in north india were studied .
serum vitamin b12 levels were measured in all patients .
diabetic neuropathy symptom score ( dns ) and diabetic neuropathy examination score ( dne ) were used to assess peripheral neuropathy while hemoglobin and mean corpuscular volume ( mcv ) were used to assess anemia.results:the serum vitamin b12 levels were 267.7 194.4 pmol / l in metformin group and 275.1 197.2
pmol / l in the no metformin group ( p = 0.78 ) . when adjusted for duration of diabetes , metformin use
was associated with a 87.7 37.7 pmol / l ( 95% confidence interval [ ci ] , 162.13.3 , p = 0.02 ) lower serum vitamin b12 levels .
no significant increase in the prevalence of neuropathy ( dns and dne scores ) , anemia , or mcv was found in the vitamin b12 deficient patients ( levels < 150
pmol / l ) as compared to patients with normal vitamin b12 . however , serum vitamin b12 levels for the entire cohort were higher by 12.2 3.0 pmol / l ( 95% ci 6.418.0 , p <
0.001 ) for every 1 year increase in the duration of diabetes.conclusions:metformin use was associated with a lower serum vitamin b12 levels when adjusted for duration of diabetes .
increasing duration of diabetes was associated with higher serum vitamin b12 levels . | I
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to report a case of visually significant band keratopathy associated with ocular inflammation and systemic hypercalcemia which markedly decreased in severity after treatment of these underlying factors .
a 53-year - old asian female with granulomatous panuveitis in the left eye presented with diffuse band keratopathy through the central cornea . the serum calcium was elevated .
the patient was treated with topical prednisolone acetate 1% and oral prednisone with marked improvement in inflammation . the band keratopathy lessened in severity with clearing of the central cornea and improvement in visual acuity .
the term band keratopathy describes the precipitation of calcium salts in bowman s layer in a band - like distribution across the central cornea .
there are several local and systemic causes of band keratopathy , the most common ocular condition being intraocular inflammation and the most common systemic condition being hypercalcemia ( najjar et al 2004 ) treatment of symptomatic band keratopathy is typically surgical ; treatment of the underlying cause can prevent further calcium deposition but does not usually reverse the corneal findings . we herein report a case of band keratopathy associated with ocular inflammation and systemic hypercalcemia , which markedly improved after treatment of the underlying factors .
a 53-year - old asian female presented with intermittent pain and decreased vision in the left eye .
her past ocular history was significant for retinal detachment repair in the right eye five months prior .
she reported a slow visual decline in her left eye starting six weeks after the surgery on her right eye .
her past medical history was significant for chronic active interstitial pneumonitis diagnosed by lung biopsy four years prior , rheumatoid arthritis , hypothyroidism , and a recent diagnosis of hypercalcemia ( 12.7 mg / dl ) believed to be secondary to excessive calcium intake . on presentation , visual acuity was 9/200 in the right eye and 20/100 in the left eye .
slit - lamp examination revealed a cataract in the right eye without uveitis ; the left eye had diffuse band keratopathy through the central cornea ( figure 1 ) , prominent mutton - fat keratic precipitates , 1 + anterior chamber cell , and 2 + vitritis .
dilated funduscopic examination revealed a cup - to - disc ratio of 0.3 in both eyes with peripapillary atrophy .
the view into the left eye was hazy due to band keratopathy and vitritis but the retina appeared flat with no areas of vasculitis or choroiditis .
a work up was obtained including a fluorescent treponemal antibody ( fta ) , rapid plasma regain ( rpr ) , human leukocyte antigen b27 ( hlab27 ) , and chest imaging but was unrevealing as to the etiology of the uveitis .
a lung biopsy was consistent with chronic active interstitial pneumonitis ; there was no evidence of sarcoidosis on biopsy .
the patient returned two months later and reported an improvement in visual acuity and resolution of ocular discomfort .
medications included prednisolone acetate 1% five times daily to the left eye and prednisone 10 mg per day , which had been initiated by the patient s primary care physician 2 weeks prior due to shortness of breath .
visual acuity was 7/200 in the right eye and 20/35 in the left eye . slit lamp examination was significant for new mild , peripheral band keratopathy in the right eye and improving band keratopathy in the left eye .
there was trace anterior chamber inflammation in the right eye and 1 + vitritis in the left eye .
dilated funduscopic examination of the left eye revealed inactive - appearing chorioretinal lesions along the superotemporal and inferotemporal arcades .
topical prednisolone acetate 1% was slowly tapered in the left eye ; no local therapy was instituted in the right eye as the patient was asymptomatic and vision was stable .
the patient returned two months and reported further improvement in vision in the left eye .
medications included prednisolone acetate 1% once daily in the left eye and prednisone 10 mg daily .
slit lamp examination was significant for stable band keratopathy in the right eye and a marked improvement in band keratopathy ( figure 2 ) in the left eye .
we herein describe a case of transient band keratopathy associated with ocular inflammation and mild systemic hypercalcemia .
although it is unclear which of the two factors was responsible for the band formation , we postulate that the ocular inflammation played a more dominant role as the uveitis was more severe and the systemic hypercalcemia was relatively mild .
in addition , the time course of the band development closely mirrored that of the ocular inflammation .
specifically , band keratopathy was noted in the right eye in the setting of ocular inflammation and normalizing calcium levels .
clearing of the left central cornea occurred 4 months after treatment of ocular inflammation and normalization of serum calcium .
transient band keratopathy has been described in patients with systemic hypercalcemia due to renal failure ( feist et al 1992 ) , sarcoidosis ( johnston et al 1995 ) , and pituitary adenoma ( alonso santiago and ramirez fe 2002 ) in the first two cases , the serum calcium level was higher than that found in our patient ( 18 mg / dl and 14.2 mg / dl , respectively ) . a mild anterior uveitis was noted in the patient with transient band keratopathy associated with sarcoidosis . the calcium level in the patient with a pituitary adenoma ( alonso santiago and ramirez fe 2002 ) was similar to that of our patient ( 12.2 mg / dl ) . in summary , we present a case of transient band keratopathy associated with moderate ocular inflammation and mild hypercalcemia .
this case suggests that early and aggressive treatment of the inflammation may result in reversal of band keratopathy .
chelation of band keratopathy should be deferred until an adequate course of anti - inflammatory therapy has been undertaken . | purposeto report a case of visually significant band keratopathy associated with ocular inflammation and systemic hypercalcemia which markedly decreased in severity after treatment of these underlying factors.methodsretrospective case report.resultsa 53-year - old asian female with granulomatous panuveitis in the left eye presented with diffuse band keratopathy through the central cornea .
the serum calcium was elevated .
the patient was treated with topical prednisolone acetate 1% and oral prednisone with marked improvement in inflammation .
the band keratopathy lessened in severity with clearing of the central cornea and improvement in visual acuity.conclusionsearly medical treatment of underlying factors may allow reversal of band keratopathy . | Purpose
Methods
Results
Conclusions
Introduction
Case report
Discussion |
unilateral vestibular hypofunction ( uvh ) is a vestibular disorder in which a total or
partial decrease in vestibular function is observed , which is characterized by abnormally
reduced responses to the caloric test unilaterally .
disorders , such as vestibular
schwannoma , vestibular neuritis , infections , and trauma affecting the inner ear , that can
lead to destruction of the sensorineural epithelium of the membranous labyrinth and/or
vestibular nerve fibers are diagnosed as uvh .
besides dizziness , the clinical picture of uvh
may include nystagmus , ocular torsion , oscillopsia , postural instability , gait disorders ,
anxiety , depression , and fear .
uvh patients commonly share the same symptoms , in spite of
differences regarding the etiology , onset , and clinical findings of dysfunction or the type
and extent of the vestibular deficit1 , 2 . for a person with vestibular hypofunction , the main aims of treatment by the health - care
providers are restoring function or preventing further dysfunction . to deliver an effective
rehabilitation program to them , these patients need to be evaluated and diagnosed properly .
routine diagnosis of vestibular hypofunction has been based on the results of conventional
methods such as the head thrust test ( htt ) , the horizontal head - shaking nystagmus test , and
the caloric test3 .
other than these tests ,
previous studies have used a variety of outcome measures including dynamic posturography ,
the motion sensitivity quotient ( for rapid head movements ) , and the self assessed dizziness
handicap inventory ( dhi - jacobson and newman , 1990 ) .
apart from these , advanced techniques
like electronystagmography ( eng ) , videonystagmography ( vng ) , and rotational chair tests have
also been used for measurement of vestibulo - ocular function , and computerized dynamic
posturography ( cdp ) incorporates all three modalities to effect an assessment of static and
dynamic stance .
the sensory organization test ( sot ) protocol of cdp evaluates the patient s
ability to use the three systems that are chiefly responsible for maintaining upright ,
volitional , balanced posture : vision , the vestibular system , and proprioception .
but the
cost and time required to perform these tests are factors that keep them from being used in
general clinics4,5,6 .
even though the sensitivity of the htt for identifying individuals with
vestibular hypofunction is relatively good , there are studies that prove that it is more
sensitive in patients with bilateral vestibular hypofunction ( bvh ) than in patients with
uvh7 .
while various clinical measures
have been developed to document uvh , the limitations of these tests are in the lack of
specificity for screening the unilateral vestibular deficit .
thus , there is a need for a
simple quantitative objective test to screen for unilateral vestibular hypofunction , and for
this reason , the sideways stepping test ( sst ) or fukuda stepping test ( fst ) could be a valid
and reliable diagnostic tool for detecting uvh .
therefore , the purpose of this study was to
investigate whether sst is both a sensitive and specific test for detection of uvh as it is
effective , safe , and economical and therefore can be used widely throughout the world .
the present study was conducted in a private otolaryngology ( ent ) department in jeddah ,
saudi arabia .
twenty - eight adults participated in the study ( 15 male and 13 females ) .
written informed consent
the
study was conducted after obtaining approval from the institutional ethics review board of
the faculty of applied medical sciences , king abdul aziz university , jeddah .
all the
participants were required to complete and clear the following comprehensive screening
tests : cervical instability test , modified vertebral artery test ( mvat ) , spontaneous
( resting ) nystagmus test , smooth pursuit test , saccadic eye movement test , and vestibular
ocular reflex ( vor ) cancellation test to rule out all central mediated problems . only
subjects complaining of peripheral dizziness who were between the ages of 25 and 55 years
old and able to ambulate independently were included in the study .
subjects with central
mediated problems such as stroke , acquired brain injury , multiple sclerosis , or peripheral
neuropathy were excluded from the study .
moreover , subjects who could not ambulate
independently , were blind , were profoundly deaf or had cervical spine pathology were also
excluded .
the major outcome variables for confirming the presence of uvh were obtained by
the sst and the gold standard test , which is head - shaking nystagmus hsn test using video
electronystagmography ( veng ) .
the patients were evaluated for uvh , starting
first with the sst . in this test
, the subject is asked to stand with their feet together and
their hands by their sides .
the subject is then asked to close his / her eyes or is
blindfolded .
the test is positive if there is involuntary leaning to one side and hip sway .
sometimes , the subject lifts a hand to compensate . in that case , the subject is asked to
take two steps sideways and stop , first with the eyes open and then with the eyes closed or
blindfolded .
the test was positive if there is any involuntary swaying and/or any steps
taken to compensate .
the suject is then asked to jog on the spot for 30 seconds with the
eyes closed or blindfolded .
the test is positive if there is
any involuntary drifting to one side or , in some cases , forward or backward .
after finishing the sst , the subject is then evaluated with the hsn test using veng , which
is the gold standard test used to determine whether a vestibular ( inner ear ) disease may be
the causative factor for a balance or dizziness problem and is one of the only tests
available today that can distinguish between a unilateral ( one ear ) and bilateral ( both
ears ) vestibular dysfunction .
veng testing consists of a series of tests designed to
document a person s ability to follow visual objects with their eyes and how well the eyes
respond to information from the vestibular system . to monitor the movements of the eyes ,
however ,
during veng testing , the therapist moves the patient s head and body into various positions
to make sure that there are no inappropriate eye movements ( nystagmus ) , when the patient s
head is in different positions .
hsn is a latent spontaneous vestibular nystagmus that can be
recorded with veng . during the test for hsn
, the examiner turns the subject s head by about
45 degrees horizontally about 20 times .
hsn is defined as the occurrence of at least 5 beats
of nystagmus immediately after the head - shaking maneuver , which should be performed with
frenzel veng goggles .
there is good evidence that hsn reflects a dynamic ( peripheral and/or
central vestibular ) asymmetry of the velocity storage mechanism . in peripheral lesions
, the
ipsilateral dynamic vor deficits can lead to an asymmetric accumulation within the velocity
storage , the discharge of which determines the direction of hsn , usually toward the
unaffected ear9 . the results of both tests
were then compared with each other ; the results should be the same in both tests .
data were analyzed using spss for windows version 19.0 ( spss , inc . , chicago , il , usa ) .
frequencies and relative frequencies were computed for nominal variables , and means and
standard deviations ( sd ) were computed for the continuous variables .
the test
of independence was used to assess the relationship between the sst and the gold standard
hsn test using veng .
sensitivity , specificity , positive predictive value and negative predictive value also were
measured .
the baseline frequency characteristics of the study group are presented in table 1table 1.baseline frequency characteristic of the study group ( n = 28)validfrequencypercentsexmale1553.6%female1346.4%hsnpositive1346.4%negative1553.6%sstpositive1657.1%negative1242.9% .
the results showed that the sst is strongly correlated with the gold standard
hsn test using veng ( r = 0.81 , p<0.001 ) .
a stepwise linear regression was conducted to
determine the effect of the sst on the gold standard ( hsn test using veng ) results
( r = 0.65 , p < 0.001 ) .
sixty - five percent of the variability in the gold
standard test result was explained by its relationship to the sst .
the data used to measure
the sensitivity and specificity in this study are presented in tables 2 and
3table 2.data for sensitivity and specificitytrue positive ( tp)false negative ( fn)13 ( 46%)0 ( 0%)false positive ( fp ) true negative ( tn)3 ( 11%)12 ( 43%)table 3.summary of sensitivity , specificity , ppv , and npv resultspatients with uvh by the hsn test using
vengcondition positivecondition negativeoutcome positivetrue positive ( tp ) = 13false positive ( fp ) = 3positive predictive value 81%outcome negativefalse negative ( fn ) = 0true negative ( tn ) = 12negative predictive value 100%sensitivity 100%specificity 80% , and the results show that sst has very high sensitivity ( 100% ) and high
specificity ( 80% ) .
the primary purpose of the current study was to examine whether the sst could be used to
identify patients with uvh as an alternate method to the hsn test using veng .
even though
there are many tests in practice for detecting vestibular hypofunction , all of them are used
to diagnose a bilateral vestibular hypofunction rather than uvh . as far as we know , this is
the first paper published based on the use of sst , which is reliable , easy to apply , and
cost effective , instead of the hsn test using veng to diagnose a patient with uvh .
uvh is currently diagnosed using veng , but some studies have shown that there is another
test that can be used for diagnosing uvh , namely , the sst . in 1959 , fukuda proposed a
variation of the tretversuch test of unterberger ( 1938 ) and the waltzing
test of hirsch ( 1940 ) named the stepping test ; that test is intended to
identify the weaker of the labyrinths ( not necessarily the side with the lesion ) by
direction of rotation of a patient while walking on the spot with the eyes closed10 . actually , some researchers have suggested
use of the sst in combination with other clinical tests ( e.g. , electronystagmography ,
rotational chair , head thrust , and head shaking tests ) in the assessment of vestibular
pathologies rather than using it in isolation11 ,
12 .
therefore , the purpose of this
study was to investigate whether the sst can be considered both a sensitive and specific
test for detection of uvh . based on our study results , we found a strong positive relationship between the sst and the
hsn test using veng ( 65% ) , which indicates that the sst is an efficient test in the
diagnosis of uvh . also , we found that the sst is both sensitive ( able to pick up the
presence of any uvh in a person who has it ) and specific ( able to pick up the absence of any
uvh in a person who does not have it )
so , it can be used to include or exclude the
dysfunction , but the results were better when it was used for inclusion . when using the hsn
test using veng as the standard reference , our results showed adequate sensitivity and
specificity to support the use of sst as a screening test for unilateral vestibular
hypofunction . a prospective study by zhang et al .
concluded that the fst was unreliable in identifying
the lesion side in acute vestibular dysfunction12 . even though some other studies also reported the same results and
conclusions13 , 14 , our study we found that the sst is a reliable and valid tool for
detection of uvh .
the possible reasons for higher sst sensitivity and specificity in our
study than in other studies is the difference in the studied populations , with more subjects
with uvh in our sample , and the previous studies not being compared with any objective
standard method of testing .
second , there was great difficulty accessing patients and veng
equipment , as there are very few otolaryngology ( ent ) departments that are specialized in
our area .
third , we were unable to take pictures of the subjects for personal privacy
reasons . therefore , we recommend that future research should be performed with a larger
number of subjects , should be experimental rather than an observational , and should measure
the sensitivity and specificity of the sst with regard to its ability to diagnose the side
of the dysfunction . in conclusion , the study results showed that the sst is useful as an objective measure in
the evaluation of uvh in parallel with the hsn test using veng .
so the sst can be used as an
alternative to the hsn test using veng for detection of uvh . | [ purpose ] this study investigated to determine whether the sideways stepping test ( sst )
is a useful test to detect unilateral vestibular hypofunction ( uvh ) . [ subjects and
methods ] twenty - eight subjects including both male and females between the ages of 25 and
55 who had been diagnosed with uvh were recruited for the study .
all the subjects were
tested with the sst and followed by the head - shaking nystagmus ( hsn ) test using video
electronystagmography ( veng ) to confirm the presence of uvh .
the results of both tests
were then compared with each other to determine the correlation , sensitivity , and
specificity .
[ results ] the results showed that the sst is strongly correlated with the
gold standard hsn test using veng and is highly sensitive and specific .
[ conclusion ] the
present study showed that the sst is a highly valid test that can be used as an
alternative method to the gold standard hsn test using veng in detecting uvh . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
the most radical example is natural - orifice transluminal endoscopic surgery ( notes ) , which intentionally disrupts a healthy organ to gain access to a diseased organ , thus avoiding incising the abdominal wall .
notes has been performed successfully in a wide range of abdominal surgical procedures , including cholecystectomy , gastrectomy , peritoneoscopy , and even nephrectomy .
notes is currently in the initial stages of development , so there are few reports demonstrating the clinical benefits of this minimally invasive technique .
the goal in developing notes techniques is that the advantages of traditional laparoscopic surgery ( ie , less pain , improved cosmesis , and more rapid return to normal activity ) will be magnified in an even less invasive surgery .
notes nephrectomies have been described through a variety of approaches , including transgastric , transvaginal , and transrectal .
compared with traditional laparoscopic techniques in which skin incisions are used to gain access to the abdomen , these approaches intentionally traverse a hollow viscus of a separate organ system , each with a unique array of microbial colonization .
contamination of the peritoneal cavity with bowel or vaginal flora and the possibility of subsequent infectious complications exist with these approaches .
recently , our center published the first report of a hybrid transureteral notes nephrectomy in a nonsurvival porcine model .
this novel notes approach does not require the intentional violation of a separate organ system as in other approaches .
after a careful review of surgical technique and technical modifications , we sought to determine the feasibility of a hybrid transureteral notes nephrectomy in a survival porcine model .
after approval was obtained from the institutional animal care and use committee , 3 female farm pigs were selected for the procedure ( weight range , 4145 kg ) .
all procedures were completed by a single surgeon with fellowship training in minimally invasive and endourologic surgery ( d.d.b . ) .
the pigs were given no food or liquid for the 12 hours prior to surgery .
general anesthesia was induced with a combination of ketamine ( 2.2 mg / kg ) , telazol ( 4.4 mg / kg ) , and xylazine ( 2.2 mg / kg ) .
anesthesia was maintained using isoflurane ( 2.5%3.5% ) under the direction of the institutional animal care facility veterinarian .
intravenous access was established in either the saphenous or ear vein with a 22-gauge angiocatheter .
the animals were given lactated ringer 's solution at a rate of 5 ml / kg / h throughout the operation .
the pigs were placed in a modified lithotomy position with sterile preparation and draping of the abdomen and perineum .
the procedure was initiated with cystoscopic ( 17-f rigid cystoscope ; gyrus acmi , southborough , massachusetts ) identification of the left ureteral orifice .
a 0.038-inch angle - tipped hydrophilic guidewire ( terumo medical corporation , somerset , new jersey ) was then placed to the level of the renal pelvis using a fluoroless technique . using a 6-f open - ended ureteral catheter ( cook medical , bloomington , indiana ) , the hydrophilic wire was then exchanged for a 0.038-inch amplatz extrastiff guidewire ( cook medical ) .
the bladder was then drained , and a novel integrated balloon dilator and sheath ( terumo medical corporation ) were placed over the superstiff guidewire ( figure 1a ) .
the urethra , ureteral orifice , and distal ureter ( to a distance 2 cm proximal to the ureteral orifice ) were then dilated to 33 f by inflating the balloon dilator to 20 atm . (
a ) in lithotomy , a superstiff guidewire is placed and used to insert an experimental bariatric balloon - dilator sheath combination .
( b ) after ureteral and urethral dilation , a 12-mm bariatric port is inserted into the distal ureter .
( c ) a hook electrode is passed through the offset hysteroscope ( with 5-mm working channel ) for dissection of retroperitoneal space and renal hilum .
( d ) a 5-mm bariatric stapling device is used to divide the renal artery and vein .
( e ) after hilar transection , the kidney is morcellated into 9-mm strips with a bipolar device and removed through the ureter using the empty housing of a bariatric stapling device .
( f ) bladder removed at necropsy and instilled with water to demonstrate absence of urine leak .
after removal of the dilating balloon , a bariatric 12-mm laparoscopic port with a previously drilled 5-f hole at the tip of the trocar ( 150 mm in length ; ethicon endo - surgery , cincinnati , ohio ) was inserted over the guidewire ( figure 1b ) . a 10-mm offset hysteroscope ( length , 10.5 in ; gyrus acmi )
laparoscopic instruments up to 5 mm in diameter were passed through the working channel of the hysteroscope .
a bariatric suction device was used to clear the ureteral lumen of blood and urine after dilation .
the ureter was opened medially with the use of a bariatric hook electrode ( megadyne , draper , utah ) to allow access to the retroperitoneal space . using the hook electrode ,
the retroperitoneal space was developed and the hilar vessels were dissected ( figure 1c ) . a 5-mm bariatric bipolar sealing device ( ligasure advance ; covidien , mansfield , massachusetts ) was used for further dissection and mobilization of the kidney .
the pig was repositioned in a 60 lateral decubitus position to allow the bowel to fall away from the kidney . a 2/3-mm transabdominal port ( tyco healthcare , norwalk , connecticut )
a second 2-mm port was placed ( tyco healthcare ) approximately 8 cm caudal to the first port in the left lower quadrant of the abdomen ( figure 2 ) .
a 2.9-mm 12 lens ( stryker endoscopy , kalamazoo , michigan ) was then passed through the 2/3-mm port , and a 2-mm grasper was passed through the caudal transabdominal port .
the renal artery and vein were divided with a bariatric stapling device ( ethicon endo - surgery ) ( figure 1d ) .
kidney morcellation was then carried out with the 5-mm bipolar sealing device through the transureteral trocar .
the transabdominal grasper and camera were used for improved renal stabilization and visualization during the morcellation process .
morcellated renal fragments were prevented from migrating by leaving the cranial end attached to apical tissue until fragment removal .
after morcellation , each renal segment was removed transureterally by using the empty housing of a 12-mm bariatric stapling device to grasp the fragment for removal ( figure 1e ) .
after complete removal of all fragments , the ureteral stump was closed using a laparoscopic suturing device ( sr 5 quick load ; lsi solutions , victor , new york ) .
the skin was reapproximated at the abdominal port sites with cyanoacrylate tissue adhesive , and a urinary catheter was left in place for 10 to 14 days postoperatively .
the animals were monitored and cared for in the institutional animal care facility under the direction of the institutional veterinarian until euthanasia on postoperative day 21 .
the primary surgeon assessed the technical aspects of each step of the operation to determine the most challenging part of each surgery .
one pig developed urinary retention when the foley catheter became plugged with a clot , which required catheter irrigation . upon subsequent removal of this catheter ,
the timing of the various steps in the operation and estimated blood loss associated with each operation are shown in table 1 . at necropsy , each of the 3 bladders was removed and instilled with saline to test the integrity of the ureteral repair .
all bladders were completely healed , without leak at the site of ureteral ligation ( figure 1f ) . with the exception of the transient clot retention , all 3 pigs displayed normal behavioral patterns and were eating and urinating expected volumes up to the day of euthanasia .
perioperative outcomes the primary surgeon rated the morcellation portion of the procedure as the most complex in each surgery .
the third most difficult step was the dissection of the retroperitoneum and hilum , because of difficulty maintaining the pneumoretroperitoneum .
notes is currently in the early stages of development . yet as technology and surgical innovation
continue to progress , notes techniques may one day become the procedure of choice for a wide variety of procedures in several different surgical disciplines .
previously , we reported the feasibility of a hybrid transureteral nephrectomy in a nonsurvival porcine model . in the current study , we demonstrate , for the first time , the feasibility of performing a hybrid transureteral notes nephrectomy in a survival porcine model .
although 1 of 3 pigs had an episode of acute postoperative urinary retention secondary to a blood clot , there were no other intraoperative or postoperative complications .
several points of entry into the peritoneal cavity for nephrectomy have been evaluated in animal models , whereas only transvaginal nephrectomy has been reported in humans .
bazzi et al recently reported successful hybrid transrectal notes nephrectomy in 5 female pigs and demonstrated similar outcomes to a transvaginal nephrectomy porcine group .
they reported an average operative time of 196 minutes , with no evidence of intra - abdominal injuries on necropsy .
the authors selected the rectum as the point of entry , citing superior space for the extraction of a large kidney when compared with a transgastric or transvesicle approach , eliminating the challenges of renal morcellation .
haber et al reported their experience with a pure transvaginal nephrectomy in 5 female pigs in a nonsurvival model .
dissection was carried out using a needle / knife electrocautery device passed through either a single- or dual - channel videogastroscope .
tissue dissection was aided by retraction supplied by a 60-cm articulated xl endogia stapling device ( us surgical , norwalk , connecticut ) which was later used for division of the renal vessels .
mean operative time was 113 minutes , and the investigators reported no intraoperative complications . there was no evidence of injury in the intrapelvic cavity on gross examination at necropsy . using a different approach from pure notes transvaginal
nephrectomy , isariyawongse et al published their experience with a combination transvaginal - transgastric technique in a nonsurvival porcine model .
after first establishing transgastric access to the peritoneal cavity for passage of a single - lumen gastroscope , a colpotomy was made under direct visualization .
a novel transvaginal port was then placed to allow passage of laparoscopic dissection tools as well as a laparoscopic stapling device for division of the hilar vessels and ureter .
the authors did not detail the closure method , but they did recognize that this was a challenge in the current state of notes nephrectomy .
all previously described methods of performing notes nephrectomy use novel techniques requiring ingenuity and considerable endoscopic skill .
yet a major limitation is the possible morbidity associated with accessing the peritoneal cavity by intentional viscerotomy of a bacterial colonized hollow viscous . in a randomized - controlled trial , guarner - argente et al randomized 40 pigs to standard laparoscopic , transvaginal , transgastric , or transrectal peritoneoscopy .
peritoneal cultures were taken at the completion of the procedure and at necropsy on postoperative day 14 .
of concern , they found that all animals had positive peritoneal fluid cultures at necropsy , although no animals exhibited signs of peritoneal infection in the 14-day postoperative study period .
the authors concluded that peritoneal contamination occurs regardless of the peritoneal access site ; however , it did not lead to septic complications in the porcine model in short - term follow - up .
donatsky et al evaluated a novel technique for obtaining peritoneal access with an ultrasound - guided gastrotomy to accommodate a gastroendoscope for peritoneoscopy .
all pigs survived to euthanasia on postoperative day 14 , yet 30% ( n = 3 of 10 ) were found to have intra - abdominal abscesses at necropsy .
sotelo et al published one of the first reports of hybrid transvaginal notes nephrectomy in 4 patients .
in addition to the transvaginal approach , they made a 2.5-cm z - plasty incision at the umbilicus for placement of a 3-channel r - port ( triport ; advanced surgical concepts , dublin , ireland ) .
the first 3 cases required conversion to standard laparoscopy because of rectal injury , difficulty with liver retraction and access to hilar vessels , and inability to adequately control upper pole bleeding in each of the 3 patients , respectively .
the fourth case was completed without conversion to standard laparoscopy ; however , the patient subsequently developed fever and a fluid collection in the renal fossa that required percutaneous drainage . in a review of 14 cases of hybrid transvaginal notes
nephrectomy performed at a single center , alcaraz et al reported that 1 patient developed abdominal pain and fever on postoperative day 2 due to a colon injury , necessitating temporary fecal diversion .
in addition to the transvaginal access , the authors used a 5-mm and a 10-mm transabdominal port .
notes in animals and hybrid notes procedures in humans demonstrate the risks to patients of using a different organ system as the entry site for notes surgery .
although designed to minimize morbidity , previously described notes techniques violate a fundamental tenet of minimally invasive surgery by intentionally injuring a second organ system .
there are few published reports of complication rates in synchronous surgical procedures performed in patients undergoing urologic surgery . however , in a review of 30 patients , somani et al reported a 15% increase in perioperative morbidity in patients who underwent nephrectomy and a second procedure on a nongenitourinary organ compared with those who underwent nephrectomy alone . during nephrectomy ,
we report a technique that offers the advantages of a notes approach without involving a nongenitourinary hollow viscus by using the ureter to access the abdomen . in the only other technique published in the literature to avoid the violation of a second organ system , lima et al reported their experience with a pure notes transvesical nephrectomy in a nonsurvival porcine model . using a ureteroscope to create a cystotomy ,
the surgeon was able to pass a wire transvesically into the peritoneal cavity , which served as a guide for dilation and subsequent passage of a 10-mm operating telescope .
transvesical nephrectomy confines the procedure to the urinary system but makes a separate opening in a second organ ( the bladder ) to allow port placement .
in contrast , our technique places the entry site through the ureteral orifice to the kidney that is being removed , avoiding the morbidity of a separate opening in the bladder .
most transvesical notes procedures place the entry point to the peritoneal cavity in the dome of the bladder ; a leak at this site would result in urinary ascites and significant patient morbidity .
in contrast , the transureteral approach has the exit of the bladder located in the retroperitoneal space , thereby potentially reducing the morbidity of a urinary leak should it occur . in an effort to avoid a viscerotomy in a second organ system ,
we have developed a technique to access the retroperitoneum transureterally . from our perspective , this is a logical application of notes surgery to remove the diseased organ through its own natural orifice .
a gastrointestinal surgical correlate would be to remove the appendix internally from the colon or to remove the gallbladder through the cystic duct .
although these operations may seem prohibitively complex at the current time when using conventional technology , downsizing of instrumentation and development of new instruments will certainly make these techniques feasible in the future .
although transureteral nephrectomy theoretically decreases the risk for bacterial contamination of the abdominal cavity compared with a transenteric or transvaginal approach , it does have certain limitations .
first , our technique requires morcellation of the kidney for removal via the ureteral access point .
this would make this technique less desirable in patients with malignancy because of the risk for possible tumor spillage into the retroperitoneal space .
additionally , this was a time - consuming and challenging portion of the surgical procedure .
however , with experience , we anticipate improved efficiency . in addition , the development of a closed system that could be deployed to isolate the kidney and allow morcellation without leakage of cells into the peritoneal cavity could allow transureteral nephrectomy to be used in oncologic applications .
second , the retroperitoneal space is limited in size , making dissection and adequate visualization challenging .
a third challenge is the limitation of currently available instrumentation . despite using the longest prototype bariatric instruments available , it was difficult to access and dissect the upper pole , even in the juvenile female pigs used in this study .
in addition , the development of an effective bariatric - length 5-mm morcellation device could simplify the procedure .
similarly , the development of a small caliber 3- to 5-mm device that reliably seals the renal hilum would allow the downsizing of instrumentation in general .
finally , this procedure would likely be more easily applied to female patients in its current state of development .
the female urethra is shorter and more accommodating of instruments that are necessary to access the retroperitoneum transureterally . to accommodate these same instruments ,
miniaturization of surgical instruments would likely address this shortcoming , potentially making this surgery feasible in the male population .
in addition , the use of these smaller caliber instruments ( < 5 mm ) would not dilate the ureteral orifice and ureter as much , potentially eliminating the need for challenging ureteral closure .
we have demonstrated for the first time the feasibility of performing a transureteral hybrid notes nephrectomy in a survival porcine model . this technique represents a novel technique that eliminates the risks associated with the intentional perforation of a separate organ system .
however , its practical application in the human population is limited by a current lack of adequate instrumentation . with modification of techniques and development of new instrumentation , notes procedures limited to a single organ system could alter how many surgical procedures are performed .
once fully developed , single - organ - system notes surgery could further reduce patient morbidity and represent the next surgical paradigm . | background and objectives : natural - orifice approaches for nephrectomy have included access via the stomach , vagina , bladder , and rectum . recently , the feasibility of using the ureter as a natural orifice for natural - orifice transluminal endoscopic surgery nephrectomy has been demonstrated in a nonsurvival porcine model . the purpose of this study was to assess the outcomes of transureteral laparoscopic natural - orifice transluminal endoscopic surgery nephrectomy in a survival porcine model.methods:three pigs underwent hybrid transureteral natural - orifice transluminal endoscopic surgery nephrectomy .
an experimental balloon / dilating sheath was inserted over a wire to dilate the urethra , ureteral orifice , and ureter . through a bariatric 12-mm laparoscopic port ,
the ureter was opened medially and the hilar dissection was performed .
next , 2 needlescopic ports were placed transabdominally to facilitate hilar transection .
the kidney was morcellated using a bipolar sealing device and extracted via the ureter using the housing of a bariatric stapling device .
the ureteral orifice was closed with a laparoscopic suturing device .
the bladder was drained by a catheter for 10 to 14 days postoperatively .
pigs were euthanized on postoperative day 21.results:all surgical procedures were successfully completed , with no intraoperative complications .
one pig had an episode of postoperative clot retention that resolved with catheter irrigation .
each pig was healthy and eating a normal diet prior to euthanasia.conclusions:this study demonstrates the feasibility of a hybrid transureteral approach to nephrectomy in a survival porcine model .
this technique avoids the intentional violation of a second organ system and the risk for peritoneal contamination .
improved instrumentation is needed prior to implementation in the human population . | INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
cervical spondylotic myelopathy ( csm ) and cervical spondylotic radiculopathy ( csr ) are classically approached by anterior single or multiple disc space decompression [ 1 , 2 ] , multilevel corpectomy , laminectomy [ 410 ] , or laminoplasty [ 1118 ] .
more recently , techniques using lateral multiple oblique corpectomy ( moc ) and/or foraminotomy [ 1928 ] have been used with increasing frequency . in general , when three or more levels are affected , the preferred techniques remain either an anterior multilevel corpectomy or a posterior route such as laminectomy , open door laminoplasty , and posterior foraminotomy .
however , the best management of such pathology ( especially if 3 or more levels are involved ) remains controversial .
the authors consider the cervical spine lateral approach a valid and safe option to treat such pathologies as it provides very good clinical results and maintains long - term spinal stability .
the goal of this paper is to further and critically present the idea and rationale of the cervical spine lateral approach with its advantages , disadvantages , complications , and pitfalls in a critical review of their last 2 decades experience .
predominant anterior compression associated with either straightening or kyphosis of the cervical spine in the absence of instability is the general indication for the proposed technique . in cases of both anterior and posterior compression ,
the posterior approach is probably the best choice as long as cervical lordosis remains preserved .
cervical ct - mri scan evidence of single / multiple level nerve roots and/or spinal cord compression , mainly anterolateral and/or myelopathy .
evidence of neutral or kyphotic cervical alignment in the lateral cervical plain x - ray , as well as absence of instability documented by cervical dynamic x - ray .
soft disc herniation documented with mri within 6 months ( only for moc ) .
the technique has already been described and reported by the authors [ 1927 , 29 ] .
the idea and rationale of the present technique is based on the evidence that anterolateral compression of the cervical spine and nerve roots may be best managed by an anterolateral approach because it provides direct exposure of the abnormal area .
experience with this technique began in 1989 , with the senior author studying the approach on cadaveric specimens .
initially , the anterolateral approach was only employed for foraminotomies to treat severe nerve root compression . over time and with greater visualization and dexterity ,
ultimately , this approach has become routine in our department for the treatment of spondylotic radiculopathy or myelopathy [ 1927 , 29 ] .
the decision of which side to perform the procedure is first based on the clinical picture and which side is most symptomatic .
if symptoms are bilateral , the side with the larger osteophytes or disc herniation is chosen .
if either clinically or radiologically there is no predominant side , the approach chosen is on the side of the smaller vertebral artery ( va ) .
the idea and rationale are that it is easier and safer when the artery is clearly visualized and under control .
the patient is positioned supine , with the head slightly extended and rotated to the contralateral side .
a longitudinal skin incision is made along the medial border of the sternocleidomastoid ( scm ) muscle at the level of the vertebral bodies to be exposed ( see also figure 1 ) .
the incision may extend to the mastoid tip to expose c2 - 3 and to the sternal notch to expose c7-t1 .
the natural space between the scm muscle and the internal jugular vein is opened by sharp dissection ( see also figures 2(a ) and 2(b ) ) .
the scm muscle is retracted laterally , while the great vessels , trachea , and esophagus are kept undissected medially and protected by a blunt retractor .
this fatty sheath surrounds the accessory nerve , which must be identified when the c2-c3 and the c3-c4 levels need to be exposed . at this point
the transverse processes are covered by the prevertebral muscles . under the aponeurosis of the longus colli muscle
the aponeurosis is divided longitudinally , medial to the sympathetic chain ; both the aponeurosis and the sympathetic chain are then retracted laterally .
the longus colli muscle is divided along the transverse processes and vertebral bodies at the decided levels and then retracted away from the field .
care must be taken to be sure the va is not entering the transverse foramen at an abnormally high level ( c5 , c4 , or even c3 ) ; in this case , the artery is running before the transverse processes and may be injured during the longus colli muscle division . at this point ,
the intervertebral foramen is opened by removing the anterior part of the transverse foramen with a kerrison rongeur after it is identified via subperiosteal dissection ; this manoeuvre helps with additional lateral va mobilization by creating a plane between the lateral aspect of the uncovertebral joint and the medial border of the va .
once both structures are separated , the hypertrophied uncovertebral joint can be safely removed with a drill and/or rongeurs . in this way
, the cervical nerve root can be completely decompressed from its dural origin up to the va lateral border . after radiological identification of the correct level
, we start the corpectomy , using a cutting drill , on the bodies on both sides of the disc .
we keep the direction of the drill parallel to the endplate ; the corpectomy continues until the cortical bone of the posterior aspect of the body is found .
it is very important to start with a vertical trench just medial to the va and then to move obliquely so as to reduce , as much as possible , the extent of bone resection ( see also figures 3(a ) and 3(b ) , figures 4(a ) , 4(b ) , and 4(c ) ) .
the intervertebral discs are incised and removed up to the posterior margin of vertebral body . at this stage
, the vertebral body is drilled obliquely from the lateral side toward the opposite posterolateral corner .
more than half of the vertebral body is preserved creating a convex - shaped posterior aspect .
next , drilling is turned towards a point of the posterior aspect of the vertebral body , which has been precisely located on the preoperative computed tomographic ( ct ) scan .
it is located at the limits of the osteophytes and often corresponds to the junction between the body and the opposite pedicle ( see also figure 5(a ) , 5(b ) , and 5(c ) and figures 6(a)-6(b ) ) .
next , the posterior longitudinal ligament must be opened longitudinally and as much as possible removed to ensure that optimal cord decompression has been obtained .
the present procedure provides the following advantages : wide anterolateral decompression of the spinal canal and foramen at single or multiple levels ( multiple levels to decompress is not a limitation ) .
there is no need for bone grafting and/or instrumentation making the technique very suitable for elderly people and heavy smokers .
the lateral approach , by using a different path , when compared to the standard anterior approach , offers an excellent visual alternative as the field between the scm muscle and the internal jugular vein is opened .
this is particularly desirable in cases of recurrence after previous anterior surgery , because there is no need to mobilize tedious postoperative scar tissue .
the present procedure has the following disadvantages : bilateral radiculopathy may not be treated in a single - staged procedure ; in these cases , an anterior midline approach remains the procedure of choice . in cases where delayed contralateral
radiculopathy appears , then selective microsurgical nerve root decompression may be advocated ; although , we suggest spine stabilization / fusion . the anterolateral technique , by exposing multiple anatomical critical structures , is a procedure with a steep learning curve .
however , surgeons should not be discouraged by initial difficulties and should keep in mind that after having performed the procedure 10 times , the operative time will be substantially reduced . stretch and potential damage of xi nerve ( when exposing c2-c3 level ) , and horner 's syndrome ( when approaching c4-c7 ) are well known but rare complications .
analysis of our experience allows the following considerations : at a mean followup of 111 months ( range 9 to 202 months ) , a global recovery rate of 87.6% was recorded for csm using the following formula :
( 1)recovery rate ( % ) = [ postop mjoa scorepreop mjoa score]10017preop mjoa score ,
and a global recovery rate of 95% for csr using a score obtained by multiplying the intensity ( vas scale 010 ) and the duration scores , ranging in this way , from 0 to 100 .
the cervical lateral approach for csm and/or csr can be extended to as many levels as required , and the number of levels is not considered a limit for this procedure .
a total of 900 levels were decompressed in 499 patients ( c2-c3 in 35 cases , c3-c4 in 122 , c4-c5 in 188 , c5-c6 in 296 , c6-c7 in 128 , and c7-t1 in 9 ) .
oblique vertebrectomy was performed at one level in 221 patients , two levels in 121 patients , three levels in 88 patients , four levels in 32 patients , and five levels in 9 patients .
the mean operation time was 118 minutes ( range 73 to 183 minutes ) ; mean intraoperative estimated blood loss was 68 ml ( range 28 to 280 ml ) and the mean hospitalization time was 6 days ( range 2 to 14 days ) .
the first developed a disc herniation ; the second had an unrecognized congenital bone malformation ; the third developed segmental instability at a level above the treated levels . no cerebrospinal fluid leakage ( 0% ) ,
infections ( 0% ) , nor c5 deficit and/or dysphagia / dysphonia were observed in our series .
a transient hs was observed in 14 patients ( 3% ) ; in almost all of them , symptoms markedly resolved within 3 months with less than 1% ( 4 cases ) retaining permanent impairment .
the lateral foraminotomy and the oblique corpectomy technique , by preserving over 50% of the vertebral body and preserving two of the three columns , do not compromise spinal stability so that bone grafts or instrumental arthrodesis are not necessary .
patient selection for these procedures is crucial ; for this reason all patients with clear spine instability ( slippage > 2 mm between at 2 adjacent vertebral bodies on dynamic x - ray ) and/or with a preoperative fixed listhesis 2 mm were excluded . in our series ,
only three patients required delayed stabilization , and aside from these three patients , a change in the spinal curvature of more than 5 degrees was never observed postoperatively at the level of the surgical decompression , regardless of the preoperative spinal curvature .
the lateral approach [ 19 , 20 , 2229 ] differs substantially from other anterolateral approaches as it leads directly to the lateral aspect of the vertebral body and the transverse process which are covered by the prevertebral muscles . when dividing the prevertebral muscles , it is important to identify and preserve the sympathetic chain running under the aponeurosis .
manipulation of the sympathetic nerves may result in a postoperative horner 's syndrome ( hs ) , but it is mild and transient if the main trunk of the sympathetic chain is preserved . in our series ,
three percent of patients experienced a transient hs as a consequence of manipulation of the sympathetic nerves . in almost all cases ,
symptoms markedly resolved within 3 months with less than 1% ( 4 cases ) retaining permanent impairment .
the majority of hs cases ( 9 cases ) occurred in the first 3 years of our practice .
horner 's syndrome can occur and constitutes a disadvantage of the technique , but as demonstrated by this series , incidence decreases significantly with increasing experience . in our experience , hs is almost always temporary , if careful identification and gentle retraction without dissection of the sympathetic chain is performed .
, who have proposed dissection of the sympathetic chain to avoid its functional damage . similarly , when exposing levels above c3 , the accessory nerve must be retracted as gently as possible and kept protected by a fat pad around it .
morbidity resulting from the dissection of the accessory nerve is very unlikely and never occurred in our experience . with the lateral approach , controlled , but not mobilized , of the va provides protection to surrounding important structures .
direct visualization of the va allows for safe drilling of the posterolateral corner of the vertebral body and the control of the distal nerve root .
troublesome venous bleeding from the perivertebral venous plexus can be prevented by preservation of the periosteal sheath around the va .
furthermore , care must be taken to identify any abnormal course of the va , especially entry of the va into the transverse canal at an abnormally high level , which may be the c5 , c4 , or even c3 level ; this can be easily achieved by carefully examining a standard preoperative mri and/or mra .
practical pearls offered by the authors while performing this technique include the placement of the suction device in front of the va to provide protection in the case of inadvertent sliding during drilling .
also with the oblique corpectomy , there is a natural tendency to drill bone inadvertently and unnecessarily in a horizontal plane which may compromise spinal stability ; therefore , the operative microscope should be set obliquely ( to have an oblique view ) to avoid this tendency .
practically , it is very important to start with a vertical trench just medial to the va and then to move horizontally , reducing in this way , as much as possible , the extent of bone resection ( see also figure 4(a ) ) ; this represents the first surgical pitfall .
another problem is represented by the absence of an anatomic landmark to define where the horizontal drilling should be stopped ; this constitutes the second pitfall , and to solve it we determine on the preoperative ct scan the extent of drilling . in general
, the limits of the osteophytes set the length of bone drilling and , very often , correspond to the junction between the body and the opposite pedicle . verifying the adequacy of the decompression in the horizontal plane is important to the success of this procedure .
therefore , the distance between the contralateral pedicle and the medial border of the ipsilateral va is measured on the preoperative ct scan , or as more recently by using the perioperative igs ( image - guided system ) which is very reliable and effective also recently confirmed by lee et al . .
in the majority of cases , the distance varies between 22 and 28 mm . in our series , no cerebrospinal fluid leakage , infections , c5 deficits / or dysphagia / dysphonia were observed .
we have no clear explanation as to why these complications were not encountered ; they may be related to the absence of any distractions during the procedure . as for the lack of c5 deficits
postoperatively , there is no clear explanation , but it could be related to this approach and the better visualization for the surgeon of the cervical spinal root at the foramen . since this technique does not require medial traction , the trachea and the esophagus are barely touched by holding a hand blade ( self - retaining retractors are never used ) . over last few years various clinical series [ 3439 ] about oblique corpectomy as well as experimental studies [ 40 , 41 ] have been published ; the formers have shown , according to our experience , the effectiveness and reliability of such technique in managing cervical myeloradiculopathy ; in all reports the authors agreed about the related good functional outcome as well as on the preservation of spinal stability with its physiological motion which represent a considerable advantage in the treatment of such pathology .
finally , we would like to emphasize that this approach is initially a demanding procedure , and the learning curve may be long for some . in the authors ' experience , it is sufficient to perform the procedure few times in the cadaver laboratory , and for the first few times , to be assisted by an experienced surgeon .
we also would like to stress that good knowledge of va anatomy and its variations is essential to performing this operation and that careful analysis of preoperative imaging is crucial .
although multilevel oblique corpectomy and/or simple foraminotomy via a lateral approach remains a rather demanding technique with a substantial learning curve , we believe it is a valid alternative for the management of multisegmental cervical spondylosis . good knowledge of va variations is essential and careful analysis of preoperative imaging is mandatory .
the incidence of early and late postoperative complications is lower , and bone grafting is not necessary , allowing for it to be used in patients with a low fusion rate such as the elderly , diabetics , and heavy smokers .
it also permits early patient mobilization with no postoperative immobilization . as often is the case , optimal results rely on scrupulous selection of patients and preservation of cervical spine stability . |
background . the authors describe a lateral approach to the cervical spine for the management of spondylotic myeloradiculopathy .
the rationale for this approach and surgical technique are discussed , as well as the advantages , disadvantages , complications , and pitfalls based on the author 's experience over the last two decades . methods .
spondylotic myelo - radiculopathy may be treated via a lateral approach to the cervical spine when there is predominant anterior compression associated with either spine straightening or kyphosis , but without vertebral instability .
results . by using a lateral approach , the lateral aspect of the cervical spine and the vertebral artery
are easily reached and visualized .
furthermore , the lateral part of the affected intervertebral disc(s ) , uncovertebral joint(s ) , vertebral body(ies ) , and posterior longitudinal ligament can be removed as needed to decompress nerve root(s ) and/or the spinal cord .
conclusion .
multilevel cervical oblique corpectomy and/or lateral foraminotomy allow wide decompression of nervous structures , while maintaining optimal stability and physiological motion of the cervical spine . | 1. Introduction
2. Technique Indication
3. Inclusion-Exclusion Criteria
4. Surgical Technique
5. Discussion
6. Conclusions |
the importance of community - based health education in undergraduate curricula has been validated in medical education and practice.(12 ) the current shift of emphasis from curative to preventive medicine makes community - based medical education of utmost importance .
even though only a small proportion of medical graduates will eventually choose public health as their specialty , a thorough knowledge - base established through robust undergraduate training programmes in community medicine is essential for all practicing doctors . despite this ,
the importance and significance of public health are often not fully appreciated,(13 ) with more emphasis being placed on hospital - based and curative medicine .
while medical curricula must be effective and relevant as they are of fundamental importance in the training of doctors,(4 ) how effective or relevant they are may be a matter of perception.(5 ) poor implementation of curricula is known to result in unfavorable student perceptions.(6 ) furthermore , positive perceptions are known to increase student motivation and , therefore , learning .
brooks , a constructivist , suggests that student opinion should be sought and valued.(7 ) thus , frequent assessments of student perceptions are recommended and many agree that they are useful in the structuring of the curriculum,(89 ) making them more acceptable and beneficial .
since the integration of public health into the medical curriculum of the faculty of medicine , university of colombo , sri lanka , the community sciences stream has maintained a dynamic approach toward its teaching .
the public health curriculum of the faculty of medicine , colombo , spans the five years of undergraduate training and is delivered as subject modules .
the introductory sessions , dealing with the concept of health promotion , are conducted by weekly lectures , small group discussions , and student presentations .
apart from conventional lectures and small group discussions , during the third and fourth years , the stream provides students with opportunities for experiential learning .
this is by way of a community - based programme , the purpose of which is to enable students to describe and assess health needs at community , family , and individual levels , become familiar with resources for health promotion , and to plan , implement , and evaluate sustainable interventions to improve health .
groups of students are allocated to specified communities ( known as the community attachment programme ) and families ( known as the family attachment programme ) for a period of 1218 months thus acquiring the competence to intervene and provide first - contact health care and promote health at various levels.(1011 ) this includes a research project , carried out in groups of three , during which students learn to develop a research idea , design a research protocol and data collection instrument , understand the ethical implications of research , conduct and analyze the results of the study , and finally , draft a research report and paper .
the final - year teaching programme concentrates on community perspectives of medical care and is conducted by way of a string of lectures and interactive case discussions on health in hospitals , relaying of health - related information , social responsibilities of doctors , and how aspects of the community impact the outcome of clinical management .
the aim of our study was to assess student perceptions of the public health curriculum and to identify factors which influence these .
data were gathered at the faculty of medicine , university of colombo , sri lanka , in 2010 .
out of 196 students , 184 students , i.e. , 94% agreed to participate in the study following completion of the five year curriculum in public health .
recruitment was carried out amongst final - year students following a compulsory student activity so as to maximize participation ; however , student participation was completely voluntary .
convenience sampling method was utilized and those not present on the day of assessment were excluded from the study .
the questionnaire was prepared following focused group discussions among graduates of the same institution , who had also completed the public health course as part of their curricula .
the questionnaire , in addition to gathering demographic data , asked students to evaluate the public health curriculum as a whole and to state their perceptions on specified components of the stream , such as the introductory programme , community - based learning , research project , and the final - year module on community perspectives of medical care through 90 separate questions .
a 4-point likert scale was used to score and collect information and students were offered the options definitely , somewhat , not really , and no . when not considered individually for purposes of analysis , the responses definitely and somewhat were grouped together as a positive response and not really and no as a negative response .
data analysis was carried out utilizing statistical package for the social sciences 18 analytical software package .
the study received ethical approval from the ethics review committee of the faculty of medicine , university of colombo .
data were gathered at the faculty of medicine , university of colombo , sri lanka , in 2010 .
all medical students in their final year were invited to participate in the study . out of 196 students , 184 students ,
i.e. , 94% agreed to participate in the study following completion of the five year curriculum in public health .
recruitment was carried out amongst final - year students following a compulsory student activity so as to maximize participation ; however , student participation was completely voluntary .
convenience sampling method was utilized and those not present on the day of assessment were excluded from the study .
the questionnaire was prepared following focused group discussions among graduates of the same institution , who had also completed the public health course as part of their curricula .
the questionnaire , in addition to gathering demographic data , asked students to evaluate the public health curriculum as a whole and to state their perceptions on specified components of the stream , such as the introductory programme , community - based learning , research project , and the final - year module on community perspectives of medical care through 90 separate questions .
a 4-point likert scale was used to score and collect information and students were offered the options definitely ,
somewhat , not really , and no . when not considered individually for purposes of analysis , the responses definitely and somewhat were grouped together as a positive response and not really and no as a negative response .
data analysis was carried out utilizing statistical package for the social sciences 18 analytical software package .
the study received ethical approval from the ethics review committee of the faculty of medicine , university of colombo .
hundred and eighty - four students ( n = 184 ) who had completed their medical undergraduate course agreed to complete the questionnaire . of those ,
48% were males and 52% were females , which is comparable to the gender distribution in our faculty .
forty - eight percent ( n = 84 out of 174 ) were residents of colombo , a district which is the urban hub of the country , while the majority ( 69.3% , n = 122 out of 176 ) had received their secondary education there as well .
forty - nine percent ( n = 89 out of 144 ) had scored more than 60% , which is considered a class grading , at the most recent public health exam . by the end of the course , 81.8% ( n = 148 out of 181 ) agreed to the question
eighty - five percent ( n = 155 out of 182 ) accepted what i learnt will benefit me in the future and the majority ( 67.5% , n = 112 out of 166 ) was satisfied that the programme has prepared me well to work in the community .
sixty - four percent ( n = 116 out of 180 ) accepted they had a good perception regarding public health prior to starting the course .
significant associations between viewing public health as an important field in medicine were found with having had a good opinion about public health prior to starting the course , perceiving the community attachment programme as enjoyable , perceiving the module on community perspectives of medicine as enjoyable , and perceiving themselves prepared and competent to work in the community at the end of the course ( p < 0.01 ) .
only 9% ( n = 16 out of 179 ) definitely agreed with the statement i am interested in taking up a career in public health , while 43% ( n = 76 out of 179 ) were definite that they were not .
there was a significant association between students considering a future career in public health care and having had a good opinion about public health prior to starting , believing public health to be an important field , perceiving the community attachment programme as enjoyable and beneficial to the community , enjoying the module on community perspectives of medicine , and perceiving themselves prepared and competent to work in the community ( p < 0.01 ) .
gender , grading obtained at the last public health examination , district of residence and district of school ( both classified as belonging to colombo , the urban hub of the country or the other districts ) did not appear to influence their choice ( p < 0.05 ) [ table 1 ] .
factors determining ( a ) the perception of public health as an important field and ( b ) the interest in choosing a career in public health as described above , this portion of the curriculum is conducted in three parts a community attachment programme , a family attachment programme , and a research project . during the community attachment programme ,
groups of 1520 students are allocated to specified areas of a community to engage in public - health - related activities .
sixty - six percent ( n = 120 out of 182 ) of students agreed to the question
65.2% ( n = 120 ) accepted i learnt a lot about public health .
sixty - four percent ( n = 117 out of 183 ) believed the community benefitted from our interventions , while 70.6% ( n = 117 out of 180 ) accepted that it was personally beneficial as they agreed to the statement i feel what i learnt through this programme will be of use to me in the future . forty - five percent (
and 34.6% ( n = 62 out of 179 ) agreed definitely to the statement the community cooperated with our efforts .
multiple site visitations are essential for the community - based programme throughout the allocated time period .
only 25% ( n = 46 ) of the students perceived the community was definitely located at a convenient location , while 47.3% ( n = 87 ) disagreed with the statement i had no difficulty in travelling to the community . of the sample questioned 54.1% ( n = 99 out of 183 ) agreed that
time had been utilized efficiently throughout the attachment and 19.7% ( n = 36 out of 183 ) were definite regarding that view . however , 56.4% ( n = 102 out of 181 ) disagreed with the statement
statistically significant associations were found between feeling satisfied about the amount they had learnt through the programme and students who had had a good opinion of public health prior to commencement of the course , those who had found the community attachment programme to be personally beneficial , those who felt that time had been utilized effectively , those who found their allocated communities cooperative and had enjoyed the attachment , and those who perceived the community benefitted ( p < 0.05 , p < 0.01 ) [ table 2 ] . factors determining satisfaction with regards to the amount learnt about public health through the community attachment programme further , enjoyment of the community attachment programme was significantly related to the convenience of the location and ease of travel to the chosen community , its residents cooperation , students perceiving the experience as personally beneficial and students believing that time had been utilized effectively ( p < 0.01 ) [ table 3 ] . factors which determine students enjoying the community attachment programme when considering the family attachment programme , during which pairs of students are allocated to provide community - based care to specified families , most students ( 66.5% , n = 121 out of 182 ) agreed that it was personally beneficial as they accepted the statement i feel what i learnt through this programme will be of use to me in the future .
fifty percent ( n = 90 out of 181 ) agreed we were able to identify significant health issues in their allocated families and 54.1% ( n = 98 out of 181 ) accepted the family was truly appreciative of our efforts . the majority ( 61.7% , n = 113 out of 183 ) firmly believed the family was welcoming and 53.3% ( n = 97 out of 182 ) definitely felt
the family was cooperative with the interventions . most ( 45.6% , n = 83 out of 182 ) agreed
sixty - four percent ( n = 117 out of 182 ) were of the opinion that time was spent effectively throughout the programme .
the research project encourages students to develop and conduct a health - related study under guidance .
seventy - four percent ( n = 133 out of 181 ) disagreed to the statement i had been involved in research prior to the community research project . by the end of the course , 50% ( n = 91 out of 182 ) agreed
eighty - seven percent ( n = 159 out of 183 ) accepted i am competent in applying for ethical clearance .
the programme also gave the students an opportunity to learn how to use data analytical packages ( e.g. , spss ) and 73.6% ( n = 134 out of 182 ) felt
majority definitely agreed to the statements i personally benefitted from this programme and i am interested in doing future research
( 60.1% , n = 110 out of 183 and 54.6% , n = 100 out of 183 , respectively ) .
however , only 36.4% ( n = 67 out of 183 ) accepted i was able to publish or present our findings .
having a sound foundation in knowledge in carrying out research ( methodology , utilizing data analysis software , and ethical considerations ) , feeling time was used effectively during the project , finding research to have been personally beneficial and having an opportunity to publish or present findings , all showed significant association with enthusiasm for future interest and involvement in research ( p < 0.01 ) [ table 4 ] .
factors shown to generate interest in future research among students following the community medicine research programme in the introductory sessions , a majority of 61.4% ( n = 113 ) accepted the topics were interesting and 85.8% ( n = 157 out of 183 ) considered the topics were useful .
however , most ( 58.7% , n = 108 ) disagreed with the statement the lectures were interesting . the majority accepted
i enjoyed working in a group and i enjoyed the discussions ( 83.2% n = 153 , 67.9% n = 125 , respectively ) and most ( 67.9% ,
n = 125 ) agreed with the opinion small group discussions were an effective method of learning .
however , only 7.1% ( n = 13 out of 183 ) definitely felt that time had been used effectively .
of those questioned , 79.3% ( n = 145 out of 183 ) and 77.5% ( n = 141 out of 182 ) respectively disagreed with the statements
i was stimulated to read more and i regularly revised what i learnt . the final year of the curriculum , based on community perspectives of medicine , is delivered via a series of lectures and interactive case discussions .
sixty percent ( n = 110 out of 182 ) felt the lectures were interesting ,
most ( 77.6% , n = 143 out of 183 ) were of the view
the topics were useful and 54.1% ( n = 99 out of 183 ) accepted the statement i enjoyed the lectures .
forty - five percent ( n = 81 out of 182 ) definitely agreed with the statement what i learnt will be beneficial to me in the future .
however , 25.3% ( n = 46 out of 182 ) definitely disagreed with the statement the lectures did not interfere with ward - learning and only 25% ( n = 45 out of 180 ) definitely agreed that we were able to concentrate on what was taught . only 16.4% ( n = 30 out of 183 ) definitely perceived
time was utilized effectively in the final year , while the majority ( 57.3% , n = 105 out of 183 ) agreed otherwise .
the majority ( 84.5% , n = 153 out of 181 ) accepted i attended lectures because attendance was recorded .
i was stimulated to read more and i regularly revised what i learnt
( 38.3% , n = 69 out of 180 and 42.8% , n = 77 out of 180 , respectively ) .
by the end of the course , 81.8% ( n = 148 out of 181 ) agreed to the question
eighty - five percent ( n = 155 out of 182 ) accepted what i learnt will benefit me in the future and the majority ( 67.5% , n = 112 out of 166 ) was satisfied that the programme has prepared me well to work in the community .
sixty - four percent ( n = 116 out of 180 ) accepted they had a good perception regarding public health prior to starting the course .
significant associations between viewing public health as an important field in medicine were found with having had a good opinion about public health prior to starting the course , perceiving the community attachment programme as enjoyable , perceiving the module on community perspectives of medicine as enjoyable , and perceiving themselves prepared and competent to work in the community at the end of the course ( p < 0.01 ) .
only 9% ( n = 16 out of 179 ) definitely agreed with the statement i am interested in taking up a career in public health , while 43% ( n = 76 out of 179 ) were definite that they were not .
there was a significant association between students considering a future career in public health care and having had a good opinion about public health prior to starting , believing public health to be an important field , perceiving the community attachment programme as enjoyable and beneficial to the community , enjoying the module on community perspectives of medicine , and perceiving themselves prepared and competent to work in the community ( p < 0.01 ) .
gender , grading obtained at the last public health examination , district of residence and district of school ( both classified as belonging to colombo , the urban hub of the country or the other districts ) did not appear to influence their choice ( p < 0.05 ) [ table 1 ] .
factors determining ( a ) the perception of public health as an important field and ( b ) the interest in choosing a career in public health
as described above , this portion of the curriculum is conducted in three parts a community attachment programme , a family attachment programme , and a research project . during the community attachment programme ,
groups of 1520 students are allocated to specified areas of a community to engage in public - health - related activities .
sixty - six percent ( n = 120 out of 182 ) of students agreed to the question
65.2% ( n = 120 ) accepted i learnt a lot about public health .
sixty - four percent ( n = 117 out of 183 ) believed the community benefitted from our interventions , while 70.6% ( n = 117 out of 180 ) accepted that it was personally beneficial as they agreed to the statement i feel what i learnt through this programme will be of use to me in the future . forty - five percent ( n = 83 ) definitely felt the community was welcoming and 34.6% ( n = 62 out of 179 ) agreed definitely to the statement the community cooperated with our efforts .
multiple site visitations are essential for the community - based programme throughout the allocated time period .
only 25% ( n = 46 ) of the students perceived the community was definitely located at a convenient location , while 47.3% ( n = 87 ) disagreed with the statement i had no difficulty in travelling to the community .
of the sample questioned 54.1% ( n = 99 out of 183 ) agreed that
time had been utilized efficiently throughout the attachment and 19.7% ( n = 36 out of 183 ) were definite regarding that view . however , 56.4% ( n = 102 out of 181 ) disagreed with the statement i am interested in working in the field in the future .
statistically significant associations were found between feeling satisfied about the amount they had learnt through the programme and students who had had a good opinion of public health prior to commencement of the course , those who had found the community attachment programme to be personally beneficial , those who felt that time had been utilized effectively , those who found their allocated communities cooperative and had enjoyed the attachment , and those who perceived the community benefitted ( p < 0.05 , p < 0.01 ) [ table 2 ] . factors determining satisfaction with regards to the amount learnt about public health through the community attachment programme further , enjoyment of the community attachment programme was significantly related to the convenience of the location and ease of travel to the chosen community , its residents cooperation , students perceiving the experience as personally beneficial and students believing that time had been utilized effectively ( p < 0.01 ) [ table 3 ] .
factors which determine students enjoying the community attachment programme when considering the family attachment programme , during which pairs of students are allocated to provide community - based care to specified families , most students ( 66.5% , n = 121 out of 182 ) agreed that it was personally beneficial as they accepted the statement i feel what i learnt through this programme will be of use to me in the future . fifty percent ( n = 90 out of 181 ) agreed we were able to identify significant health issues in their allocated families and 54.1% ( n = 98 out of 181 ) accepted the family was truly appreciative of our efforts . the majority ( 61.7% , n = 113 out of 183 ) firmly believed the family was welcoming and 53.3% ( n = 97 out of 182 ) definitely felt
the family was cooperative with the interventions . most ( 45.6% , n = 83 out of 182 ) agreed
sixty - four percent ( n = 117 out of 182 ) were of the opinion that time was spent effectively throughout the programme .
the research project encourages students to develop and conduct a health - related study under guidance .
seventy - four percent ( n = 133 out of 181 ) disagreed to the statement i had been involved in research prior to the community research project . by the end of the course , 50% ( n = 91 out of 182 ) agreed
eighty - seven percent ( n = 159 out of 183 ) accepted i am competent in applying for ethical clearance .
the programme also gave the students an opportunity to learn how to use data analytical packages ( e.g. , spss ) and 73.6% ( n = 134 out of 182 ) felt
majority definitely agreed to the statements i personally benefitted from this programme and i am interested in doing future research
( 60.1% , n = 110 out of 183 and 54.6% , n = 100 out of 183 , respectively ) .
however , only 36.4% ( n = 67 out of 183 ) accepted i was able to publish or present our findings .
having a sound foundation in knowledge in carrying out research ( methodology , utilizing data analysis software , and ethical considerations ) , feeling time was used effectively during the project , finding research to have been personally beneficial and having an opportunity to publish or present findings , all showed significant association with enthusiasm for future interest and involvement in research ( p < 0.01 ) [ table 4 ] . factors shown to generate interest in future research among students following the community medicine research programme
in the introductory sessions , a majority of 61.4% ( n = 113 ) accepted the topics were interesting and
85.8% ( n = 157 out of 183 ) considered the topics were useful .
however , most ( 58.7% , n = 108 ) disagreed with the statement the lectures were interesting . the majority accepted
( 83.2% n = 153 , 67.9% n = 125 , respectively ) and most ( 67.9% ,
n = 125 ) agreed with the opinion small group discussions were an effective method of learning . eighty - one percent ( n = 142 out of 175 ) agreed with
however , only 7.1% ( n = 13 out of 183 ) definitely felt that time had been used effectively .
of those questioned , 79.3% ( n = 145 out of 183 ) and 77.5% ( n = 141 out of 182 ) respectively disagreed with the statements
i was stimulated to read more and i regularly revised what i learnt . the final year of the curriculum , based on community perspectives of medicine , is delivered via a series of lectures and interactive case discussions .
sixty percent ( n = 110 out of 182 ) felt the lectures were interesting , most ( 77.6% , n = 143 out of 183 ) were of the view
and 54.1% ( n = 99 out of 183 ) accepted the statement i enjoyed the lectures . forty - five percent ( n = 81 out of 182 ) definitely agreed with the statement what i learnt will be beneficial to me in the future .
however , 25.3% ( n = 46 out of 182 ) definitely disagreed with the statement the lectures did not interfere with ward - learning and only 25% ( n = 45 out of 180 ) definitely agreed that we were able to concentrate on what was taught . only 16.4% ( n = 30 out of 183 ) definitely perceived
time was utilized effectively in the final year , while the majority ( 57.3% , n = 105 out of 183 ) agreed otherwise .
the majority ( 84.5% , n = 153 out of 181 ) accepted i attended lectures because attendance was recorded .
i was stimulated to read more and i regularly revised what i learnt ( 38.3% , n = 69 out of 180 and 42.8% , n = 77 out of 180 , respectively ) .
as described above , well - implemented curricula are received positively by students and are more likely to facilitate learning .
the method that we used to assess student satisfaction with the curriculum was measurement of student perceptions .
it may be argued that students perceptions of various aspects of the course are interdependent , and therefore , can not be considered in isolation or that students who view a certain area of the curriculum positively are more likely to view others in a similar manner due to characteristics of their personality . while this may be true
, it does not detract from the evidence that perceptions influence learning , whatever their basis may be .
it is clear that perceptions alone may be inadequate in aiding assessment as they are subjective and liable to be affected by various situations and variables .
however , when combined with an additional objective measure it holds great value.(12 ) the results of our study acknowledge the importance that students place in the field of public health as most students believe it will be of value in later practice .
although public health was not a popular choice of career , this is likely to have had many other contributory factors , and it is thus difficult to conclude that this was a reflection of how important the teaching programme was perceived to be . in our study , the region of residence and schooling did not show an association with public health being a career choice .
it would have been interesting to compare the backgrounds of students interested in a career in community medicine with those who were not in greater detail , as mcauliffe and barnett suggest that there is a relationship between an individual 's background and choosing to work in a similar setting.(13 ) however , analysis did reveal that career choice is affected by various perceptions on community - based learning , including perceiving the experience as mutually beneficial . a similar view is shared by modipa who , in his study on career choice , hypothesizes that individuals must be motivated by a need to work and serve for the good of the community.(14 ) thus , allocating students to communities with obvious health - needs may help students identify important health concerns and observe tangible improvements following interventions , rendering the experience more exciting and inspiring while providing valuable service to the community .
making the process of learning active , enjoyable and obviously personally beneficial , encourages students to be more involved in the education they receive .
for example , field - based activities ( community and family attachment programmes ) which are mostly student - oriented and engaging were found to enjoyable .
these ideas are further strengthened by a study on medical students by duke et al . , which , at its core , tests the same notion .
he suggests that greater engagement will improve knowledge and a fun and structured experience will enhance learning.(15 ) nosek et al .
, in a similar study states that attention is held longer when learning is made fun , and students prefer enjoyable teaching methods.(16 ) that being said students appeared to have problems with the access to their allocated community and since students appeared to find community - based learning less productive and enjoyable , when they perceived the location inconvenient , making field - based training accessible and enjoyable may ensure students better appreciate the community attachment programme and the field of public health .
the research programme which utilized some of the above - mentioned principles was received very positively by students . directly integrating the programme with a student scientific forum or student - based journal , which allows students to present their findings , may increase student perception of achievement and render the programme more productive . in the final year , most perceived that lectures had a tendency to disrupt their clinical work and claimed they were not able to fully concentrate on the subject matter , even though most agreed they found the lectures interesting which is in contrast to their response in regard to the introductory lectures in public health .
it appears that students still have a tendency to give greater priority to clinical work over activities related to the public health curriculum .
these observations have also been made by dare and bullen who state that there is a perception that population health is considered a poor cousin of the clinically orientated medical specialties.(11 ) if public health can not at this point compete with its clinical counterpart a possible compromise would be to reduce the likelihood of any negative perceptions .
therefore , a suggestion would be to either conclude all components of the course prior to the final year , when students are likely to be more receptive , or to integrate community - based medicine with ward activities in the final year .
one of the notable pitfalls in the curriculum was its failure to stimulate independent learning as large numbers admitted that they did not feel inspired to read up more on the topics taught , even though most found lecture topics useful and interesting .
however , group and discussion - centered activities were received positively among our study populace .
suggest the combination of sgds followed by presentations is an efficacious method of learning(17 ) and our students , too , appeared to agree .
a similar opinion , regarding problem - based learning ( pbl ) sessions , is shared by kwan , who hypothesised that a pbl - based curriculum is better than typical didactic teaching and will make students lifelong learners.(18 ) both these methods utilize group - based teaching at their core , a feature already present in the current system .
constructivists argue that while the responsibility of learning should be borne unto to the learner , instructors should be facilitators and not teachers , thus taking on a more passive role . to be effective , learning environments should guide students to reach their own understanding , while increasing students sense of autonomy and engagement in their education.(1922 ) based on these principles , we propose that introducing more problem - based activities , in preference of lectures , for delivery of factual knowledge , may , apart from being interesting , have the added advantage of promoting lifelong learning by motivating students to learn both independently and from one another .
this study utilized one hundred and eighty - four final year students who had completed their undergraduate course in medicine at the faculty of medicine colombo , one of eight medical faculties in the country which have a combined annual output of 1100 doctors .
however , the curriculum in public health and how it is implemented varies in each faculty and this study utilized convenience sampling for recruitment of its participants due to low sample numbers as each academic year has only 200 students 10 and only final years were considered ; thus , it could be argued that the findings can not be generalized to represent all the medical graduates of the country .
however with a recruitment rate of 94% the findings of this study give a clear picture of the perceptions of students of the faculty of medicine , colombo .
it would have been interesting to analyze each faculty individually and to understand variations in perception as per the curricula in place and it is our recommendation that similar studies be carried in other faculties as well .
the curriculum in place has been able create a positive opinion about public health among medical students of the faculty of medicine , colombo , who understand its significance .
however , students lack interest and motivation to learn independently and clinical components of medicine still take preference over public health .
active , engaging , and enjoyable methods of group - based teaching and a constructivist approach will likely be better received than traditional methods such as lectures , and we recommend the use of problem - based learning for delivery of factual knowledge . ensuring teaching activities and programmes are structured , time - efficient , and student friendly will make the course more enjoyable and productive . while community - based learning is viewed positively by students , making these experiences mutually beneficial to both community and students is likely to inspire students to think favorably about a future in public health .
we recommend that student opinions and perceptions should be assessed periodically to aid in the development of medical curricula . | background : the faculty of medicine , university of colombo , has an integrated curriculum in which teaching of public health takes place through a series of modules which span the full five - year study programme.aim:to assess final year medical student perceptions regarding the public health curriculum and to identify factors which influence this.materials and methods : the study was cross sectional . convenience sampling was utilized on final - year students of the faculty of medicine , university of colombo , sri lanka . a self - administered 4-point likert scale questionnaire covered general opinion on public healthcare and perceptions about the curriculum .
data were analyzed using descriptive statistics and chi - square tests.results:one hundred and eighty four students ( 94% ) participated in the study .
eighty - two percent ( 148 ) viewed public health as an important field .
only 9% ( 16 ) were interested in a career in public health .
a significant association was found between choosing public health as career and the following : perception of public health as an important field ; holding a good opinion about public health prior to commencement of the course ; having found the field - based experience enjoyable and beneficial to the community ; and feeling competent to work in the community at the end of the course ( p < 0.01 ) .
with regard to teaching methods , group activities and discussion - centered activities were identified positively ( 153 , 83% and 125 , 68% respectively ) .
the majority of students indicated that they were not stimulated to read more on the subject or regularly revise what they have learnt , both during the introductory public health programme and during the final year.conclusions:the curriculum has been able to create a positive opinion about public health .
however , students lack enthusiasm to learn independently .
experiential , group - centered teaching activities and a constructivist approach may be more effective in promoting independent learning .
perceptions are important and should aid in structuring the curricula . | Introduction
Materials and Methods
Design
Participants
Data collection
Statistical analysis
Ethical considerations
Results
The general view of students regarding public health
Student perceptions on community-based learning
Student views regarding the research project
Perceptions regarding the introductory phase and final year programme of the public health curriculum
Discussion
Conclusions |
american ginseng ( panax quinquefolium ) is an important medicinal plant belonging to the araliaceae family .
it is native to the hardwood - forested regions of the eastern part of north america , but it has been successfully cultivated in new zealand , china , australia , holland , france , and poland ( koodziej et al .
2006 ) . however , ginseng is a slow - growing plant , difficult to cultivate in the field , and prone to disease .
saponins , a type of ginsenoside , the pharmacologically active ingredient , can be obtained only after 47 yr of cultivation .
therefore , hairy root in vitro cultures of p. quinquefolium provide an attractive alternative for obtaining the biologically active compounds .
the two major groups of ginsenosides are rb and rg groups , derived from the 20(s ) protopanaxadiol and 20(s ) protopanaxartiol structures , respectively .
the main compounds are the ginsenosides rb1 ( 20(s ) protopanaxadiol-3-[o--d - glucopyranosyl(l2)--d - glucopyranoside]-20-o--d - glucopyranosyl(16)--d - glucopyranoside ) , rb2 ( 20(s)-protopanaxadiol-3-[o--d - glucopyranosyl(l-2)--d - glucopyranoside]-20-[o--l - arabinopyranosyl(l6)--d - glucopyranoside ] ) , rc ( 20(s)-protopanaxadiol-3-[o--d - glucopyranosyl(l2)--d - glucopyranoside]-20-o--l - arabinofuranosyl ( l6)--d - glucopyranoside ) , and rd ( 20(s)-protopanaxadiol-3-[o--d - glucopyranosyl(l2)--d - glucopyranoside]-20-(o--d - glucopyranoside ) from the rb group , and re ( re-20(s)-protopanaxatriol-6-[o--l - rhamnopyranosyl(l2)--d - glucopyranoside]-20-o--d - glucopyranoside ) , rg 1 ( rg120(s)-protopanaxatriol-6,20-di - o--d - glucoside ) from the rg group ( figs . 1 and 2 ; table 1 ) .
ginsenosides are responsible for most of the therapeutic action of ginseng ( yuan et al . 2010).figure 1.chemical structure of protpanaxadiol and protopanaxartiol.figure 2.biosynthetic pathway of ginsenosides from squalene in p. ginseng .
triterpene undergoes oxidation , glycosylation and is finally converted into triterpene saponins ( ginsenosides ) , according to kim et al ( 2009).table 1.sugar molecules in the structures of ginsenosides examined in this studymetaboliter1r220(s)-protopanaxadiolh h rb1glc
triterpene undergoes oxidation , glycosylation and is finally converted into triterpene saponins ( ginsenosides ) , according to kim et al ( 2009 ) .
sugar molecules in the structures of ginsenosides examined in this study experimental research has identified a range of pharmaceutical activities possessed by many secondary plant metabolites including polysaccharides , flavonoids , coumarins , glycosides , phenolic acids , saponins , and also essential oils .
many have strong antibacterial , antifungal , antiviral , anti - inflammatory , antioxidant , and also anticancerogenic properties ( sparg et al .
2004 ; chanwitheesuk et al . 2005 ; ahn et al . 2006 ; sienkiewicz et al .
, enterococcus spp . , and gram - negative bacteria , mainly of the klebsiella spp .
in addition , the broad and complex activity of plant metabolites , as well as their synergy of action , can make them a valued weapon against multidrug resistant bacterial strains ( tan and vanitha 2004 ; mahesh et al .
currently , there is no literature concerning the antibacterial and antifungal activity of the extracts obtained from p. quinquefolium hairy root cultures . in this study , we investigated antimicrobial properties associated with ginseng hairy root extracts .
p. quinquefolium l. hairy root cultures were established from seedlings ( obtained from field cultivation in the agriculture university of lublin , lodz , poland ) after infection with an agropine - type strain of agrobacterium rhizogenes atcc 15834 .
after 68 wk , the roots emerged from the site of infection . when the roots were 1.52 cm long , they were individually excised and transferred into a hormone - free b-5 liquid medium with addition of 500 mg / l ampicillin .
an aseptic culture of hairy roots was obtained , which grew rapidly on the same medium without antibiotic supplementation .
three clones , a , b , and g , were obtained and transformation was verified by pcr analysis ( kochan et al .
hairy root cultures were grown in 300 ml shaken erlenmeyer flasks with 80 ml of hormone - free liquid b-5 medium ( gamborg et al .
the average inoculum size was about 300 mg fresh weight and 0.30 mg dry weight ( dw ) .
the cultures were maintained in the dark at 26c on a rotary shaker ( 100 rpm ) and subcultured every 28 d. fresh roots , after drying on absorbent paper , were dried at room temperature and were processed for ginsenoside extraction and hplc analysis as previously described ( kochan et al .
five standard strains of gram - positive bacteria were used : staphylococcus aureus atcc 433000 , s. aureus atcc mr3 , enterococcus faecalis van b atcc 51299 , e. faecalis , vancomicin - sensitive atcc 29212 , enterococcus faecium , and vancomicin - sensitive atcc 35667 .
in addition , four gram - negative bacteria were used : escherichia coli atcc 35218 , e. coli atcc 25922 , pseudomonas aeruginosa atcc 27853 , and the yeast strain candida albicans atcc 10231 .
these standard bacterial and yeast strains used in the agar dilution method came from the collection of the medical and sanitary microbiology department , medical university of lodz , poland .
three antimicrobial agents were used : 5 g / ml ciprofloxacin ( graso ) , a mixture of 20 g / ml amoxicillin 10 g / ml clavulanic acid ( graso ) ; and 25 g / ml fluconazole ( graso ) . the bacteriological media were columbia agar ( biomerieux ) , mueller hinton ii agar ( emapol ) , and sabouroud dextrose agar ( graso ) .
the standard strains were cultivated in columbia agar medium , incubated at 37c for 48 h ( bacteria ) and in sabouroud dextrose agar at 28c for 48 h ( yeast ) in aerobic conditions .
bacterial and yeast suspensions were prepared with an optical density of 0.5 on the mcfarland scale using a bio merieux densitometer .
susceptibility testing was carried out using the disk diffusion method ( jorgensen and turnidge 2007 ) .
cultures were incubated at 37c for 1618 h ( bacterial strains ) and 28c for 24 h ( yeast ) in aerobic conditions .
the results were interpreted according to clinical and laboratory standard institute guidelines ( clsi 2009 ) . for antimicrobial testing , a1 , a2 , b1 , b2 , g1 , g2 independent weighted samples
each of the extracts from the hairy root clones was weighed , diluted in ethanol a concentration of 97% w / v of extracts and used as a stock solution .
an appropriate amount of this solution was mixed with columbia agar medium ( bacteria ) and sabouroud dextrose agar ( yeast ) to obtain concentrations from 0.8 to 1.4 mg / ml , and these were dispensed into petri dishes .
an inoculum containing 1.510 cfu ( 0.1 ml ) per spot was seeded either upon the surface of an agar plate with an extract from the hairy root clones ( at various concentrations ) , or a plate with no extracts added ( strains growth control ) . the minimal inhibitory concentration ( mic )
was determined after 24 h of incubation at 37c for bacteria and at 28c for yeast in aerobic conditions .
the analyses of the antibacterial and antifungal activity of the extracts were performed independently three times .
control media containing only alcohol ( at concentrations used in the dilutions of extracts ) did not inhibit the growth of any of the bacterial or yeast strains .
three independently generated hairy root in vitro cultures were established from sterile seedlings of p. quinquefolium that had be transformed with a. rhizogenes . for the 28-d culture period , the highest increase of dry biomass ( above eightfold ) was recorded for line a of p. quinquefolium hairy root culture , with a slightly lower increase for line g ( sevenfold ) , and the lowest for line b ( fivefold ) .
the obtained root cultures synthesized six identifiable saponins : rb1 , rb2 , rc , rd , ( derivatives of protopanaxadiol ) , and rg1 and re ( derivatives of protopanaxatriol ) .
ginsenoside production ( expressed in mg / g dw ) after 28 d of culture is shown in table 2 and fig .
3.table 2.ginsenoside content in three hairy root culture lines of p. quinquefoliumlines of hairy rootsginsenoside [ mg / g dw]rb1rb2rcrdrg1retotala3.934 0.080.296 0.0190.9477 0.110.066
0.0211.216 0.143.662 0.3610.12 0.52b1.933 0.080.346 0.0380.6133 0.030.037 0.010.785 0.072.384 0.366.097 0.44g3.169 0.350.073 0.0150.7553 0.070.085
0.0360.992 0.073.006 0.098.079 0.34each value is a mean of six replicates sdfigure 3.ginsenoside contents of rb group and rg group in three lines of p. quinquefolium hairy roots .
ginsenoside content in three hairy root culture lines of p. quinquefolium each value is a mean of six replicates sd ginsenoside contents of rb group and rg group in three lines of p. quinquefolium hairy roots .
clone a of the hairy roots achieved the highest total ginsenoside level ( 10 mg / g dw ) followed by clones g and b. the content of individual metabolites in clones a , b , and g differed .
the quantitative level of rb1 , rb2 , rc , rd , re , and rg1 saponins shows the pattern rb1 > re > rg1 > rc > rb2 > rd for clone a , with rb1 being the most abundant and rd the least .
clone g has similar pattern , but the content of ginsenoside rd was slightly higher than rb2 .
clone b followed the same sequence as clone a , but the main ginsenoside was the metabolite re ( table 2 ) .
metabolite rb was dominant among the protopanaxadiol derivatives , and metabolite re was dominant among the protopanaxatriol derivatives in all examined lines .
the sum of the main components ( rb1 , re ) was more than 70% of total ginsenosides .
the mics of the hairy root extracts ( a1 , a2 , b1 , b2 , g1 , and g2 ) against the tested microorganisms and susceptibility to standard strains of bacteria and yeast are shown in table 3.table 3.mic ( milligrams per milliliter ) for extracts of the hairy root of p. quinquefoliumstandard strainmic ( mg / ml ) of hairy root clone extractcontrol of standard strains , susceptibility to antibiotics ( mean dimeter zone of inhibiton ( mm))ethanola1a2b1b2g1g2cipamcflus .
albicans atcc 102311.01.01.41.31.21.2ndnd28nicip ciprofloxacin ( 5 g ) ( r 15 , 16 i 20 , s 21 ) , amc amoxicillin / clavulanic acid ( 20 g/10 g ) ( r 13 , 14 i 17 , s 18 ) , flu fluconazole ( 25 g / ml ) ( r 14 , 14 i 18 , s > 18 ) , nd not determined , ni not inhibited mic ( milligrams per milliliter ) for extracts of the hairy root of p. quinquefolium cip ciprofloxacin ( 5 g ) ( r 15 , 16 i 20 , s 21 ) , amc amoxicillin / clavulanic acid ( 20 g/10 g ) ( r 13 , 14 i 17 , s 18 ) , flu fluconazole ( 25 g / ml ) ( r 14 , 14 i 18 , s > 18 ) , nd not determined , ni not inhibited the tested hairy root extracts were the most active against gram - negative standard strains : e. coli atcc 35218 and e. coli atcc 25922 .
the mic values were between 0.8 and 1.3 mg / ml . the extracts from clones a were found to be very effective against the standard e. coli strains ( mic 0.80.9 mg / ml ) .
however , the mic values of the growth inhibition factors in the extracts from all the clones were higher against p. aeruginosa ( 1.01.4 mg / ml ) .
the highest concentrations of extracts , between 1.1 and 1.3 mg / ml for a clones and 1.4 mg / ml for b and g hairy root clones , were effective against the vancomycin - resistant standard strain , e. faecalis van b atcc 51299 . however , the mic values for standard strain s. aureus atcc 4330 were between 0.9 and 1.2 mg / ml and for s. aureus atcc mr3 were between 0.8 and 1.4 mg / ml for the all clones .
the mic of the hairy root extracts against c. albicans atcc 10231 was 1.0 mg / ml for the a clones , 1.2 mg / ml for the g clones and 1.31.4
overall , the strongest activity was observed by the a clones of the hairy root extracts .
the total ginsenoside level in lines a , g , and b of the hairy roots was found to be about 10 , 8 , and 6 mg / g dw , respectively . a lower level of total ginsenoside ( 2.585.44 mg / g dw ) was reported by mallol et al .
( 2001 ) in different phenotypes of hairy roots of p. ginseng . in the present study , ginsenosides rb1 and re were seen be the major component .
mathur et al . ( 2010 ) demonstrated that extracts of p. quinquefolium hairy roots also contained the highest level of metabolite rb1 having protopanaxadiol as sapogenin and re having protopanaxatriol as sapogenin after 4 wk of culture .
the same metabolites , rb1 and re , dominated all 18 lines of p. ginseng hairy roots studied by woo et al .
hairy roots of hybrid p. ginseng and p. quinquefolium growing on b-5 medium also synthesized six of the ginsenosides examined in this study , but only metabolite rb1 significantly exceeded the level of the other saponins ( washida et al .
the lowest level of saponin rd , found in this study , was similar to that observed by washida et al ( 1998 , 2004 ) and woo et al .
( 2004 ) . in our tests , standard strains of s. aureus ( n = 2 ) , enterococcus spp .
( n = 2 ) , p. aeruginosa ( n = 1 ) , and c. albicans ( n = 1 ) were sensitive to hairy root extracts at 0.81.4 mg / ml concentrations .
methanolic extracts from the leaves of cussonia sp . from the araliaceae family were active against p. aeruginosa ( mic of 1.01.5 mg / ml ) and s. aureus ( mic of 1.8 mg / ml ) .
in accordance with the literature , extracts from hairy root and untransformed roots of maytenus senegalensis showed an inhibitory effect against the growth of s. aureus at concentrations in the range of 0.651.25 mg / ml .
however , the root extracts of both untransformed and transformed root cultures were active against gram - positive bacterial strains only ( jain et al .
glycyrrhiza glabra root extracts were found to demonstrate significant antibacterial activity against two gram - positive ( bacillus subtilis and s. aureus ) and two gram - negative ( e. coli and p. aeruginosa ) bacterial strains ( nitalikar et al .
studies on the antimicrobial properties of the essential oils obtained from the cultured hairy roots of salvia miltiorrhiza bunge containing four diterpenoid tanshinones and three phenolic acids revealed that the compounds have very strong activity against gram - positive and gram - negative bacteria , and one fungal species ( zhao et al .
additionally , studies on the antimicrobial properties of korean red ginseng confirmed that ginsenosides possess antibacterial activities toward pathogenic gram - positive and gram - negative bacteria . in our tests ,
the highest antimicrobial activity was demonstrated by clone a hairy root culture , which was specifically correlated with the high content of ginsenosides .
the literature reports confirm not only the antimicrobial properties of ginsenosides , but also their synergy of action with commercial antibiotics , such as kanamycin and cefotaxime , on antibacterial activity against methicillin - resistant s. aureus strains ( sung and lee 2008 ) .
three studied lines of hairy root cultures of p. quinquefolium a , b , g synthesize six types of known ginsenosides .
the highest level of saponins is reported for line a.the tested extracts from hairy root clones inhibit the growth of standard bacteria and yeast strains .
these have the potential to be used in combination with antibiotics in fighting infectious diseases .
three studied lines of hairy root cultures of p. quinquefolium a , b , g synthesize six types of known ginsenosides .
the highest level of saponins is reported for line a. the tested extracts from hairy root clones inhibit the growth of standard bacteria and yeast strains .
these have the potential to be used in combination with antibiotics in fighting infectious diseases . | panax quinquefolium , american ginseng , is valued for its triterpene saponins , known as ginsenosides .
these constituents possess a number of pharmacological properties and hairy root cultures can synthesize similar saponins to those of field - cultivated roots .
the antibacterial activity of extracts from three hairy root clones of p. quinquefolium l. was tested against a range of standard bacterial and yeast strains .
the agar diffusion method was used to evaluate inhibition of microbial growth at various extract concentrations .
commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains .
susceptibility testing to antibiotics was also tested using the disk diffusion method .
the minimal inhibitory concentration values of the extracts , obtained by agar diffusion , ranged from 0.8 to 1.4 mg / ml . the results showed that extracts from hairy root cultures inhibited the growth of bacteria and yeast strains and suggest that they may be useful in the treatment of infections caused by pathogenic microorganisms . | Introduction
Materials and Methods
Results
Discussion
Conclusions |
aki is associated with short - term morbidity , a long - term risk of chronic kidney disease ( ckd ) and cardiovascular events and decreases survival [ 38 ] .
hospitalized patients , particularly those with comorbidities and those undergoing complex procedures are at high risk for developing aki [ 7 , 9 ] . despite increasing attention in recent years [ 1 , 2 , 8 ,
10 ] , little improvement in outcomes for aki has occurred and many authors have speculated that this is largely due to the inability to identify aki early , in a potentially easily reversible stage [ 1 , 2 ] .
common functional markers such as serum creatinine or urine output are late indicators of aki .
there are numerous potential strategies that could be deployed to mitigate aki if sufficient warning was provided . in this review
, we will summarize the current evidence that supports the use of cell - cycle arrest biomarkers for risk assessment of aki , we will consider various responses to this warning and we will speculate on how this mechanism could be exploited to protect the kidney .
serum creatinine ( scr ) , the most widely used marker of kidney function , is a component of the definition of aki , and yet as a single measurement is completely useless in differentiating aki from ckd .
while scr is an adequate marker of glomerular filtration rate ( gfr ) , scr itself does not correlate with hospital survival whether measured at the time of presentation or the start of dialysis .
what predicts short- and long - term outcomes is the change in renal function and herein lies the problem .
changes in scr require several hours to days before they reach steady state following an injury to the kidney .
thus the change in scr is an excellent tool for defining when a change in function has occurred but not particularly good for detecting that it is occurring ( or about to occur ) .
worse yet , scr , like all functional markers , may be both insensitive to and non - specific for injury .
uo will often decrease before scr increases making it a more time sensitive marker of gfr .
while in the absence of obstruction if there is no uo , there can be no gfr , not all reductions in uo signal aki .
sustained oliguria is invariably associated with aki but then , the timeliness of uo as an early indicator is lost .
importantly , functional change is neither necessary nor sufficient to define aki as it is occurring . over time , a persistent renal functional change can be used to infer kidney injury in the same way that regional wall motion abnormalities or a decrement in ejection fraction can be used to infer myocardial injury . however
conversely , the absence of functional change can not be used to exclude injury , especially in previously healthy individuals with normal renal reserve . as such , markers that can detect actual damage or warn of impending damage
are required in the same way that functional measures alone ( e.g. left ventricular ejection fraction ) are insufficient to manage acute coronary syndrome .
in 2013 , we reported the results of a prospective , observational , international investigation ( sapphire study ) of tissue inhibitor of metalloproteinases-2 ( timp-2 ) and insulin - like growth factor - binding protein 7 ( igfbp7 ) in a heterogeneous group of critically ill ( evidence of respiratory or cardiovascular failure ) patients .
this report actually included two multi - center studies discovery and validation . in the discovery phase we enrolled 522 adults in three distinct cohorts including patients with sepsis , shock , major surgery and trauma and examined over 300 markers in urine and blood . in the validation study we enrolled 744 adults with critical illness and without evidence of aki at enrollment ; the final analysis cohort was a heterogeneous sample of 728 critically ill patients .
the primary end point was moderate - severe aki [ kidney disease : improving global outcomes ( kdigo ) stage 23 ] within 12 h of sample collection which occurred in 14% of sapphire subjects .
area under the receiver operating characteristic curve ( auc ) of 0.80 together ( 0.76 and 0.79 alone ) .
[ timp-2][igfbp7 ] was significantly superior to all previously described markers of aki ( p < 0.002 ) including neutrophil gelatinase - associated lipocalin ( ngal ) and kidney injury molecule ( kim)-1 , none of which achieved an auc > 0.72 .
however , this result might be due in part to varying kinetics of different early aki biomarkers .
[ timp-2][igfbp7 ] significantly improved risk stratification when added to a 9-variable clinical model when analyzed using cox proportional hazards model , generalized estimating equation , integrated discrimination improvement or net reclassification improvement . finally , in sensitivity analyses [ timp-2][igfbp7 ] remained significant and superior to all other markers regardless of changes in reference creatinine method .
other investigators have confirmed that [ timp-2 ] and [ igfbp7 ] are useful in the detection of aki . in a small study of patients undergoing cardiac surgery , wetz et al . found that levels of urinary [ timp-2][igfbp7 ] were higher in the patients who developed aki than those that did not .
yamashita demonstrated that urinary [ timp-2 ] performed well to predict severe aki in critically ill patients with and without sepsis with a roc auc range of 0.810.84 .
next , we developed and separately evaluated , two clinical cutoffs for a test using urinary [ timp-2][igfbp7 ] . in order to do so , we first had to consider the intended use of the test
. there are no specific means to prevent aki , no equivalent to thrombolytic therapy and recommended actions are low - risk and generally applicable to most if not all critically ill patients ( see figure 1 ) . however , these actions are difficult to implement in all patients and some are time - consuming and potentially expensive .
thus , the main purpose of the test would be to identify the majority of high - risk patients , and to ensure all such patients receive these recommended actions . as such , the test would need to have a primary cutoff that provides high sensitivity and high negative predictive value ( npv)the proportion of true negatives over all patients testing negative . although this would be the main use for the test , a cutoff with a high specificity and high positive predictive value ( ppv ) would have value as well .
for example , in judging the risk - benefit to a patient for a decision to postpone some form of therapy that carries a risk of kidney damage ( e.g. cardiac surgery ) one might wish to have a test with a high ppv .
shading of boxes indicates priority of action solid shading indicates actions that are equally appropriate at all stages whereas graded shading indicates increasing priority as intensity increases .
aki , acute kidney injury ; icu , intensive care unit . source : www.kdigo.org .
shading of boxes indicates priority of action solid shading indicates actions that are equally appropriate at all stages whereas graded shading indicates increasing priority as intensity increases .
thus , we derived cutoffs for urinary [ timp-2][igfbp7 ] based on sensitivity and specificity ( as well as npv and ppv ) for prediction of aki using data from the sapphire study .
we set the high sensitivity / high npv cutoff at 0.3 ( ng / ml)/1000 and the high specificity / high ppv cutoff at 2.0 ( ng / ml)/1000 .
next , we verified these cutoffs in a new study ( opal ) enrolling 154 critically ill adults from six sites in the united states . in total
, 27 subjects met the primary end point ( stage 23 aki within 12 h ) .
the results of the opal study replicated those of sapphire ( figure 2 ) where the sensitivity at the 0.3 cutoff was 89% , and npv was 97% . for 2.0 , specificity was 95% and ppv was 49% .
relative risk of aki for subjects testing in each strata is shown in figure 2 .
compared with baseline risk , subjects in the middle strata ( > 0.32 ) were more than 4-fold and those in the highest strata ( > 2 ) were more than 10-fold more likely to manifest moderate to severe aki ( kdigo stage 23 ) in the next 12 h. in opal , 46% of patients tested 0.3 while the upper two strata included 39% and 16% of patients .
we also examined these cutoffs for urinary [ timp-2][igfbp7 ] in sapphire with respect to subsequent development of major adverse kidney events at 30 days ( make30 ) , which included death , dialysis or persistence of renal dysfunction ( creatinine twice baseline ) .
risk for make30 was lowest when [ timp-2][igfbp7 ] was 0.3 ( raw risk 18% ) and increased to 23% for > 0.32.0 and 40% for > 2.0 ( p < 0.001 ) .
figure 2:top : [ timp-2][igfbp7 ] roc curves for the opal ( solid ) , sapphire ( short dash ) and topaz ( long dash ) cohorts . closed circles and triangles indicate [ timp-2][igfbp7 ] cutoffs of 0.3 and 2.0 , respectively .
area under the roc curve ( 95% ci ) = 0.79 ( 0.690.88 ) , 0.80 ( 0.740.84 ) and 0.82 ( 0.760.88 ) for opal , sapphire and topaz , respectively .
bottom : relative risk of aki stage 2 or 3 within 12 h in the opal ( light gray ) , sapphire ( medium gray ) and topaz ( dark gray ) cohort .
top : [ timp-2][igfbp7 ] roc curves for the opal ( solid ) , sapphire ( short dash ) and topaz ( long dash ) cohorts . closed
area under the roc curve ( 95% ci ) = 0.79 ( 0.690.88 ) , 0.80 ( 0.740.84 ) and 0.82 ( 0.760.88 ) for opal , sapphire and topaz , respectively .
bottom : relative risk of aki stage 2 or 3 within 12 h in the opal ( light gray ) , sapphire ( medium gray ) and topaz ( dark gray ) cohort .
finally we conducted a third study , topaz , and enrolled another heterogeneous cohort of 420 critically ill patients in order to prospectively validate the lower ( 0.3 ) biomarker cutoff value for risk assessment of aki .
this was the first study to evaluate urinary [ timp-2][igfbp7 ] where the end point was determined by clinical adjudication by aki experts blinded to the results of the test .
we found that the urinary [ timp-2][igfbp7 ] test significantly improved risk assessment by stratifying patients into distinct risk categories , with a 7-fold increase in risk for patients with a [ timp-2][igfbp7 ] test value > 0.3 compared with those 0.3 .
when two cutoffs are used , relative risk for > 0.32.0 was 5 , and > 2.0 relative risk was 17 ( p < 0.002 ) .
furthermore , using a multivariate model including clinical information , urinary [ timp-2][igfbp7 ] remained statistically significant and a strong predictor of aki . the clinical model alone exhibited an auc 0.70 , 95% ci 0.630.76 while the auc was 0.86 , 95% ci 0.800.90 for clinical variables plus [ timp-2][igfbp7 ] ) .
thus , urinary [ timp-2][igfbp7 ] has now been shown to provide early risk stratification for imminent aki in over 1200 critically ill patients in three multi - center studies enrolling diverse groups of patients ( table 1 ) with the prevalence of major exposures for aki , such as sepsis , similar to other reports in the literature [ 19 , 20 ] .
table 1.baseline characteristics of subjects included in three large multi - center trials evaluating cell - cycle arrest markerssapphireopaltopazall patients728153408male449 ( 62%)87 ( 57%)219 ( 54%)age , years64 ( 5373)65 ( 5477)65 ( 5476)race white573 ( 79%)119 ( 78%)339 ( 83% ) black87 ( 12%)13 ( 8%)56 ( 14% ) other / unknown68 ( 9%)21 ( 14%)13 ( 3%)history of ckd65 ( 9%)13 ( 8%)32 ( 8%)icu type medical225 ( 31%)53 ( 35%)180 ( 44% ) surgical179 ( 25%)13 ( 8%)70 ( 17% ) combined icu147 ( 20%)43 ( 28%)62 ( 15% ) cardiac surgery61 ( 8%)1 ( 1%)38 ( 9% ) neurologic39 ( 5%)2 ( 1%)14 ( 3% ) coronary care unit30 ( 4%)27 ( 18%)10 ( 2% ) trauma24 ( 3%)9 ( 6%)27 ( 7% ) other / unknown23 ( 3%)5 ( 3%)7 ( 2%)reason for icu admission respiratory310 ( 43%)81 ( 53%)206 ( 50% ) surgery247 ( 34%)23 ( 15%)128 ( 31% ) cardiovascular243 ( 33%)64 ( 42%)165 ( 40% ) sepsis136 ( 19%)29 ( 19%)97 ( 24% ) neurological70 ( 10%)15 ( 10%)52 ( 13% ) trauma55 ( 8%)12 ( 8%)44 ( 11% ) other126 ( 17%)57 ( 37%)120 ( 29%)enrollment scr , mg / dl0.9 ( 0.71.2)1.1 ( 0.81.6)0.9 ( 0.71.3)shown are n ( proportion ) or median ( interquartile range).results reproduced from table 1 of .results reproduced from table s1 of .results partially reproduced from table 1 of .subjects may have multiple reasons for icu admission . baseline characteristics of subjects included in three large multi - center trials evaluating cell - cycle arrest markers shown are n ( proportion ) or median ( interquartile range ) .
taken together the results of these studies of urinary [ timp-2][igfbp7 ] should enable early triage and risk stratification in a wide range of critically ill patients from the emergency room , acute admission areas and intensive care .
early identification of these at - risk patients should improve delivery of kdigo - recommended interventions and might also enable interventional studies for the treatment of aki in the not - so - distant future .
importantly , both timp-2 and igfbp7 may increase in response to a wide variety of insults ( inflammation , oxidative stress , ultraviolet radiation , drugs and toxins ) [ 2123 ] .
this may help explain why they correspond to risk for aki , a syndrome known for its multiple etiologies even in the same patient .
however , these insults may not actually destroy cells and these molecules appear to be able to signal in autocrine and paracrine fashions [ 23 , 24 ] thus behaving more like an
alarm spreading to adjacent cells ( figure 3 ) . in terms of timing , this signal could represent the earliest point of cellular stress .
biomarkers that can detect cellular stress ( or conversely cell health ) may be more useful than markers of injury or cell death .
figure 3:proposed mechanistic involvement of the novel biomarkers in aki : initial tubular cells sustain injury by various insults . in response to dna and possibly other forms of damage igfbp7 and timp-2
these effects are conducted in an autocrine and paracrine manner via igfbp7 and timp-2 receptors . the p proteins in turn , block the effect of the cyclin - dependent protein kinase complexes ( cycld - cdk4 and cycle - cdk2 ) on the cell - cycle promotion , thereby resulting in g1 cell - cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing .
proposed mechanistic involvement of the novel biomarkers in aki : initial tubular cells sustain injury by various insults . in response to dna and possibly other forms of damage igfbp7 and timp-2
these effects are conducted in an autocrine and paracrine manner via igfbp7 and timp-2 receptors . the p proteins in turn ,
block the effect of the cyclin - dependent protein kinase complexes ( cycld - cdk4 and cycle - cdk2 ) on the cell - cycle promotion , thereby resulting in g1 cell - cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing .
as urinary [ timp-2][igfbp7 ] can aid in the risk assessment for aki , it is important to recognize that , like all diagnostic tests , it does not take the place of clinical judgment .
furthermore , while the test was designed to risk assess for stage 23 aki , the biomarker concentrations correspond to severity of aki across all three stages .
however , the markers do not appear to persist in the urine for long after aki has occurred and thus they may be normal in patients who have already manifest aki by functional criteria ( e.g. scr ) .
finally , the cutoffs for the test are based on overall behavior of the biomarkers in the majority of patients .
while this performance appears to be very consistent across various exposures and susceptibilities for aki , specific groups of patients may require more fine - tuning .
for example , meersch et al . examined sensitivity and specificity of [ timp-2][igfbp7 ] for aki ( stage 1 or greater ) in a high - severity group of patients undergoing cardiac surgery .
these investigators found a sensitivity of 0.92 and specificity of 0.81 for a cutoff value of 0.5 ( auc 0.90 ) using the maximum urinary [ timp-2][igfbp7 ] concentration achieved in the first 24 h following surgery ( composite time point ) . taking the cardiac surgery example ,
we propose a set of actions based on pre - test assessment of risk and the urinary [ timp-2][igfbp7 ] test result ( figure 4 ) .
low , moderate and high risk are based on a combination of ( i ) underlying clinical predisposition ( susceptibility ) which includes most of the same variables that determine sts predicted risk of mortality ; ( ii ) acute evidence of aki ( oliguria or increasing scr ) ; ( iii ) clinical suspicion of aki based on exposures and ( iv ) urinary [ timp-2][igfbp7 ] .
when [ timp-2][igfbp7 ] is 0.3 ( ng / ml)/1000 , the high npv means that patients are low - risk unless clinical suspicion is high or unless they already have clinical evidence of aki .
when [ timp-2][igfbp7 ] is > 0.3 but < 2.0 ( ng / ml)/1000 , risk is moderate but should be increased to high if any of the following exist : clinical evidence of aki , sts predicted risk of mortality 4% , clinical suspicion for aki high .
finally , given the high ppv when urinary [ timp-2][igfbp7 ] > 2.0 ( ng / ml)/1000 , anyone regardless of clinical risk assessment should be considered to be at high risk .
figure 4:proposed clinical application of risk assessment for patients immediately after cardiac surgery .
sts , society of thoracic surgery ; i / o , input and output ; scr , serum creatinine ; cvp , central venous pressure ; nsaids , non - steroidal anti - inflammatory drug ; ace , angiotenson converting enzyme inhibitor ; scvo2 , central verous oxygen saturation ; h / o , history of ; lv fx , left ventricular function ; pa , pulmonary artery ; ci , cardiac index .
sts , society of thoracic surgery ; i / o , input and output ; scr , serum creatinine ; cvp , central venous pressure ; nsaids , non - steroidal anti - inflammatory drug ; ace , angiotenson converting enzyme inhibitor ; scvo2 , central verous oxygen saturation ; h / o , history of ; lv fx , left ventricular function ; pa , pulmonary artery ; ci , cardiac index .
each phase of the cell cycle has a specific function that is required for appropriate cell proliferation .
begin the process of repair , they must enter and exit each phase of the cell cycle on schedule .
if the cell exits a phase too soon , or stays in a phase too long , the normal repair and recovery process can become maladaptive .
for instance , if epithelial cells remain arrested in g1 or g2 , it favors a hypertrophic and fibrotic phenotype [ 27 , 28 ] .
cyclins and cyclin - dependent kinases , and inhibitors control each phase of the cell cycle
. the cell uses cell - cycle arrest as a protective mechanism to avoid cell - division when potentially damaged . by initiating cell - cycle arrest , cells can thus avoid cell division during stress and injury , which is protective .
however , if the cells do not re - initiate the cell - cycle and remain arrested at g1 or g2 ( or possibly other phases of cell cycle ) , a fibrotic phenotype can ensue .
both timp2 and igfbp7 have been implicated in the g1 cell - cycle arrest phase noted to occur during the very early phases of cellular stress ( figure 3 ) [ 2123 ] .
specifically , it has also been shown that renal tubular cells also go through this g1 cell - cycle arrest phase following stress due to a variety of insults .
induction of cell - cycle arrest is not only associated with increased risk for aki but may also serve as a mechanistic link between aki and ckd .
sustained cell - cycle arrest will result in a senescent cell phenotype and lead to fibrosis .
interestingly , the various sub - types of timp proteins may play different roles in the kidney .
have shown that timp-3 protects the cells from damage , whereas timp-2 appears to promote injury through matrix metalloproteinase activation .
similarly , research studies in renal transplant allografts show that matrix metalloproteinase activity is important in mediating scarring in chronic allograft nephropathy .
indeed there is already evidence that urinary [ timp-2][igfbp7 ] is strongly associated with a composite end point of death or need for dialysis .
we found that [ timp-2][igfbp7 ] > 2.0 ( ng / ml)/1000 was associated with increased risk for mortality or receipt of rrt over the next 9 months ( hazard ratio [ hr ] , 2.11 ; 95% confidence interval [ 95% ci ] , 1.37 to 3.23 ; p,0.001 ) .
interestingly , in a multivariate analysis adjusted for the clinical model , [ timp-2][igfbp7 ] > 0.3 ( ng / ml)/1000 were associated with death or rrt only in subjects who developed aki ( p = 0.002 ) .
additionally , aregger et al . recently demonstrated that igfbp7 levels predicted mortality , renal recovery and severity / duration of aki in a small cohort of critically ill adults .
as discussed above , cell - cycle arrest is nonetheless a protective mechanism to avoid the cell entering the cell - cycle when it is injured or even in an adverse environment .
thus , temporary g1 cell - cycle arrest should reduce kidney damage . using an animal model of septic aki secondary to cecal ligation and
puncture we hypothesized that a pharmacologically induced early cell - cycle arrest would be associated with less aki .
we used cyclosporine a , a known inducer of cell - cycle arrest and previously shown to attenuate kidney damage in the setting of folic acid - induced aki , and found that a single dose given 18 h after cecal ligation and puncture and along with initial antibiotics was successful in reducing aki .
thus manipulation of cell - cycle may represent a new therapeutic strategy in the prevention and treatment of aki .
these results are also important because they have implications for how we understand the pathogenesis of aki .
there is a growing appreciation for the concept of secondary injury to the kidney as danger signaling molecules ( damage and pathogen - associated molecular patterns ) are delivered to the renal tubule via both glomerular filtration and the blood stream .
these molecules are detected by pattern recognition receptors on the tubular cell surface where they initiate a cascade leading to inflammation and/or apoptosis .
in essence , the available data suggest that cell - cycle arrest signaling is a protective response , but when engaged by multiple cells such that increases in markers like timp-2 and igfbp7 can be detected in the urine , it is often followed by aki .
furthermore , if cell - cycle arrest persists the result can become maladaptive and lead to a fibrosis phenotype .
early protection of cells might be achievable by supporting the cell 's own self - preservation mechanisms including cell - cycle arrest .
conversely , once the danger is past , it may be important to rapidly reverse this process so that the adverse consequences including cell senescence and fibrosis are avoided .
thus , cell - cycle arrest activation and deactivation at critical clinical time points for a patient may prove to be targets of therapeutic intervention in the future .
dark side where it heralds the onset of aki and persistence of this signaling may lead to maladaptive repair processes ultimately favoring fibrosis over regeneration .
light side in that its detection can be used as an early warning prior to actual damage
intriguingly though , we might also be able to manipulate cell - cycle in ways that can foster the innate protective aspects of this response while avoiding the maladaptive aspects turning the dark toward the light .
cell - cycle arrest biomarkers : the light at the end of the acute kidney injury tunnel . | acute kidney injury ( aki ) is a common consequence of systemic illness or injury and it complicates several forms of major surgery .
two major difficulties have hampered progress in aki research and clinical management .
aki is difficult to detect early and its pathogenesis is still poorly understood .
we recently reported results from multi - center studies where two urinary markers of cell - cycle arrest , tissue inhibitor of metalloproteinases-2 ( timp-2 ) and insulin - like growth factor - binding protein 7 ( igfbp7 ) were validated for development of aki well ahead of clinical manifestations azotemia and oliguria .
cell - cycle arrest is known to be involved in the pathogenesis of aki and this
dark side may also involve progression to chronic kidney disease .
however , cell - cycle arrest has a
light side as well , since this mechanism can protect cells from the disastrous consequences of entering cell division with damaged dna or insufficient bioenergetic resources during injury or stress .
whether we can use the light side to help prevent aki remains to be seen , but there is already evidence that cell - cycle arrest biomarkers are indicators of both sides of this complex physiology . | INTRODUCTION
WHY ARE BIOMARKERS NEEDED?
DISCOVERY AND VALIDATION OF CELL-CYCLE ARREST MARKERS
CLINICAL APPLICATION OF AKI BIOMARKERS
TWO SIDES TO CELL-CYCLE ARREST
CONCLUSIONS
CONFLICT OF INTEREST STATEMENT |
smoking is one of the important risk factors and also one of the increasing factors of diseases in the world , especially chronic and noncommunicable diseases such as heart diseases , cardiovascular , respiratory , cancer , and stroke .
it is regarded as one of the health problems and an obstacle to be healthy in some developing countries . as a result ,
tobacco epidemic is one of the most serious public health threats that the world has ever faced .
it is estimated that , by 2030 , the number of deaths caused by smoking will reach to more than ten million of which 70% will be in developing countries .
the prevalence of smoking in our country , based on a meta - analysis , is 13.9% ( 21.7% in male and 3.6% in female ) .
smoking does not belong to a particular class , but the most vulnerable groups are adolescents and youth and that problem targets children too .
this is one of the public health problems and it has become a principle problem in tobacco control , because there is a tendency of continuation or repetition of consumption behavior in adulthood which highlights the need for preventing smoking .
health belief model ( hbm ) is widely used in the research studies to predict health - related behaviors such as smoking and is one of the most functional models of health education in the field of prevention .
according to this model , if people feel that they are exposed and sensitive to a situation ( perceived susceptibility ) , and believe that the situation is potentially dangerous and it has negative consequences ( perceived severity ) and have this idea that through a series of operations , they can reduce the risks and side effects of the situation ; and believe that the benefits of these actions ( perceived benefits ) are more than the barriers of doing that behavior ( such as time and money ) ( perceived barriers ) , they will do the preventive behaviors in order to avoid the risk .
meantime , stimulus can act as a trigger for behavior and they can be regarded as a guide and motive for the action ( cues to action ) , in this sense the individuals have a sense of efficiency and adequacy due to overcoming the barriers of behaviors ( self - efficacy ) .
in fact , the hbm is a psychological model that attempts to explain and predict health behaviors .
this is done by focusing on the attitudes and beliefs of individuals . according to the history of the model that was used in many studies and the importance of its use in public health issues in order to predicting the health problems ,
this study was aimed to determine the predictors of smoking among the secondary high school students in marivan ( kurdistan - iran ) in 2015 , based on the constructs of hbm .
it was conducted among the secondary high school boys in the spring of 2015 in marivan city of kurdistan province of iran .
the samples were selected using multistage random cluster sampling among the six secondary high schools in the city .
twelve secondary boy high schools of the city were considered as clusters of the study ; six schools were selected among them ; two to three classes were selected from each school in proportion to the number of students .
the criteria for inclusion in this study were being a 1- , 2- , and 3-year students and filling a written consent to participate in the study .
sample size was 470 subjects . to collect data , the questionnaire of reisi et al .
the first part contained 11 questions to assess demographic variables ; the second part contained 11 questions to assess knowledge about smoking disadvantages .
the questions of this section were designed by means of three answers : yes , no , and i do not know .
two scores were considered for each correct answer , and one point for each i do not know answer and any points for wrong answers ( range of score 022 ) .
the third part consisted of 10 questions to measure the attitude ( as a one structure of hbm ) with five - level likert scale ( from absolutely agree to absolutely disagree ) and the fourth part of the questionnaire consisted of 33 questions related to the constructs of hbm with five option ( from strongly agree to strongly disagree ) .
twelve questions were about perceived susceptibility ( with a minimum score of 12 and a maximum score of 60 ) , 6 questions about perceived benefits ( with a minimum score of 6 and a maximum score of 30 ) , 6 questions about perceived barriers ( with a minimum score of 6 and a maximum score of 30 ) , 4 self - efficacy question ( with a minimum score of 4 and a maximum score of 20 ) , and 5 questions about cues to action ( with a minimum score of 5 and a maximum score of 25 ) .
the content validity of the questionnaire was calculated using the opinions of four experts in health education and health promotion from the university of medical sciences of kurdistan and isfahan in 2015 .
in addition , the reliability of the questionnaire was calculated by internal consistency cronbach 's alpha among 58 students , the reliability for knowledge questions was 0.75 , perceived susceptibility 0.81 , perceived barriers 0.77 , perceived benefits 0.79 , perceived self - efficacy 0.82 and tips for action 0.80 .
after the approval of the proposal , the study was confirmed by the research committee of the university of medical sciences of kurdistan . then arrangements were made with education management of marivan , school principals , teachers , and students , and the objectives of the study were explained to them clearly .
data were analyzed using descriptive statistics such as frequency and percentage , and analytical statistics such as chi - square test , spearman correlation coefficient , and logistic regression .
it was conducted among the secondary high school boys in the spring of 2015 in marivan city of kurdistan province of iran .
the samples were selected using multistage random cluster sampling among the six secondary high schools in the city .
twelve secondary boy high schools of the city were considered as clusters of the study ; six schools were selected among them ; two to three classes were selected from each school in proportion to the number of students .
the criteria for inclusion in this study were being a 1- , 2- , and 3-year students and filling a written consent to participate in the study .
the first part contained 11 questions to assess demographic variables ; the second part contained 11 questions to assess knowledge about smoking disadvantages .
the questions of this section were designed by means of three answers : yes , no , and i do not know .
two scores were considered for each correct answer , and one point for each i do not know answer and any points for wrong answers ( range of score 022 ) .
the third part consisted of 10 questions to measure the attitude ( as a one structure of hbm ) with five - level likert scale ( from absolutely agree to absolutely disagree ) and the fourth part of the questionnaire consisted of 33 questions related to the constructs of hbm with five option ( from strongly agree to strongly disagree ) .
twelve questions were about perceived susceptibility ( with a minimum score of 12 and a maximum score of 60 ) , 6 questions about perceived benefits ( with a minimum score of 6 and a maximum score of 30 ) , 6 questions about perceived barriers ( with a minimum score of 6 and a maximum score of 30 ) , 4 self - efficacy question ( with a minimum score of 4 and a maximum score of 20 ) , and 5 questions about cues to action ( with a minimum score of 5 and a maximum score of 25 ) . the content validity of the questionnaire was calculated using the opinions of four experts in health education and health promotion from the university of medical sciences of kurdistan and isfahan in 2015 . in addition , the reliability of the questionnaire was calculated by internal consistency cronbach 's alpha among 58 students , the reliability for knowledge questions was 0.75 , perceived susceptibility 0.81 , perceived barriers 0.77 , perceived benefits 0.79 , perceived self - efficacy 0.82 and tips for action 0.80 .
after the approval of the proposal , the study was confirmed by the research committee of the university of medical sciences of kurdistan . then arrangements were made with education management of marivan , school principals , teachers , and students , and the objectives of the study were explained to them clearly .
data were analyzed using descriptive statistics such as frequency and percentage , and analytical statistics such as chi - square test , spearman correlation coefficient , and logistic regression .
in this study , all of 470 secondary high school students participated ( 163 people ( 34.7% ) in the first grade , 140 ( 30% ) in the second grade and 167 ( 35.3% ) in the third grade ) .
mean age of student was 16.2 0.25 ( rang of age 1417 ) years .
the education of the majority of the parents of the samples was diploma and under high school diploma ( 78.1 and 81.9 respectively ) .
there was not a significant relationship between the education of students parents and smoking ( p = 0.637 ) .
the results of the study about smoking showed that 4.7% of the samples of the study (
about 6.4% ( n = 31 ) had smoked in the last 30 days and 34.7% ( n = 163 ) of them have experienced smoking at least once in their lifetime .
fifty - three percent of students had friends who smoke cigarette and 6.29% of the students reported that there were smokers in their families , 80% of fathers , 18% of brothers , and 42/1% of their mothers were smoking .
smoking had a significant difference in terms of mother 's jobs , smokers in the family , having friends who smoke and living with their parents ( p < 0.05 ) .
however , there was not a significant correlation between smoking status of the students and their grade of school , their fathers jobs , the education of their parents , birth order , household size , and history of their failing ( p > 0.05 ) . according to results of spearman correlation coefficient test
, smoking had a significant correlation with the constructs of the model and the attitudes toward smoking .
however , there was not a significant correlation between smoking behavior and awareness of its harms . among the constructs that had a significant correlation , the correlation between cigarette smoking and cues to action was stronger , but the correlation between cigarette smoking and perceived barriers is weaker [ table 1 ] .
the relationship between smoking and health belief model constructs based on the results of the analysis of the logistic regression method , the structure of attitudes , self - efficacy , and cues to action were the significant predictors of smoking behavior and among these significant predictors , attitude ( odds ratio [ or ] = 0.87 ) is the strongest predictor of the smoking behavior [ table 2 ] .
severity did not evaluate summary of regression analysis for the variables of knowledge , attitudes and constructs of health belief model as the predictors of smoking
this study aimed to investigate the prevalence of smokers in students and relationship among structures of health belief model in the 1- , 2- , and 3-year students of marivan city .
studies in iran and around the world showed different results of the prevalence of smoking .
for example , smoking rates in the study of mohammadpourasl et al . on the students of tabriz
the results showed that there is a significant relationship between the mother 's occupation and smoking in teenagers .
hence , the students whose mothers were housewives had higher rates of smoking . similar to these findings , kassiri et al . in their study on smoking in ahvaz and namakinand et al . on high school students in birjand reported the highest prevalence of smoking in teenagers whose mothers were housewives .
these findings may indicate low awareness of housewives from the harms of the risky behaviors such as smoking on their children or they have less control over their behavior . conducting more comprehensive studies are essential to investigate the reasons of this relationship more closely .
significant positive correlation means that as much the people have positive feelings toward cigarettes , their tendency to smoke also increases . in confirming the findings of this study , karimi and et al . in their study on the students in zarandieh , rise
et al . in norway and moini and et al . on the students in hamedan reported that positive attitude toward smoking can be a factor that increases this behavior among the students .
according to the findings of this study , modification of students attitudes toward smoking by educational programs can be a factor in preventing its use .
shahnaz et al . in their study on preuniversity students between smokers and nonsmokers reported that there is a significant difference regarding the perceived benefits .
this means that in nonsmokers , the perceived benefits ( awareness about advantage of avoiding from cigarette ) of smoking is higher .
thus , according to these findings , the emphasis on the advantages of not smoking in educational interventions can have an important role in preventing smoking .
another significant constructs that is negatively correlated with smoking behavior is the self - efficacy to nonsmoking .
bandura believed that self - efficacy , as an independent part of the individual 's basic skills , is an important factor in behavior .
moreover , self - efficacy is an important prerequisite to manage a learned behavior that can improve healthy behaviors .
based on the findings of this study , self - efficacy to nonsmoking significantly reduces smoking behaviors .
the findings of this study is aligned with moeini et al . and bashirian et al . , in terms of self - efficacy effect .
hence , paying attention to self - efficacy is necessary to reduce unhealthy behaviors such as smoking . according to the findings of this study , a cues to action compared to other hbms has a stronger correlation with smoking .
this means that a person 's symptoms and triggers in the environment is an important factor in stimulating students to smoke .
as studies show , parents and friends who smoke can be an important factor that motivates students to smoke . according to these results
, it is recommended that educational interventions designed to prevent smoking in young adults also pay special attention to the people around them .
these results are similar to the results of li and kay , mokhtari et al . and some other studies .
it is possible that potentially negative factors of a behavior such as the usefulness of action against the costs , risks , and time consuming can be a barriers to adopt healthy behavior .
thus , positive healthy behaviors can also be promoted by reducing the barriers of smoking .
the findings of this study is consistent with the study conducted by gharghani et al . on secondary school male students of shiraz .
these findings suggest that it may be possible to sensitize students about the consequences of risky behavior and increase adoption of healthy behaviors in them . according to the results of logistic regression method , the significant and meaningful predictors of smoking behavior are as the followings : the constructs of attitudes , self - efficacy , and cues to action . among these predictors , attitudes ( or 0.87 )
attitude is considered as the most important predictors factor in adolescent drug use behavior that is consistent with the findings of this study . in another study conducted by umeh and patel on ecstasy ,
it is shown that attitudes toward drug , is a strong predictor of ecstasy consuming behavior .
these results suggest that attitude must be considered as the first priority in educational programs for preventing the risky behaviors such as smoking .
moreover , the protective behaviors were investigated in males ; therefore , to compare smoking in both sexes , other studies on females are also required .
it is suggested that smoking behavior among female students will also be examined in future studies .
moreover , the protective behaviors were investigated in males ; therefore , to compare smoking in both sexes , other studies on females are also required .
it is suggested that smoking behavior among female students will also be examined in future studies .
the findings of this study show that the prevalence of smoking in the studied sample is somewhat lower than other regions of iran , but it should be noted that if no interventions are done to prevent smoking in this age group , it is possible that nonsmokers start smoking influenced by their friends in the future .
as mentioned before , mean age of participants was 16/2 0.25 years , considering that authorities can make change in policies of cigarette selling only for over 18 years .
the findings of the study also show that the structure of attitudes , self - efficacy , and cues to action are the strongest predictors of smoking among students .
therefore , the educational programs designed to promote understanding of this health problem and prevent smoking should pay more attention to these variables . | background : smoking is one of the most important risk factors for health and also health problems , such as heart diseases , especially for young people .
this study aimed to investigate the effect of factors related to smoking among the secondary high school students in the city of marivan ( kurdistan - iran ) , in 2015 , based on the constructs of health belief model ( hbm).methods : this cross - sectional study was conducted in 470 secondary high school students in marivan in 2015 .
the samples were selected by random cluster sampling . a question with four sections
was used to collect data ( demographic questions , knowledge section , attitude section , and questions related to e constructs of hbm).results : according to the results , the correlation of smoking was stronger with attitude ( r = 0.269 and odds ratio = 0.89 ) but weaker with perceived barriers ( r = 0.101 ) .
there was not a significant correlation between smoking behavior and knowledge of the harms of smoking ( r = 0.005 ) .
moreover , cues to action was effective predictor of smoking behavior ( r = 0.259).conclusions : the findings of this study show that the prevalence of smoking in the studied sample is somewhat lower than other regions of iran , but it should be noted that if no interventions are done to prevent smoking in this age group .
the findings of the study also showed that the structure of attitudes , self - efficacy , and cues to action are the strongest predictors of smoking among students .
albeit , attitude was strongest predictor of smoking that shows the prevalence of smoking can be reduced by focusing in this part . considering the mean age of participants ( 16/2 0.25 years ) , that shows
the riskiest period for smoking is 16 years and authorities can make change in policies of cigarette selling only for over 18 years . | Introduction
Methods
Study design
Data collection instrument
Statistical analysis
Results
Discussion
Limitations
Conclusions
Financial support and sponsorship
Conflicts of interest |
kinked silicon nanowires with nanofets encoded near the kink were synthesized using gold nanocluster catalyzed vapor - liquid - solid growth method as previously described .
probe fabrication was carried out on si substrates ( nova electronic materials , flower mound , tx ) with 600 nm sio2 coated with 100 nm ni , which serves as relief layer , and pre - baked ( 65 c for 2 min ) su8 2000.5 photoresist . kinked nanowires were deposited on desired region from ethanol dispersion using a micropipette , and then ebl was used to define nanowire end of the probe and pl used to pattern the remainder of the 4.5 mm long su8 probe body , which also serves as lower passivation of metal interconnects .
subsequently , a combination of ebl and pl steps were used to define the metal connections scaling from the kinked nanowire arms to the millimeter - scale end of the probe , where cr / pd / cr ( 1.5/120/60 nm ) was used for contacts to the nanowire , and cr / au ( 5/200 nm ) for the rest of the interconnections .
top su8 passivation / structural layers were defined using a 500 nm thick su8 2000.5 for the tip region covering the metal contacts , and a 50 m thick low - stress su8 layer ( glm 2060 , gersteltec ) for the majority of the probe body , which can increase the overall mechanical strength .
the kinked nanowire end of the probe was protected with 300 and 500 nm thick layers of lor 3a and s1805 ( microchem ) defined by pl .
after etching the ni layer ( 40% fecl3:39% hcl : h2o=1:1:20 ) , the floating probe structure was removed using tweezers and glued on the printed circuit board connector portion of the probe body that is mounted in the 3d manipulator
. the protective cap is removed immediately prior to measurements following uv exposure , and measurements were made in similar manner to previous chip - based device studies .
additional fabrication details , as well as cell culture and measurement protocols are described in the supplementary information . | recording intracellular bioelectrical signals is central to understanding the fundamental behaviour of cells and cell - networks in , for example , neural and cardiac systems14 .
the standard tool for intracellular recording , the patch - clamp micropipette5 is widely applied , yet remains limited in terms of reducing the tip size , the ability to reuse the pipette5 , and ion exchange with the cytoplasm6 .
recent efforts have been directed towards developing new chip - based tools14,713 , including micro - to - nanoscale metal pillars79 , transistor - based kinked nanowire10,11 and nanotube devices12,13 .
these nanoscale tools are interesting with respect to chip - based multiplexing , but , to date , preclude targeted recording from specific cell regions and/or subcellular structures . here
we overcome this limitation in a general manner by fabricating free - standing probes where a kinked silicon nanowire with encoded field - effect transistor detector serves as the tip end .
these probes can be manipulated in three dimensions ( 3d ) within a standard microscope to target specific cells / cell regions , and record stable full - amplitude intracellular action potentials from different targeted cells without the need to clean or change the tip .
simultaneous measurements from the same cell made with free - standing nanowire and patch - clamp probes show that the same action potential amplitude and temporal properties are recorded without corrections to the raw nanowire signal .
in addition , we demonstrate real - time monitoring of changes in the action potential as different ion - channel blockers are applied to cells , and multiplexed recording from cells by independent manipulation of two free - standing nanowire probes . | Methods Summary
Supplementary Material |
trichomoniasis , caused by parasitic protozoan trichomonas vaginalis , is the most common non - viral sexually transmitted infection ( sti ) .
diagnosis of t. vaginalis infection based solely on clinical symptoms and signs is unreliable because the spectrum of infection is broad and other sexually transmitted pathogens cause similar signs and symptoms .
therefore , the laboratory plays a key role in the diagnosis of this infection ; and more so , it is compelling to identify t. vaginalis infection reliably in resource poor settings with the readily available tests .
our study was undertaken to study the frequency of t. vaginalis infection in women presenting with vaginal discharge , to characterize the clinical features , and to study the sensitivity and specificity of microbiological investigations in the diagnosis of the same .
this was a hospital - based descriptive study done on 400 female patients with vaginal discharge attending the gynecology out - patient department ( opd ) of jipmer , puducherry , from may 2010 to july 2011 .
the institute ethics committee clearance was obtained and informed consent was taken from the recruited women .
women of age between 20 years and 50 years presenting with vaginal discharge , irrespective of marital status , were included , and detailed history was elicited and thorough examination was performed . women who had used antibiotics or vaginal medication in the previous 14 days and pregnant women were excluded from the study . the vaginal ph was measured and a sterile cotton swab was used to collect five samples of vaginal discharge from the posterior fornix under direct vision . in virgins , the specimen was obtained from the introitus .
the papanicolaou smears ( pap smears ) were taken and a bimanual examination was done in all , except in virgins , to look for adnexal tenderness .
the first swab was subjected immediately to wet mount microscopy to observe for motile trichomonads under 100 and 400 magnifications . with the second sample , a 10% potassium hydroxide ( koh )
mount was prepared and whiff test was done and the same was examined for the presence of budding yeast cells under 100 and 400 magnifications .
the third sample was immediately inoculated directly and swirled into diamond medium ( himedia labs , mumbai , india ) for t. vaginalis culture .
the culture tubes with 5 ml of the broth were incubated in an anaerobic atmosphere at 35c .
the fourth sample was inoculated on to sabouraud 's dextrose agar medium for candidia culture .
the fifth sample was streaked onto a microscopic slide for gram staining to check for the presence of clue cells , yeast cells , and pus cells under 1000 magnification .
women with high - risk sexual behavior were counseled and tested for hiv using enzyme - linked immunosorbent assay ( elisa ) .
a diagnosis of trichomoniasis was made based if the wet mount showed pear - shaped organisms with characteristic jerky movements or if the pap smear showed blue or grey pear - shaped organisms with bright - red granules . from the culture ,
diagnosis was made by performing wet mounts for evidence of motile trichomonads by examining cultures after 24 h of incubation and then daily up to 7 days .
bacterial vaginosis was diagnosed based on amsel 's criteria , and a diagnosis of candidiasis was made based on positive microscopy and/or culture .
a diagnosis of pelvic inflammatory disease was made if , in addition to the presenting symptoms of abnormal vaginal discharge and lower abdominal pain , adnexal tenderness was elicited on examination .
the data collected was tabulated in microsoft excel worksheet and computer - based analysis was performed using the spss 13.0 software ( spss , chicago , il , usa ) . for comparison of means ,
unpaired t - test and one - way analysis of variance ( anova ) were used for two and more groups , respectively .
for comparison of proportions , chi - square test was used . in cases where any one of cell value was < 5 ; fisher 's exact test was used .
among the 400 women enrolled in our study , 27 ( 6.75% ) had infection with t. vaginalis , identified by culture technique , the reference standard , the mean age being 35.37 7.66 years .
eighteen ( 66.6% ) were positive for t. vaginalis on screening with the wet mount of vaginal discharge ; 19 of 26 ( 73.1% ) available papanicolaou smear had evidence of trophozoites of trichomonas .
table 1 shows the sensitivity , specificity , and positive and negative predictive values of the abovementioned tests and the ancillary tests in the diagnosis of t. vaginalis infection .
of the 27 women with trichomoniasis , 12 ( 44.4% ) had trichomoniasis alone and the other 15 ( 55.5% ) had trichomoniasis with concurrent infections .
bacterial vaginosis was the most common concurrent infection with trichomoniasis with an odds ratio of 1.864 ( 95% confidence interval : 0.8453 - 4.112 ) .
the demographic and risk behavior characteristics of patients with trichomoniasis are summarized in table 2 .
the sensitivity , specificity , and positive predictive and negative predictive values of the various symptoms and signs are presented in table 3 .
performance of diagnostics tests for trichomoniasis ( individual tests and combinations ) demographic and risk behavior characteristics of patients with trichomonas vaginalis infection as compared to those without infection diagnostic value of clinical features associated with trichomonas vaginalis infection ( n=27 )
t. vaginalis , a pathogenic protozoan of urogenital tract causes vaginitis in women , although a considerable proportion of infections are asymptomatic . prevalence estimates of t. vaginalis infection varies considerably in female population around the world , depending on the population studied and techniques employed in diagnosis , ranging from 5% to 74% . our study , which recruited women from the gynecology opd revealed that 27 out of the 400 women ( 6.75% ) with vaginal discharge had infection with t. vaginalis by culture . amongst the south - asian studies ,
the rate observed in our study was comparable with the population - based study on community prevalence of stis and hiv infections in rural south india done by thomas et al . , which reported a prevalence of 5.25% for trichomoniasis by direct wet mount of vaginal swabs . among mixed infections occurring with t. vaginalis ,
, probably because t. vaginalis alters the vaginal ecology and facilitates the development of bacterial vaginosis or women with bacterial vaginosis have lost the natural protection against genital tract infection , leading to the acquisition of stis like t. vaginalis infection . unlike chlamydia and gonorrhea , the prevalence of t. vaginalis has been shown to increase with age .
the increased prevalence of trichomonal infection in older women suggests longer duration of infectiousness and the predominant asymptomatic nature of infection . this was evident from our study as the mean age at presentation for women with t. vaginalis infection was 35.37 years
, who reported a mean age of 33 years . infection with t. vaginalis can be a marker for high - risk behavior , and the predominant asymptomatic nature of the infection underscores the need for identification of risk factors associated with the infection .
significant risk factors associated with t. vaginalis infection in our study were a history of pre / extramarital sexual contact in the women ( or = 8.250 , 95% ci 2.594 - 26.234 ) , husband having extramarital contact ( or = 5.916 , 95% ci 2.559 - 13.679 ) , a history of symptomatic partner in the form of dysuria and urethral discharge suggestive of trichomoniasis in men ( or = 6.899 , 95% ci 1.974 - 24.110 ) , and alcohol consumption by women ( or = 106.71 , 95% ci 5.356 - 2126.3 ) .
kaur et al . , in their study in north india to assess the prevalence of t. vaginalis infection in symptomatic women as well as in women with carcinoma cervix and hiv infection , observed that being a housewife , belonging to the middle socioeconomic status , and non - use of contraception were significantly associated with trichomoniasis .
the signs and symptoms associated with the infection have a relatively low - positive predictive values for trichomoniasis because of the frequent occurrence of these signs and symptoms among women with other infections .
, in their study on women attending a sti clinic in georgia demonstrated that , if the clinical features alone were used to diagnose trichomoniasis , 88% of the infected women would be missed and 29% would be falsely indicated as having infection . in our study on women presenting with vaginal discharge ,
the reported symptoms had low - positive predictive values for trichomoniasis . among the clinical signs in women with vaginal discharge , having a profuse vaginal discharge ( or = 4.426 , 95% ci 1.994 - 9.823 )
, malodorous discharge ( or = 3.554 , 95% ci 1.507 - 8.381 ) , frothy discharge ( or = 273.29 , 95% ci 15.071 - 4955.7 ) , and mucopurulent or purulent discharge ( or = 27.013 , 95% ci 6.292 - 115.98 ) were significantly associated with trichomoniasis .
frothy discharge was observed in only 25.9% women in our study , but this was the most specific sign with a positive predictive value of 100% .
colpitis macularis was not observed in any of the women by naked eye examination in our study as colposcopy was not done on a routine basis .
women with t. vaginalis infection have a significantly higher risk of pelvic inflammatory disease ( pid ) than women without trichomoniasis .
adnexal tenderness , as an evidence of pid was present in 40.7% patients , which was significantly higher than that seen in women without trichomoniasis .
the sensitivity and specificity of microscopic and cytologic examination were assessed considering culture of t. vaginalis as the reference standard .
whiff test and ph testing are simple tests recommended in the evaluation of vaginal discharge and can be used as screening tools .
ph testing was reported to be a highly sensitive , but less specific screening tool for diagnosis of trichomoniasis .
a similar finding was observed in our study , where ph of > 4.5 had 100% sensitivity , but a low specificity of 41.3% in the diagnosis of trichomoniasis .
whiff test was observed to have low sensitivity ( 51.8% ) and low specificity ( 78.8% ) in predicting t. vaginalis infection in our study .
women with trichomoniasis have a significantly higher concentration of white blood cells in the vaginal wet mount than those without trichomoniasis . in our study , 55.6% cases with trichomoniasis had pus cells in gram - stained smear of vaginal discharge .
in our study , we attempted to increase the specificity of these simple screening tests in the diagnosis of trichomoniasis by studying the performances of the combinations of these tests [ table 1 ] .
on combining positive whiff test with ph > 4.5 and including cases that were positive for both tests , the specificity was increased by two folds ( 78.8% ) .
similar finding was observed on combining pus cells in gram - stained smear with ph > 4.5 . when all three ( ph > 4.5 , whiff test , pus cells in gram - stained smear ) tests were combined , the specificity ( 97.3% ) approached to that of our reference standard diagnostic test .
this offers an advantage in settings where culture is not available and where the patient may be lost to follow - up due to it being time - consuming .
the diagnosis of trichomoniasis in women is most commonly made by wet mount examination ; the papanicolaou smear represents a common , readily available cytological technique for primary screening of trichomoniasis . in our study ,
the sensitivity of wet mount and papanicolaou smear was observed to be 66.7% and 76% , respectively ; both being 100% specific with 100% positive predictive value in the diagnosis of t. vaginalis infection .
thus , culture of direct wet mount positive vaginal specimens may be unnecessary owing to the 100% specificity of the test . since our study was restricted to women with symptomatic t. vaginalis infection
, it is probable that these women harbored greater numbers of organisms and thus had a higher rate of positivity for wet mount . on combining wet mount with papanicolaou smear ,
the sensitivity increased to 92.6% ; this has an implication in resource - limited setting where culture may not be available , combining papanicolaou smear with wet mount would help identify few more cases of trichomoniasis that would have been missed by wet mount alone .
algorithm for diagnosis of trichomoniasis derived from our study to conclude , in our study , a moderate prevalence of t. vaginalis infection was observed , though the prevalence in the community might be higher as we had included only symptomatic women when majority of the infection is asymptomatic .
the poor clinical value of symptoms and signs in the diagnosis of t. vaginalis infection could be corrected by appropriate combination of simple tests that increases the performance of these screening tests comparable to that of the diagnostic tests , especially , in the setting of non - availability of culture for t. vaginalis . | background : diagnosis of trichomonas vaginalis vaginalis infection based solely on clinical symptoms and signs is unreliable because the spectrum of infection is broad and other sexually transmitted pathogens cause similar signs and symptoms.aims:our study was undertaken to study the frequency of t. vaginalis infection in women presenting with vaginal discharge , to characterize the clinical features , and to study the sensitivity and specificity of microbiological investigations in the diagnosis of the same.materials and methods : this was a hospital - based descriptive study done on 400 female patients with vaginal discharge attending the gynecology out - patient department ( opd ) of jipmer , puducherry , from may 2010 to july 2011 .
women of age between 20 years and 50 years presenting with vaginal discharge irrespective of marital status , were included , and detailed history was elicited and thorough examination was performed.results:in 400 women presenting with vaginal discharge from gynecology out - patient department ( opd ) included in the study , t. vaginalis infection was found in 27 ( 6.75% ) women .
the risk factors for trichomoniasis included history of pre- or extramarital sexual contact in the woman or her partner , symptomatic partner , and alcohol consumption . a positive association with pelvic inflammatory disease was also observed .
the most frequent symptoms included lower abdominal pain , dysuria , and dyspareunia .
combining of whiff test , ph > 4.5 , and pus cells in gram - stained smear , the specificity in diagnosing the infection ( 97.3% ) approached that of the reference standard , i.e. , culture . on combining wet mount with papanicolaou smear ,
the sensitivity increased to 92.6% , which was higher than that individually done.conclusion:to conclude , diagnosis of t. vaginalis infection based solely on clinical symptoms and signs is unreliable , and combination of simple laboratory tests increases the diagnostic performance close to the reference standard ( culture ) , especially in resource poor settings . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION |
interocclusal recording materials are used to transfer the interocclusal relationship from patient mouth to the lab .
these are impression plaster , compound wax , resin , metal oxide paste , rubber base , and silicone materials .
any inaccuracy in the interocclusal record leads to occlusal errors in the final prosthesis . to minimize the necessity of occlusal adjustments ,
failure to capture an accuracy lead to a time - consuming chair - side adjustments , the need for remounting casts and possible refabrication of prosthesis .
bite registration record acts as a significant source for cross - contamination ; so the american dental association ( ada ) issued guidelines for disinfecting impressions in 1988 , 1991 , and 1996 .
there are two important factors to consider when choosing a disinfectant namely , its ability to eliminate microbial contamination and its effect on the resultant material .
the literature varies markedly in the concentration , type and immersion time of disinfection protocols , making it difficult to assess the appropriate method .
there is also very little guidance provided by the manufacturers as to the most suitable disinfectants to use with their products .
although numerous studies have been made on the accuracy and stability of impression , few studies on the dimensional stability of different interocclusal recording materials have been investigated but the effect of disinfection and its method on the dimensional accuracy of interocclusal recording materials have not been reported previously .
three commercially available dental interocclusal recording materials and three commonly available disinfectants were selected for this in vitro study [ tables 1a and b ] . a stainless steel die
was prepared in accordance with the american national standards institute / ada council on scientific affairs specification no . 19 for linear dimensional changes that consisted of ruled block ,
the ruled block contained three horizontal lines x , y , z ( 50 8 , 20 4 , 75 8 m ) perpendicular to two vertical lines cd and cd of 75 8 m each . the lines cd and cd were separated from each other by 25 mm .
the test mold was a cylinder with an inner and outer diameter of 30 and 38 mm .
the riser was a stainless steel disk of diameter 29.9 mm and thickness of 3 mm [ figure 2 ] to retrieve specimen .
test block and ring mold were placed in an incubator ( set at 35 1c ) for conditioning for 15 min before manipulation of specimens .
interocclusal recording materials various disinfecting agents tested american national standards institute / american dental association no .
19 specified stainless steel die systematic diagram of stainless steel ( transverse and front view ) the cartridge along with mixing tip was attached to an auto - mixing gun .
the material was spread and pushed ahead of the syringe tip in a zigzag pattern with the tip buried in the material . a glass plate ( 4 4 inches square ) covered with polyethylene sheet was placed on the die over which a weight of 1 kg was kept and allowed to set for 45 min in a thermostatically controlled water bath to stimulate mouth condition [ figure 3 ] .
manipulation of polyvinylsiloxane ( a ) jetbite ( addition silicone ) supplied in the form of catridge with automixing gun and tips .
( c and d ) glass plate with sheet placed and 1 kg weight kept over die for the aluwax [ figure 4 ] , the method was modified by submerging it in a 45c water bath for 5 min using 5 ml glass syringe .
this was carried out by breaking the wax and putting it into the syringe before melting .
( d ) 1 kg weight kept over the die the required amount of equal length of pastes were dispensed on the mixing pad and mixed for 45 s to get homogenous streak free mix .
the mix was injected into the mold and spread same as other material [ figure 5 ] .
( d ) specimen separated from the mold by using riser their testing were conducted in controlled laboratory conditions .
these conditions were entailed the temperature 24 2c and relative humidity of 55 10% .
specimens [ figure 6 ] were grouped as : group a ( polyvinylsiloxane ) , group b ( aluwax ) , and group c ( polyether ) . these were subjected to the following disinfecting treatments based on ada recommendations 0.5% chlorhexidine gluconate ( subgroup i ) , 1% sodium hypochlorite ( subgroup ii ) , and 2% glutaraldehyde ( subgroup iii ) solutions that were used by two techniques ; immersion and spray atomization .
60 specimens from group a , b , and c were immersed in 0.5% chlorhexidine ( subgroup i ) solutions for 30 min ( n = 10 , total = 30 ) and 60 min ( n = 10 , total = 30 ) separately at room temperature [ figure 7a ] . test samples . silicone ( yellow ) , aluwax ( grayish green ) and polyether ( light yellow ) ( a ) test samples immersed in disinfected solution .
( b ) test samples were sprayed to wet all the surfaces sixty specimens from group a , b , and c were immersed in 1% sodium hypochlorite ( subgroup ii ) solutions for 30 min ( n = 10 , total = 30 ) and 60 min ( n = 10 , total = 30 ) separately at room temperature .
sixty specimens from group a , b , and c were immersed in 2% glutaraldehyde ( subgroup iii ) solutions for 30 min ( n = 10 , total = 30 ) and 60 min ( n = 10 , total = 30 ) separately at room temperature . specimens ( n = 10 , total = 180 )
were sprayed same as mentioned above ( spray atomization technique ) with all disinfectants until the complete surface of the specimens became wetted and sealed in a plastic bag for 30 and 60 min separately [ figure 7b ] .
it consisted of plain orifice nozzle with finger sprayer to propel contents from the container with no external source of compressed air being used .
specimens ( n = 10 , total = 60 ) from each group were treated with distilled water ( control ) for 30 and 60 min separately .
distribution of sample specimen for each material : jet bite ( group a ) , aluwax ( group b ) and ramitec ( group c ) in three disinfectants : 0.5% chlorhexidine gluconate ( subgroup i ) , 1% sodium hypochlorite ( subgroup ii ) , 2% glutaraldehyde ( subgroup iii ) and control are after disinfection , both control and test specimens were rinsed again under tap water for 10 s. this was done to stimulate rinsing the impression after removal from the oral cavity and also after removal from the disinfectant solution .
measurements of the specimens were taken after 24 1 h. all the measurements were performed by the same operator .
the distance between the cross lines cd and cd reproduced in the samples were measured at the intersection of these lines with the xyz lines by measuring microscope ( stm 6-lm , olympus ) [ figure 8 ] with 10 magnification ( 0.0001 mm submicron precision ) .
specimen observed under measuring microscope the test block was measured five times to produce a mean value of 24.9150 mm . according to iso 4823
, the following equation was used to calculate the mean percentage of change for each specimen .
the data obtained were analyzed with two - way anova followed by post - hoc tukey test ( = 0.05 ) .
in addition , a qualitative analysis was done using scanning electron microscope ( hitachi s-3700n sem , unified engineering inc . ,
the cartridge along with mixing tip was attached to an auto - mixing gun . the material was spread and pushed ahead of the syringe tip in a zigzag pattern with the tip buried in the material .
a glass plate ( 4 4 inches square ) covered with polyethylene sheet was placed on the die over which a weight of 1 kg was kept and allowed to set for 45 min in a thermostatically controlled water bath to stimulate mouth condition [ figure 3 ] .
manipulation of polyvinylsiloxane ( a ) jetbite ( addition silicone ) supplied in the form of catridge with automixing gun and tips .
( c and d ) glass plate with sheet placed and 1 kg weight kept over die
for the aluwax [ figure 4 ] , the method was modified by submerging it in a 45c water bath for 5 min using 5 ml glass syringe .
this was carried out by breaking the wax and putting it into the syringe before melting .
the required amount of equal length of pastes were dispensed on the mixing pad and mixed for 45 s to get homogenous streak free mix .
the mix was injected into the mold and spread same as other material [ figure 5 ] .
( a ) apparatus for mixing ramitec ( polyether ) material . ( b ) material spread over the surface of the die . ( c )
these conditions were entailed the temperature 24 2c and relative humidity of 55 10% .
specimens [ figure 6 ] were grouped as : group a ( polyvinylsiloxane ) , group b ( aluwax ) , and group c ( polyether ) .
these were subjected to the following disinfecting treatments based on ada recommendations 0.5% chlorhexidine gluconate ( subgroup i ) , 1% sodium hypochlorite ( subgroup ii ) , and 2% glutaraldehyde ( subgroup iii ) solutions that were used by two techniques ; immersion and spray atomization .
60 specimens from group a , b , and c were immersed in 0.5% chlorhexidine ( subgroup i ) solutions for 30 min ( n = 10 , total = 30 ) and 60 min ( n = 10 , total = 30 ) separately at room temperature [ figure 7a ] .
silicone ( yellow ) , aluwax ( grayish green ) and polyether ( light yellow ) ( a ) test samples immersed in disinfected solution .
( b ) test samples were sprayed to wet all the surfaces sixty specimens from group a , b , and c were immersed in 1% sodium hypochlorite ( subgroup ii ) solutions for 30 min ( n = 10 , total = 30 ) and 60 min ( n = 10 , total = 30 ) separately at room temperature .
sixty specimens from group a , b , and c were immersed in 2% glutaraldehyde ( subgroup iii ) solutions for 30 min ( n = 10 , total = 30 ) and 60 min ( n = 10 , total = 30 ) separately at room temperature .
specimens ( n = 10 , total = 180 ) were sprayed same as mentioned above ( spray atomization technique ) with all disinfectants until the complete surface of the specimens became wetted and sealed in a plastic bag for 30 and 60 min separately [ figure 7b ] .
it consisted of plain orifice nozzle with finger sprayer to propel contents from the container with no external source of compressed air being used .
specimens ( n = 10 , total = 60 ) from each group were treated with distilled water ( control ) for 30 and 60 min separately .
distribution of sample specimen for each material : jet bite ( group a ) , aluwax ( group b ) and ramitec ( group c ) in three disinfectants : 0.5% chlorhexidine gluconate ( subgroup i ) , 1% sodium hypochlorite ( subgroup ii ) , 2% glutaraldehyde ( subgroup iii ) and control are after disinfection , both control and test specimens were rinsed again under tap water for 10 s. this was done to stimulate rinsing the impression after removal from the oral cavity and also after removal from the disinfectant solution .
measurements of the specimens were taken after 24 1 h. all the measurements were performed by the same operator .
the distance between the cross lines cd and cd reproduced in the samples were measured at the intersection of these lines with the xyz lines by measuring microscope ( stm 6-lm , olympus ) [ figure 8 ] with 10 magnification ( 0.0001 mm submicron precision ) .
specimen observed under measuring microscope the test block was measured five times to produce a mean value of 24.9150 mm . according to iso 4823 ,
the following equation was used to calculate the mean percentage of change for each specimen .
the data obtained were analyzed with two - way anova followed by post - hoc tukey test ( = 0.05 ) .
in addition , a qualitative analysis was done using scanning electron microscope ( hitachi s-3700n sem , unified engineering inc . ,
comparison of 3 different groups by immersion technique for 30 min showed no statistically significant difference by anova .
there was a statistically significant difference in the mean values between the group and the subgroups for each group when immersed for 60 min [ table 3 ] .
post - hoc result showed that there was a significant interaction between subgroups of different groups . within group c ( polyether ) , there was a statistically significant difference between subgroups and control [ table 4 ] .
subgroups for each material group showed no results when sprayed for 30 and 60 min .
immersion and spray techniques were compared for 30 and 60 min by t - test , which showed no statistically significant difference except for the sodium hypochlorite in group c for 60 min [ table 5 ] .
comparison of three material groups ( group a , group b , and group c ) and subgroups by immersion technique for 60 min with dimensional change by two - way anova procedure pairwise comparison of subgroups by immersion technique for 60 min for dimensional changes by tukey 's post - hoc procedure comparison of immersion and spray techniques with dimensional changes in all disinfectants of three materials at 60 min by t - test figure 9 showed statistically significant difference between 30 and 60 min ( p = 0.05 ) by immersion technique for each group , while spray technique did not showed any significant interaction when time period was increased except for group b. addition silicone showed [ figure 10 ] the least dimensional change which ranged from 0.024% to 0.050% .
specimens were gold coated for 90 s with a plasma current of 1820 ma . at low magnification ,
all disinfected and nondisinfected samples were able to produce the full length of 20 m wide line . at 600 , trough and
ridge was appreciable for polyether and silicone with clean demarcation while aluwax showed poor demarcation [ figure 11a c ] .
further magnification at 2.5 [ figure 12 ] , 60 min immersed polyether samples showed the micro - pits and voids which clearly demarcates the interferences in the polymerization reaction .
comparison of 30 and 60 min of immersion and spray techniques in three material groups comparison of immersion and spray techniques with dimensional changes of three materials in disinfectants at 60 min .
asterisk indicates significant difference , p < 0.05 ( a ) disinfected specimen of addition silicone at 600 .
( c ) disinfected specimens of polyether at 600 polyether specimen treated with 1% sodium hypochlorite solution by immersion technique at 2.50 kx
according to this study , all the interocclusal record materials showed variations when disinfected by immersion so null hypothesis were rejected .
based on spray technique for 30 and 60 min , subgroups for each material group showed no results , which is consistent with results of studies by oderinu et al .
chlorhexidine is a cationic bis - guanide which consists of two symmetric 4-chlorophenyl rings and two biguanide groups connected by central hexamethylene chain .
its efficacy is due to the interaction of positive charge of the molecule and negatively charged phosphate groups on the microbial cell walls .
glutaraldehyde solution acts by fixating cell membranes , blocking the release of cellular components and consequently killing the micro - organisms .
the performance of sodium hypochlorite is based on cell oxidation . a study by silva and salvador frederick et al . and drennon et al .
showed that disinfection of impression by spray appeared to be as efficacious as immersion and unlike the immersion method , it does not cause any dimensional changes . in this study ,
polyether showed varied dimension with sodium hypochlorite when immersed for 60 min . since sodium hypochlorite is both an oxidizing and hydrolyzing agent .
they are strongly alkaline , hypertonic , and typically have normal concentrations of 1014% available chlorine .
they deteriorate with time , exposure to light , temperature , and contamination with metallic ions .
the dimensional change might be due to reaction of chlorine compound with sulfonic ether which interferes with the polymerization reaction and produces distortion .
addition silicone showed the least dimensional variations among all , but showed significant difference in dimensional change with time when disinfected for longer period ( 60 min ) in disinfectant solutions .
it might be due to the addition of surfactants to improve its ability to reproduce details .
the presence of these agents improves the compatibility with water and increases the sorption of water when impressions are immersed for longer period .
aluwax has gained wide acceptance for interocclusal record transfer , however studies showed that waxes contain aluminum or copper particles which have flow rate of 2.522% at 37.5c so that they are susceptible to distortion upon removal from the mouth .
variation in the dimension was might be attributed to the greater coefficient of thermal expansion and distortion due to stress release .
the total percentage dimensional changes that occurred during the disinfection process ranged from 0.03% to 0.30% . to put the current result in context
, the ada specifies that impression materials should not exhibit more than 0.5% dimensional change in the first 24 h and all the materials investigated in this study satisfied the criteria .
future research should make an attempt to measure the linear measurements directly on the impressions to see if there are any errors that could be introduced during articulation of the casts .
within the limitation of the study , the dimensional change of addition silicone , aluwax and polyether after disinfection in 0.5% chlorhexidine gluconate , 1% sodium hypochlorite and 2% glutaraldehyde for 30 or 60 min is clinically acceptable .
the tested specimens gave the results which are within the limits of ada specification no . 19 .
for 60 min immersion disinfection time period , sodium hypochlorite had significantly affected polyether ( p = 0.0005 ) .
therefore , spray atomization technique can be recommended for polyether disinfected with sodium hypochlorite , to preserve dimensional stability , whereas both spray or immersion technique can be safely used with the other two disinfectants for addition silicone and aluwax .
in addition , dimensional stability of interocclusal recording materials was found to decrease with the increase in immersion time period . for polyether interocclusal record , restrictions based on type , duration , and method of disinfection must be applied to preserve the accuracy of the impression and effective microbial elimination .
for polyether interocclusal record , restrictions based on type , duration , and method of disinfection must be applied to preserve the accuracy of the impression and effective microbial elimination . | objective : to evaluate and compare the effect of 0.5% chlorhexidine gluconate , 1% sodium hypochlorite , and 2% glutaraldehyde by immersion and spray atomization technique on the linear dimensional stability of jet bite , aluwax and ramitec interocclusal recording materials.materials and methods : three representative materials : jet bite ( addition silicone ) , aluwax and ramitec ( polyether ) were mixed according to manufacturer 's instructions and then specimens were prepared according to the specifications of iso 4823 .
all the specimens except the control ( distilled water ) were treated with disinfectant solutions ( 0.5% chlorhexidine gluconate , 1% sodium hypochlorite , and 2% glutaraldehyde ) for 30 and 60 min ( n = 10 ) by spray and immersion technique .
once removed from the solutions , the test samples were washed in water for 15 s , dried and measured after 24 h 3 times using a measuring microscope with an accuracy of 0.0001 mm .
two - way anova and tukey 's test with significance level of 5% were used to assess the statistical data ( = 0.05).result : all groups showed no significant difference statistically , in linear dimension when disinfected for 30 min by spray or immersion technique .
polyether had significantly higher dimensional variation when immersed in sodium hypochlorite for 60 min .
addition silicone showed the least dimensional change which ranged from 0.024% to 0.05% , followed by polyether from 0.004% to 0.171% and aluwax from 0.146% to 0.228%.conclusion : to preserve the dimensions and surface of the recording materials and effective microbial elimination , restrictions should be applied in the method of disinfection and time duration . however , using the disinfectants either by spray or immersion technique , the dimensional change was < 0.5% which was not clinically significant according to the american dental association specification no .
19 criteria within the first 24 h. | INTRODUCTION
MATERIALS AND METHODS
Manipulation of polyvinylsiloxane bite registration material
Manipulation of Aluwax bite registration material
Manipulation of polyether bite material
Preparation of samples
RESULTS
DISCUSSION
CONCLUSION
Clinical implications
Financial support and sponsorship
Conflicts of interest |
approximately one third of patients with renal cell carcinoma ( rcc ) have metastatic disease at the time of initial diagnosis ( synchronous metastatic disease ) , and 40% to 50% will develop distant metastases after the initial diagnosis .
synchronous metastasis of rcc to the ureter or the bladder , however , is extremely rare .
a 73-year - old woman complained of gross hematuria for the past 1 week . her medical history and family history were negative for gross hematuria .
she underwent a cystoscopy that revealed a sessile mass on the right lateral bladder wall ( fig .
1 ) . abdominopelvic computerized tomography showed a 0.9 cm sized enhancing lesion on the right lateral bladder wall .
a 6.45.3 cm sized heterogenous enhancing mass in the lower pole of the left kidney with left renal vein thrombosis , multiple small enhancing nodules in the pancreas parenchyma , and nodular thickening of both adrenal glands were also found ( fig .
chest computed tomography ( ct ) showed hematogenous lung metastasis and left mediastinal and hilar lymph node metastasis .
the mass was found to be sessile , 1.01.0 cm in size , and with indistinct demarcation .
the pathological analysis of the resected biopsy specimen revealed metastatic rcc of the clear cell type ( fig .
3 ) . the patient underwent target therapy with sorafenib 200 mg owing to the multiple metastatic sites .
five weeks after the target therapy , she fell down in the bathroom , sustained an intracranial hemorrhage , and died of accompanying hyponatremia and aspiration pneumonia .
metastatic bladder cancers usually come from gastric adenocarcinoma , melanoma , and adenocarcinoma of the breast and colon .
rcc is an uncommon source of bladder metastases , with fewer than 40 such reported cases .
frequent metastatic sites of rcc are the regional lymph nodes , lung , liver , bone , adrenal gland , brain , and skin . reported
metastatic sites in the genitourinary tract include the ipsilateral ureter , contralateral ureter , ureteric stump , bladder , and prostatic fossa .
patients with rcc metastatic to the bladder typically present with gross hematuria . in the majority of cases , there is a well - established history of rcc .
however , infrequently , the primary renal tumor may present initially as a bleeding bladder lesion . in this case , the renal tumor was found during the staging work - up of the bladder cancer .
bladder lesions are generally sessile , spherical protuberances into the bladder lumen . in this case , the bladder tumor was sessile . histological evaluation of the resected tissue is usually consistent with metastatic rcc . in this case , the pathological analysis of the resected biopsy specimen revealed metastatic rcc of the clear cell type .
the mechanisms underlying the spread of rcc to the bladder remain a matter of debate .
several possible mechanisms have been proposed , including hematogenous metastasis through the general circulation , retrograde spread of the tumor from the renal vein or renal hilar lymphatics down the periureteral veins or lymphatics that connect with pelvic organs , and direct intraluminal transit of tumor cells with seeding of the distal urothelium [ 1 - 6 ] .
rcc commonly metastasizes through the bloodstream , leading to the synchronous discovery of a widespread area of metastasis . in this case , the multiple metastatic sites , including the regional lymph nodes , lung , bladder , adrenal gland , and pancreas , lend support to hematogenous metastasis through the general circulation .
treatment options for rcc , particularly when metastatic , are limited owing to poor treatment response to chemotherapy and radiation therapy .
conventional surgical treatment remains controversial regarding the establishment of good criteria for its application and often results in poor treatment effects . before the development of target treatment agents ,
target therapy is now accepted as the standard therapy for metastatic rcc because of the superior effect of sunitinib and sorafenib , which target vascular endothelial growth factor receptor and platelet - derived growth factor receptor . for cases in which rcc is metastatic at the time of diagnosis , as in this patient 's case
, the decision of whether to perform nephrectomy is debatable . for severe symptoms due to primary carcinoma
, palliative nephrectomy can be performed , and nephrectomy is performed with an expectation of spontaneous regression or to improve the treatment effect and survival rate by reducing the total volume of primary carcinoma when systemic treatment such as immune therapy or chemotherapy is given . in doing so , careful consideration should be given to the patient 's performance status , position and degree of remote metastasis , types and degree of differentiation of carcinoma cells , size of carcinoma , patient 's age , and gender .
for the treatment of metastatic lesions in the bladder , transurethral resection , partial cystectomy , and radical cystectomy have been used .
although genitourinary metastases are generally considered to have a poor prognosis , long - term survival is occasionally reported .
prognosis has been reported to be good when only a single metastasis exists in the bladder , and follow - up without additional systemic treatment is possible after the surgical removal of the metastatic lesion in the bladder .
in addition , when carcinoma has metastasized to other organs at the time when rcc metastasis is found in the bladder , additional systemic treatment such as immune therapy is required . in this case , considering the patient 's performance status and the degree of remote metastasis , we decided that nephrectomy was not indicated .
thus , transurethral resection of the bladder tumor was performed and metastatic bladder cancer was confirmed .
chest and abdominopelvic ct and a whole - body bone scan showed adrenal and lung metastasis .
the patient was treated with target therapy with sorafenib owing to the multiple metastatic sites .
however , she fell in the bathroom and suffered an intracranial hemorrhage that resulted in her death . | renal cell carcinoma is an uncommon source of bladder metastases . here
we report a case of renal cell carcinoma that metastasized to the bladder .
a 73-year - old woman complained of gross hematuria .
abdominopelvic computerized tomography showed a bladder mass and a heterogenous enhancing mass in the lower pole of the left kidney with left vein thrombosis .
the pathological analysis of the resected bladder specimen revealed metastatic renal cell carcinoma of the clear cell type . | INTRODUCTION
CASE REPORT
DISCUSSION |
moyamoya disease ( mmd ) is an infrequent disease that is characterized by progressive occlusive or stenotic lesions at the distal portions of internal carotid arteries and an aberrant vascular network at the base of the brain that resembles puffs of smoke on angiography .
mmd has captured increasing and intensive attention from neurosurgeons because it has been deemed to be a major cause of stroke in adults and particularly in children despite its relative low incidence .
although a large number of studies have been conducted , the actual etiology and pathogenesis remain extremely unclear .
however , in recent years , ring finger protein 213 ( rnf213 ) was identified as a susceptibility gene among east asian populations , which resulted in a shift of people 's attention to the relevant genetic factors .
rnf213 encodes a 596,000 protein that includes an alpha-2-macroglobulin , an aaa - type atpase and ring finger domains from its amino to carboxyl termini .
stated that the rnf213 c.14576g > a variant is detected in 95% of familial mmd cases and 79% of sporadic patients .
nevertheless , a portion of mmd patients do not carry the c.14576g > a variant and this portion is higher in western countries .
it is disappointing that we have been unable to determine whether mmd is caused by a synergy of genetic and environmental factors or some other unknown causes .
further research should be directed toward illuminating the cause of mmd and identifying a more effective therapeutic strategy .
the aim of this literature review is to help people comprehensively understand the research advances related to rnf213 in mmd patients .
because the rnf213 c.14576g > a variant is detected in 95% of familial mmd cases and 79% of sporadic patients , an increasing number of researchers have focused on mimicking mmd in mice via knock - in and knock - out technologies .
however , sonobe et al . did not discover any modification of angiogenesis after they generated mice lacking rnf213 .
soon after , this same team generated mice with the r4859k mutation of rnf213 and obtained results similar to those of the earlier study . to determine whether ischemia can result in cerebrovascular abnormalities in knockout mice ,
consequently , numerous researchers have insisted that mmd is primarily triggered by both genetic and environmental factors despite the ambiguous causes .
previous studies of environmental factors and the development of the mmd have emphasized the latent role of varicella zoster virus infection .
a study of mmd and inflammatory signals suggested that rnf213 is associated with the immune response .
in addition , two groups have recently demonstrated that interferon , which is invariably induced by inflammatory and immune responses , can stimulate the expression of rnf213 .
based on the two studies mentioned above , we have adequate reason to believe that the mmd is not triggered by a single genetic factor but rather is triggered by both environmental and genetic factors . another examination conducted by sato - maeda et al . indicated that the rnf213 gene is also expressed during transient middle cerebral artery occlusion , particularly in neurons , and this result provided new insight into the role of rnf213 in neuroprotection .
several studies have reported that rnf213 c.14576g > a variant carriers have reduced angiogenesis abilities , which contrast sharply with the pathologic characteristics of mmd .
however , one study suggested that transient middle cerebral artery occlusion can activate the expression of rnf213 .
these findings indicate the possible occurrence of a vicious cycle in which the expression of rnf213 aggravates ischemia , and ischemia induces the expression of rnf213 . further research is certainly indispensable to confirming this hypothesis .
induced pluripotent stem cells ( ipscs ) using the fibroblasts of mmd patients and healthy controls in an effort to detect the associated angiogenic activities and discovered that the proliferation abilities of the cells from the patients and carriers were reduced compared with those of the cells from the controls .
therefore , how the rnf213 c.14576g > a variant gives rise to reduced angiogenesis has become an important question . from our perspective , this reduced angiogenesis ability might be caused by a mitotic abnormality , the pattern described by hitomi et al . or through the regulation of the expression of matrix metalloproteinase 1 .
although we know of the critical connection between rnf213 and angiogenesis abnormalities , we do not possess sufficient evidence to interpret how reduced angiogenesis results in an aberrant vascular network at the base of the brain .
miyawaki et al . studied patients with non - mmd intracranial major artery stenosis / occlusion ( icaso ) and found that 9 of 41 patients ( 21.9% ) carried the c.14576g > a variant . to confirm their previous research , the following year
consistent with the results of the previous study , the c.14576g > a variant was found to be present in 20/84 patients in a non - mmd icaso group , which indicated that the c.14576g > a variant is significantly associated with non - mmd icaso .
analyzed 352 consecutive patients with relevant intracranial arterial stenosis and discovered that 176 of the 352 patients with intracranial arterial stenosis carried the c.14429g > a variant , which is also termed c.14576g > a .
in addition , the mutation genotypes of the rnf213 gene in an mmd population from taiwan ( china ) revealed that half of the carriers of the c.14576g > a variant had intracranial arterial stenosis .
liu et al . generated rnf213-knockdown zebrafish and discovered irregular wall formations in major arteries and abnormally sprouting vessels .
interestingly , some other vascular diseases , such as premature coronary artery disease and stroke , aortic coarctation , thoracic aortic aneurysm , and stenosis of other arteries , have also been reported to be associated with rnf213 variants .
in addition , two patients with co - occurring pulmonary hypertension and mmd were reported to have homozygous p.r4810k mutations in rnf213 .
interestingly , diabetes and blood pressure have also been found to be associated with rnf213 ; the ablation of rnf213 blocks the development of diabetes in mice , and the rnf213 c.14576g > a variant increases the risk of hypertension .
although the specific mechanisms remain unknown , we predict that rnf213 variants are indeed correlated with angiocardiopathy and cerebrovascular diseases .
based on the above research , we draw the following conclusions : ( 1 ) rnf213 is associated with non - mmd icaso and other cerebrovascular diseases , and ( 2 ) rnf213 is not associated with non - mmd icaso ; however , mmd has been misclassified as icaso due to the late onset and the absence of one or two of the diagnostic criteria .
these conclusions suggest that rnf213 genotype should be included in the diagnostic criteria for mmd because the treatment strategies for mmd and icaso are completely different .
if mmd is treated with strategies designed for icaso , the actual result may be the opposite of the intended result .
there is no doubt that rnf213 is a strong susceptibility gene for mmd among east asian people .
liu et al . reported that the minor allele frequencies of p.r4810k are 1.4% , 1.3% , and 1.0% among the general populations of japan , korea , and china , respectively , but this does not explain the relatively lower frequency of mmd among the chinese .
however , a subsequent large - scale screening for p.r4810k among east and southeast asians demonstrated that this contradiction was attributable to selection bias and suggested that carriers of the c.14576g > a ( p.r4810k ) variant are indeed less frequent among the chinese than the japanese and korean populations .
moreover , the c.14576g > a variant was detected in only 4 of 11 locations in china and was not detected in southeast asia , which indicates that environment factors might play a role in mmd .
an investigation of the frequency of the rnf213 c.14576g > a variant in two korean populations revealed that the estimated frequencies of the variant allele were 1.13% and 1.32% in cord blood samples and adult samples , respectively , which again confirms that the frequencies of the variant allele in japan and korea are higher than the frequency in china and that rnf213 variants exhibit ethnic diversity .
given that there is no large - scale research on the association between rnf213 and mmd in the chinese han population , wu et al .
analyzed 170 mmd cases and 507 controls and discovered that the c.14576g > a variant is related to mmd and that the frequencies of this variant allele are much lower among the chinese han population than the populations of japan and korea .
intriguingly , this group stated that male chinese patients are more likely to be adversely affected than females ( 1.3:1 ) , which contrasts with the opposite pattern in japan ( 1:18 ) .
in addition , the incidence of mmd in east asian is much higher than that in european countries , which could be partly be due to the ethnic diversity of rnf213 mutations .
one study reported that no p.r4859k carriers were detected among 400 caucasian controls and five caucasian mmd patients .
moreover , cecchi et al . also reported that the c.14576g > a variant was identified in 9/16 mmd patients of asian descent and in 0 of 94 patients of non - asian descent . in conclusion ,
the c.14576g > a variant is mainly detected in japanese , korean , and chinese populations .
however , the frequency in the latter population is much lower than those of the former two populations .
in addition , the rare rnf213 variants mainly exist in china and other non - asian countries ; however , a recent study argued that the japanese also carry some rare variants .
there are numerous differences in the clinical manifestations of mmd between young children and adults .
in addition , a portion of patients with mmd have severe symptoms and early - onset whereas some patients present with slight headache or are asymptomatic .
although a clear understanding of the pathogenesis of mmd has not yet been achieved , the heterogeneity of rnf213 , which has significant associations with mmd , provides a clue that indicates that the clinical manifestations of mmd may be associated with genetic background .
although two turkish siblings who were homozygous for the c.14576g > a rnf213 variant exhibited distinct clinical features and inoue et al . also reported a family case of mmd that involved different phenotypes among family members with the heterozygous c.14576g > a variant , these authors have been unable to demonstrate that there is no relation between the rnf213 genotypes and phenotypes .
a study conducted by miyatake et al . discovered that patients who were homozygous for the c.14576g > a variant of rnf213 presented with earlier onsets and more serious symptoms than mmd patients who carried were heterozygous for the c.14576g > a variant .
in addition , miyatake et al . reported sibling cases of mmd in which the homozygous c.14576g > a variant manifested with an early - onset and severe clinical manifestation whereas the heterozygous c.14576g > a variant manifested with a relatively late onset and mild symptoms . in accordance with the above results
, a recent study also confirmed that the homozygous c.14576g > a variant is associated with early - onset , severe symptoms , and an unfavorable prognosis .
in addition , according to this report , the rnf213 c.14576g > a variant mainly causes mmd that presents with ischemia whereas the p.a4399 t variant is primarily associated with hemorrhaging in mmd .
importantly , kobayashi et al . reported that rnf213 r4810k carriers have lower angiogenic capacities and are prone to cerebral hypoxia insults .
it has been reported that chinese heterozygous carriers of the p. r4810k variant are younger at diagnosis , have more familial cases , more ischemic cases , and more frequently exhibit involvement of the posterior cerebral artery .
further research with large - scale populations should be performed to prove the conclusions of these authors .
overall , strong evidence demonstrates that various genotypes can lead to distinct phenotypes of mmd .
this finding motivates us to reacquaint ourselves with the roles played by genetics in the mechanism of mmd and to routinely detect the genotypes of mmd patients to identify those who likely to experience early - onsets , severe symptoms , and bad prognoses because early diagnoses and interventions could prevent malignant outcomes of mmd .
the guidelines for the diagnosis of mmd created in 1997 clarify the diagnostic criteria for mmd as follows : ( 1 ) stenosis or occlusion of the distal internal carotid artery , ( 2 ) an aberrant vascular network , and ( 3 ) bilateral lesions .
the absence of any of these criteria excluded a patient from the spectrum of mmd .
for example , unilateral lesions can only be called unilateral moyamoya phenomena and not mmd . furthermore ,
if a patient meets all three of the above criteria and has other relevant basic diseases , such as hyperthyroidism , turner syndrome , meningitis , behcet disease , idiopathic pachymeningitis , and neurofibromatosis type 1 , the patient should be given a diagnosis of moyamoya syndrome or quasi - mmd but not mmd .
given that the srnf213 c.14576g > a variant has been identified as a susceptibility gene for mmd , miyawaki et al .
analyzed the genotypes of nine patients with quasi - mmd to clarify whether moyamoya syndrome , which characteristics similar to those of mmd , exhibits an identical etiology or genetic background .
these authors discovered that none of the patients with quasi - mmd had the rnf213 c.14576g > a variant whereas 66 of 78 patients with definite mmd had the variant .
these findings indicate that mmd and quasi - mmd may be two completely separate diseases .
although these conditions have similar imaging manifestation , their pathogeneses might be totally different because the latter is more similar to the complications of other associated basic diseases , whereas the former is mainly associated with the genetic background .
in addition , some individuals argue that the unilateral moyamoya phenomenon , especially combined with the rnf213 c.14576g > a variant , should be classified as mmd . mineharu et al
. also reported on a patient with the c.14576g > a variant who exhibited rapidly progressing unilateral mmd .
all of the above findings hint that the rnf213 c.14576g > a variant should be considered in the diagnosis of mmd .
the growing literature demonstrates that the mmd is mainly caused by the synergy of genetic and environmental factors .
we believe that an unknown genetic modifier might play a role in the etiology of mmd .
in addition , from our perspective , genotype should be considered in the diagnosis of mmd to enable the application of therapy through the relevant effective surgery as soon as possible and to prevent misdiagnoses .
certainly , we all anticipate an easier but effective therapeutic strategy to cure this disease as predicted by some authors .
this study was supported by the grants from the national science and technology supporting plan ( the 11 five - year plan ) ( no .
this study was supported by the grants from the national science and technology supporting plan ( the 11 five - year plan ) ( no . | objective : the aim of this study was to help people comprehensively understand the research advances related to ring finger protein 213 ( rnf213 ) in moyamoya disease ( mmd ) and to understand the disease at the molecular level to provide a new perspective of the diagnosis of the disease.data sources : this review was based on data in articles published between 2005 and 2015 that were retrieved from the pubmed database .
the search terms included rnf213 , mmd , intracranial major artery stenosis / occlusion ( icaso ) , genotype , phenotype , mutant and variants , and the combinations of these terms.study selection : articles related to mmd and rnf213 were selected for review , and we also reviewed publications related to icaso.results:rnf213 is not only associated with mmd but also associated with intracranial major artery stenosis .
in addition , rnf213 variants exhibit apparent ethnic diversity ; specifically , the c.14576g > a variant is mainly detected in korean , chinese , and japanese populations , particularly the latter population .
the genotypes of rnf213 correlate with the phenotypes of mmd ; for example , the homozygous c.14576g > a variant is associated with early - onset , severe symptoms , and an unfavorable prognosis .
furthermore , the rnf213 c.14576g > a variant should be considered during the diagnosis of mmd because no patients with quasi - mmd have been reported to carry the rnf213 c.14576g > a variant whereas 66 of 78 patients with definite mmd have been found to carry this variant.conclusions:the growing literature demonstrates that mmd is primarily caused by the synergy of genetic and environmental factors , and unknown genetic modifiers might play roles in the etiology of mmd .
further research should be conducted to clarify the pathogenic mechanism of mmd . | I
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Conflicts of interest |
a 48-year - old chinese female was admitted to department of oncological surgery ( zhejiang cancer hospital , hangzhou ) with left upper abdominal pain , accompanying with belching and weight loss of 2.5 kg over the previous 3 months .
prior to this presentation , she had been healthy , without any previous medical illness . on physical examination ,
the left upper quadrant was slight tender but no abdominal mass was detected by abdominal palpation .
laboratory data were all normal . computed tomography ( ct ) demonstrated a low - density mass with a ct value of 1 hounsfield unit ( hu ) , measuring about 3.2 2.2 cm in the right lobe of liver ( figure 1a ) , and a solid low - density mass with a ct value of 8hu , measur - ing about 5.2 cm 4.6 cm in the upper pole of the spleen ( figure 1b ) .
ct demonstrated a low - density mass measuring about 3.2 cm 2.2 cm in the right lobe of liver ( a ) , and a solid low - density mass measuring about 5.2 cm 4.6 cm in the upper pole of the spleen ( b ) .
based on the physical examination and ct , the preoperative diagnosis of the hepatic cyst was definite but the preoperative diagnosis of the splenic tumor was difficult .
then a laparo - scopic splenectomy was performed under a presumed diagnosis of a malignancy of the spleen .
intraoperatively , a solid mass ap - proximately 5 cm in diameter was found in the upper pole of the spleen .
there were not any signs of metasta - ses or any visible neoplastic tissue remnant at the operation site .
the re - sected spleen measured 11 10 8 cm , weighed 740 g. histopathology showed that tumor cells were pleomorphic and consisted of fibroblasts , histiocytes , and myofibroblast - like cells arranged in a storiform pattern ( figure 2a ) .
immunohistochemistry showed these tu - mor cells were positive for vimentin ( vim ) , -smooth muscle actin ( -act ) and cd68 ( figure 2b ) .
based on these results of histopathology and immunohistochemistry , the final diagnosis of mfh of the spleen was conformed .
the patient was discharged on the 7th day after the initial operation and was not given any additional treatment .
she was followed up for 13 months without recurrence or metastases after the initial surgery at the time of reporting .
histopathology showed tumor cells were pleomorphic and consisted of fibroblasts , histiocytes , and myofibroblast - like cells arranged in a storiform pattern ( a ) .
mfh is the most common soft tissue sarcoma mainly occurring in the extremities ( 70% ) , followed by the abdominal or retroperitoneum ( 16%).1 the origin of mfh is still controversial .
primary mfh of the spleen is extremely rare , although the incidence of mfh of the spleen remains undetermined . since the first description of primary splenic mfh reported by govoni et al.6 in 1982 ,
only 13 cases have been reported in the english language literature in medline.617 compared with the 13 previously reported cases of mfh of the spleen , our case was the first case of mfh of the spleen accompanied with other disease , and also the first case treated successfully by laparoscopic splenectomy .
the spleen , for poorly documented reasons , generally shows relative resistance to malignant tumors .
accordingly , the preoperative diagnosis of mfh of the spleen is difficult because the cases are extremely rare , the clinical symptoms are nonspecific , and the imaging patterns are not suggestive .
these tumor cells consist of histiocyte - like and fibroblast - like cells with other multinucleated giant cells.11318 there are positive stainings for lysozyme , vim , cd68 , and -1 antitrypsin.13 in our case , we could not get a definite preoperative diagnosis of the splenic tumor according to the physical examination and ct scanning .
a final diagnosis of mfh of the spleen was confirmed by the histopathology and immunohistochemistry . in review of the english language literature in medline
, we found 13 previously reported cases of the mfh of the spleen ( table 1 ) . among the 14 cases of mfh of the spleen , including our case , six cases were females and eight cases were males with the age from 35 to 82 years old , mainly in the fourth or fifth decade of life ( 9/14 )
the presenting symptoms were usually nonspecific , but the clinical characteristics such as abdominal pain , fever , weight loss , and splenomegaly were prominent features in about 70% of the patients.17 about 35.7% ( 5/14 ) of the cases with splenic mfh presented metastases at the initial surgery including liver ( 3 cases ) , stomach and pancreas ( 1 case ) , and omentum ( 1 case ) ; and the liver was the most common metastatic site .
mfh has been categorized into five types , based on the histopathologic subtype , including storiform - pleomorphic , myxoid , inflammatory , giant cell and angiomatoid variants.16913 however , only the subtype of storiformpleomorphic and inflammatory variants have been reported in the mfh of the spleen in the previously reported literature . in review of the 14 cases , although 4 cases were not mentioned , eight cases were storiform - pleomorphic types including our case and two cases were inflammatory types .
all of the 14 patients underwent the surgery of splenectomy , while our case was the first case treated successfully by laparoscopic splenectomy .
six patients died of local recurrence with multiple metastasis , 3 , 6 , 7 , 8 , 15 , and 19 months postoperatively .
one patient was dead after splenectomy with hepatic involvement and the other was alive at 18 months with multiple hepatic metastases at the time of reporting .
five cases , including our case , survived for 3 , 13 , 17 , 18 , and 46 months , without local recurrence and distant metastasis at the time of reporting . reported cases of malignant fibrous histiocytoma of the spleen in the english language literature in medline
the incidence of splenic mfh remains undetermined due to limited data on a rare malignancy .
the clinical symptoms of primary splenic mfh are usually nonspecific , but the characterics such as abdominal pain , fever , weight loss , and splenomegaly are prominent features .
chemotherapy and radiotherapy are other therapeutic modalities for patients with metastasis , but the therapeutic effect is poor .
jff carried out the design and coordinated this study , hl and jl collected and analyzed the clinicopathologic data . | primary malignant fibrous histiocytoma ( mfh ) of the spleen is extremely rare . since the first description of primary splenic mfh reported by govoni et al in 1982 , to the best of our knowledge ,
only 13 cases of mfh of the spleen have been reported in the english language literature in medline .
we herein report a rare case of primary splenic mfh accompanying with hepatic cyst in a 48-year - old chinese female who treated successfully by laparoscopic splenectomy and fenestration , which has not yet been reported in the literature . compared with the 13 previously cases of mfh of the spleen ,
our case is the first case accompanied with other disease , and also the first case treated successfully by laparoscopic splenectomy .
a literature review of mfh of the spleen previously reported in the english language literature in medline is also provided . | Case Report
Discussion
Conclusion
Authors Contributions |
among striking developments in history writing towards the end of the twentieth century were the moves to the spatial and the visual in the reconstruction of historical consciousness . global history , accelerated through american hype on
globalisation , and visual culture studies propelled by society s increasing reliance on visual communications , became fashionable projects .
global history,1 grounded in contemporary economics and partly pitched in reaction to western parochialism , came seriously to challenge nationalistic and structuralist approaches to history.2 at the same time , the pictorial turn , with its epistemological claim for vision as the prime sense in knowledge production , came to defy not only conventional history of art , but the status and value of the discipline of history itself.3 wrestling with these turns proved enormously productive .
in the history of medicine it led to foregrounding disease in its global dimension , revivifying , at the same time as challenging , older narratives on the world wide spread of disease.4 in the history of science it led to heightened attention to visual representations in struggles over the production of scientific knowledge and authority.5 both turnings did more than merely provide historians with exciting new conceptual frameworks for comprehending the past .
as illustrated through the contemporaneous growth of a literature on the history of material objects in all their global distribution , they also helped broaden the range of objects deemed worthy of historical attention.6 this paper draws on these moves in relation to one particular material object that is both
global and visual : the aids poster.7 their worldwide production from the mid-1980s hugely reinvigorated the whole genre of the health poster and , according to one expert , restored the genre s original function as a communications medium.8 certainly , as never before was so much money , aesthetic effort and psychological marketing put into this particular media on the part of voluntary bodies , national governments and international health agencies.9 our concern , however , is with the wider conceptual frameworks that were mobilised to make these objects meaningful . in the 1980s and 1990s ,
as we have outlined elsewhere , a cohort of western intellectuals concerned themselves with them along with other representations of aids in order to talk about the politics of identity.10 those concerns , in turn , were linked to broader ones emerging at the time over the rights of citizens to equal access to health care , the privatisation of medicine , and the role of the international pharmaceutical industry in the commercialisation of health care . in this paper
we focus on another aspect of these ephemeral mass - produced objects : not their active life on the streets and in the corridors of learning but their
afterlife when they were turned into items to be collected , exchanged and stored in museums and archives .
it is well known that the social life of material objects in such places is not the same as that of their initial culture of production , circulation and consumption.11 museums and archives , like other depositories for images and artifacts , have particular collecting agendas and particular institutional and intellectual traditions into which new acquisitions are fitted .
they also inhabit the present , embracing wider conceptual contexts that serve further to shape the organisation and meaning of their artifacts . here , we explore one such
afterlife for aids posters : an exhibition entitled against aids : posters from around the world , which was held at the museum fr kunst und gewerbe in hamburg between february and april 2006 .
impact of the exhibition ; our interest in it is , rather , as an illustration of the more general trend towards the
the museum fr kunst und gewerbe was founded in 1877 in a spirit of aesthetic modernism and german nationalism , and by the 1890s was host one of germany s largest and most prestigious poster collections .
the peoples arts and crafts , much like the south kensington museum in london , established in 1852 and renamed the victoria and albert museum in 1899 at the height of british jingoism .
as at the hamburg museum fr kunst und gewerbe , so at the victoria and albert museum over the past few decades , objects have been reorganised for exhibitions accentuating the global. the representation of aids posters at the hamburg exhibition provides us with a means to discuss the politics of such
global assemblages.12 on the one hand , it permits us to draw out the inherent contradictions and tensions that can be involved in any such institutional mobilisation of the concept of the unity of the globe.13 on the other , it allows us to underline important continuities hidden under the more apparent or insisted upon
discontinuities between national and global discourses , and between modern and postmodern politics of aesthetics
continuities rooted , we argue , in shared aesthetic values.14 as important , the example permits us to reflect on how the discourse of the global affects the work of historians using material objects in their constructions of historical consciousness .
as these aims and objectives should suggest , we are not concerned here with how viewers might have responded to the images or to the exhibition as a whole ( an almost impossible task given the uniqueness of individual psychology and experience).15 nor are we interested in providing a walk - through critique of the exhibition ; our main interest is in the historical context of the museum and how this bears on the politics of aesthetics implicated in its exhibition of aids posters .
against aids : posters from around the world was a modest , low - budget affair .
primarily , it was staged in order to exploit the museum s recent acquisition of over a thousand hiv / aids and safe sex posters from a private dealer , a purchase that enabled it to join the club of institutions harbouring such collections.16 the organisers of the exhibition selected only a hundred of the posters to display , choosing those that were most visually arresting , and others that , even after three decades in some cases , still had the power to shock , titillate , and/or amuse ( such as that used on the front of the flyer for the exhibition , figure 1 , or
condoman , figure 2 ) . in part to enhance these effects , realist anti - homophobic posters ( figure 3 ) , were mixed with erotic
semen kit , for example , joined nautical space with a poster of a condom disguised as a life - saving ring,17 which , in turn , was hung alongside a russian poster advert for rubber tyres .
these striking juxtapositions were intended to reveal the variety of aesthetic choices that governments , charities , commercial bodies , and private artists employed in their efforts to inform the public of the threat of hiv / aids and incite onlookers to ethical behaviour ( safer sex ) .
dramatically , at the entrance to the exhibition , the visitor was confronted with a full billboard - size reproduction of oliviero toscani s iconic image of 1992 : his re - conception of the prize - winning photograph from life depicting the death of the american aids activist david kirby , turned into an advertisement for the united colours of benetton ( figure 5).18 other less dramatic images played on popular solidarities around aids , as prefigured in the socially integrative against aids in the title of the exhibition .
these images could serve to counter any charge that might be levelled at the museum for its use of the more erotic and more humorous and ironic ones , namely , that it was denying the pain and suffering of hiv / aids victims , or trivialising the world s most devastating disease . against aids
it was through the display of these posters in particular that the exhibition sought to exemplify regional variety and similarities in aesthetic styles .
condoms , the viewer might come to see , were globally an unambiguous symbol for , and the warning against , unsafe sex.19figure 1the leaflet ( 10 21 cm ) for against aids : posters from around the world , museum fr kunst and gewerbe , hamburg , 2006 . by permission of the museum fr kunst and gewerbe , hamburg.figure 2issued by the commonwealth department of community services , aboriginal health workers of australia ( queensland ) , 1991 ( 41 28 cm ) .
this poster was reproduced in several countries with the english translated.figure 3produced by terrence higgins trust , 1999 . by permission of the terrence higgins trust.figure 4issued by gay men fighting aids , london , 1994 ( 59 42 cm ) , hywel williams photographer .
the image was reproduced on a 15 cm card , which was handed out at the gay pride festival in july 1994 .
the text on the card mentions the uk government 's poor funding of safe sex campaigns and parliament 's homophobic reaction to the proposal of an equal age of consent of 16 for both hetero- and homosexuals.figure 5oliviero toscani s 1992 billboard image of the death of david kirby for benetton s shock of reality advertising campaign . by permission of the united colors of benetton .
the leaflet ( 10 21 cm ) for against aids : posters from around the world , museum fr kunst and gewerbe , hamburg , 2006 . by permission of the museum fr kunst and gewerbe , hamburg
. issued by the commonwealth department of community services , aboriginal health workers of australia ( queensland ) , 1991 ( 41 28 cm ) .
this poster was reproduced in several countries with the english translated . produced by terrence higgins trust , 1999 . by permission of the terrence higgins trust
. issued by gay men fighting aids , london , 1994 ( 59 42 cm ) , hywel williams photographer .
the image was reproduced on a 15 cm card , which was handed out at the gay pride festival in july 1994 .
the text on the card mentions the uk government 's poor funding of safe sex campaigns and parliament 's homophobic reaction to the proposal of an equal age of consent of 16 for both hetero- and homosexuals .
oliviero toscani s 1992 billboard image of the death of david kirby for benetton s shock of reality advertising campaign . by permission of the united colors of benetton .
although a flyer ( figure 1 ) , but no catalogue was produced for the show , the aesthetic and intellectual motives behind it are discernable through the catalogue that accompanied a much larger exhibition of posters held at the museum in 1996 ( which was curated by the same person , jrgen dring , and displayed some of same posters).20 the 1996 exhibition marked the centenary of the museum s first - ever exhibition of posters a late nineteenth century entertainment that was in fact the first of its kind in germany and one of the first in europe.21 in many ways the 1996 exhibition was faithful to that of 1896 , its agenda being more or less the same as that articulated by the main advocate and co - founder of the museum fr kunst und gewerbe , the hamburg lawyer and art critic justus brinckmann ( 18431915).22 in his catalogue for the 1896 exhibition , brinckmann opined that posters and their display in museums and galleries performed the ethical task of elevating the masses to aesthetic appreciation :
art should be accessible to everyone .
it should bring elevation and joy to all ; not only to those who buy it or have the time to visit art galleries . in order to fulfil
this purpose art has to go on the street , and has to cross as if by accident the path of the many thousands going to work who have neither time nor money to devote to it otherwise.23 art should be accessible to everyone
. it should bring elevation and joy to all ; not only to those who buy it or have the time to visit art galleries . in order to fulfil
this purpose art has to go on the street , and has to cross as if by accident the path of the many thousands going to work who have neither time nor money to devote to it otherwise.23 these were not the only politics in the 1896 exhibition with resonance for the one in 1996
. the wider context at the end of the nineteenth century was that of flourishing national rivalries as well as attitudes to competition as a virtue in itself
the posters in the 1896 exhibition , from italy , france , russia , the usa and elsewhere , were organised accordingly and , typical of most exhibitions at the time displaying products from different countries , were competitively judged by an international panel .
the exhibition thus served to cohere national identities at the same time as sell the ideology of competition along with the virtues of the products of mass - production for mass ( visual ) consumption .
more than this , while on the one hand the 1896 exhibition promoted the promise of democracy embedded in the notion of arts and crafts by and for the people , uniting people through aesthetics education , on the other , it sold an litist aesthetic ethics that simultaneously challenged traditional litist views of what constitutes art and who properly can access and comment on it.24 a century later many of brinckmann s views were dusted off .
jrgen dring , the curator of the 1996 exhibition ( and subsequently the 2006 one ) similarly revelled in the non - litist engagement of poster art , at the same time as celebrating the particular aesthetic distinction that brinckmann drew between mundane commercial advertising and sophisticated poster design.25 the 1996 exhibition visually illustrated this difference by juxtaposing examples of each .
above all , dring celebrated and reproduced brinckmann s ethical mission to teach people how to appreciate and enjoy the visual world through a better understanding of its underlying aesthetic principles and techniques of production.26 this ethical educational work of the poster is all the more important today , he argued in the catalogue , because the fine arts have lost the main task they had when brinckmann was living , namely , to interpret the world in its visual parts and provide the onlooker with an edifying understanding of it.27 the 2006 exhibition of aids posters was conceived in the same intellectual framework , according to dring.28 it , too , was intended to elicit an emotional response from the viewer and , from that , heighten their sensitivity to art.29 but while brinkmann in his day had looked forward to a brighter future for the poster and its onlookers in museums such as the kunst und gewerbe in hamburg , dring and his colleagues found it hard to be quite so optimistic . in their view as maintained in the catalogue for the 1996 exhibition
the general quality of the visual language of posters had dramatically decreased due to the increasing flood of pictures in everyday life .
discriminating aesthetic appreciation , they felt , was less and less in evidence in contemporary culture because modern education failed to teach the classics and its iconography .
consequently , despite the fact that posters were intimately a part of the revolution in graphic design and design technology of the 1980s and 1990s,30 they had become impoverished superficial , because they could no longer be
the apparent proof of this lay in the popular media s focus on the human body or , more precisely , on superficial beauty and feelings around the human body . according to dring and
his colleagues this was the zeitgeist of our times that exceeds rational understanding.31 today s fashionable heroes hardly transmit any moral values , they lamented ; indeed , they did the opposite dissipating , through preoccupations with individual self - fulfilment and superficial gratification , the cannon of christian ethics and traditions of virtue behind
good art.32 aids posters apparently shared this fate in having behind them no shared aesthetic appreciation . according to the 1996 catalogue , they could communicate no meaningful visual language for the deadly disease they referred to because the health educationalists who issued them were themselves unable to formulate one . hence , the iconography of aids posters appealed predominantly only to the feelings of onlookers .
all that could really be said in their favour was that they sponsored a positive moral practice , namely caution against the spread of hiv : the onlooker feels attracted by the picture and decides to use a condom.33 although it seems odd that the curators of the museum fr kunst und gewerbe disdained the very objects they were celebrating , they justified themselves on the grounds that such posters were expressive of their times and therefore wholly within the remit of an institution whose agenda was to archive and exhibit the art of the streets and the people . by the 2006 exhibition ,
the times were global , and hence a new justification was established through the parade of moral values within that discourse . in maintaining that contemporary culture and advertising were saturating the world with meaningless , morally deprived , corporeally fixated images ,
the curators of the museum fr kunst und gewerbe were maintaining a distinctly modernistas opposed to postmodernist view of art. in fact , in believing that the mechanics of perception operated along the lines of emotional attraction , followed by rational thought , and then enlightened responsible behaviour , they were following nineteenth - century notions of the physiology of seeing and sense perception.34 they were also assuming that onlookers were simply passive vessels for ethical education in visual good taste and , moreover , that good taste was the highest form of human awareness .
interestingly , in many of these respects as well as in connection with the understanding of disease they were sharing an outlook with one of the few intellectuals in the twentieth century ever to provide sustained commentary on posters , the late susan sontag ( 19332004)despite the fact that sontag , as a theorist , was to their left in embracing the marxism of the frankfurt school of critical theory.35 sontag s views were made clear in 1970 in posters : advertisement , art , political artifact , commodity.36 posters , inhabiting the low end of high art , she explained , originated in the effort of expanding capitalist productivity to sell surplus or luxury goods. they could not exist :
[ b]efore the specific historic conditions of modern capitalism .
sociologically , the advent of the poster reflects the development of an industrialised economy whose goal is ever - increasing mass consumption , and ( somewhat later , when posters turned political ) of the modern secular centralised nation - state , with its peculiarly diffuse conception of ideological consensus and its rhetoric of mass political participation.37 [ b]efore the specific historic conditions of modern capitalism .
sociologically , the advent of the poster reflects the development of an industrialised economy whose goal is ever - increasing mass consumption , and ( somewhat later , when posters turned political ) of the modern secular centralised nation - state , with its peculiarly diffuse conception of ideological consensus and its rhetoric of mass political participation.37 posters thus served the purpose of aggressively pushing consumption or , in politics , of selling national i d entities and ideologies .
the modern concept of public space as a theatre of persuasion.38 this regard of posters , as aim[ing ] to seduce , to exhort , to sell , to educate , to convince , to appeal,39 was predicated on a view of advertising ( of any sort ) as psychologically dangerous . for sontag , posters blinded people to the real world , a view ( shared by the curators of hamburg s museum fur kunst und gewerbe ) that presupposes some unmediated or
, one has only to break through the constructions mediated by language or by images to experience a direct understanding of the world though senses and perceptions .
although sontag had no reason to comment on health posters until she came to write aids and its metaphors in 1988 , her thinking on them was indistinguishable from her earlier thoughts .
aids posters , she maintained , indict the ideology of consumer capitalism that celebrates recreational risk - free sexuality in the name of individual liberty.40 furthermore , as in her illness as metaphor ( 1978 ) , there was no doubt about the biological essence of such a disease . just as posters mediated false - consciousness , so behind the distorting metaphors of any widely feared disease there lurked a real entity .
one had only to strip away the metaphors to get at the underlying value - neutral , non - stigmatising nature of any disease.41 the organisers of the exhibitions of aids posters at hamburg s museum fr kunst und gewerbe similarly believed aids to be realthat is , to be a universal medical problem that was simply yet to be solved .
as if to underline this understanding of hiv / aids as medical and nothing more , the 2006 exhibition was accompanied by health education information issued by various private and national hiv / aids agencies .
significantly , in both the flyer for the 2006 exhibition and in the section on aids in the 1996 catalogue , a medical discussion of hiv / aids preceded that on the aesthetics of the posters .
thus , a distinctly modernist view of art was accompanied by a distinctly modernist , as opposed to postmodernist , view of science and medicine as unquestionably superior forms of consciousness and practice , even to art .
how people interacted with aids posters during the images active life on the streets on buses , billboards , underground trains , and so on and how the power and fear of hiv / aids operated in relation to identity were simply not parts of the 2006 exhibition in hamburg . through the literal framing of the posters ,
their hanging according to the conventions of art galleries , their arrangement ( three or four to a single wall ) , and the choice of them in terms of the quality of their visual language ( bildersprache ) , the organisers denuded them of the local contexts in which they were created and in which they were engaged with politically , intellectually and emotionally .
this absenting can probably be illustrated by paying close attention to the history of any one of the posters in the exhibition .
here , we focus on only a few of them to illustrate the point : semen kit ( figure 4 ) , stand up against homophobia ( figure 3 ) , and toscani s advertisement for benetton ( figure 5 ) .
the first two of these were among the five british examples of posters in the exhibition .
both were issued by hiv / aids charities ( as were the other three british examples in the show ) , and were relatively recent .
semen kit ( 1994 ) was produced by the gmfa [ gay men fighting aids ] ( established in 1992 ) , whilst stand up against homophobia ( 1999 ) was distributed by the terrence higgins trust ( established in 1982 and , by the 1990s , britain s leading hiv and sexual health charity ) . that they were not issued by the state
in 1986 , when thatcher s government announced its intention to spend 20 million over the next twelve months on hiv / aids health information , and commissioned the advertising agency tbwa to undertake it , the resulting iceberg and
tombstone images were crafted to sell a health message to a general audience and to meet the government s inflated interest in family values , heterosexual sex , and nationhood.42 condoms were not then in the frame , nor were gay men , and nor was
the global. the story of how the patriotic heterosexual imaging43 began to be turned around in the 1990s need not concern us here.44 as in most western countries , it was gay men the first victims of the disease who initially confronted the benign , sexually prudish and denying images of the establishment , and then inverted the anti - liberal homophobic rhetoric generated by the gay plague in celebration of their own collective identity ( the gay community ) .
it only needs observing that in the uk this development was significantly different from elsewhere ; bearing upon it were the particularities of social and cultural traditions as well as prevailing political , medical and legal circumstances .
pre - existing affirmative representation in the public media of gays were far less pronounced than in some other places , while the ability of the tabloid press and the conservative government of the day to stir homophobia was greater.45 prime minister margaret thatcher s homophobic legislation of 1988 ( section 28 of the local government act forbidding the promotion of homosexuality ) in particular , did much for heightening alterity , struggle and confrontation .
yet it was precisely these features that the hamburg exhibition eclipsed by having the images hung alongside foreign advertisements and alongside aids posters from china and elsewhere .
the exhibition thereby performed not for the struggle for gay identity within a national context , but for world solidarity .
stand up against homophobia , by virtue of being both a product of 1999 , and by presenting hiv / aids as a part of a wider social issue , served in itself to mask the historical significance of these images .
semen kit performed similarly through its play to a would - be unproblematic history of overt sexual behaviour among men ( inconised in the image of the sailor ) , and by its appropriation from some american aids posters of the by then more - than - a - decade - old image of the gay
body beautiful.46 that gay men in britain , well into the 1990s , were still struggling for a viable visual public identity could not be guessed from the hanging of this poster at hamburg .
nor could it be known from either of these images that the changes in gay identity that occurred in britain in the 1990s were , in part , largely attributable to the uptake of a slick visual language borrowed from the culture of madison avenue - driven international advertising the same visual language , from the same source , that was simultaneously taken up by thatcher and other politicians around the world in electioneering .
but it was not only governments and gays in the 1980s and 1990s who struggled to capture the meaning of aids ; so too did western medicine .
it was its lack of success at turning aids into a meaningful scientific category that , in fact , opened the medical profession to pointed confrontation , and opened up aids to the visual politics of identity . in effect , through aids , and in particular through the early debate over whether hiv caused aids , the medical profession was pulled off its pulpit as the authoritative arbiter of modern secular identity.47 arguably , toscani s poster advertisement for benetton reflects this by blurring the normative boundaries between public art and private anatomy / medicine , as well as by confusing the conventional distinction between commercial marketing and medical humanitarianism.48 for toscani , who regarded the david kirby image as the most significant of those he designed for benetton s shock of reality advertising campaign of 1992,49 it was supposed to show how an international corporation was open to the world s influences and engaged in a continuing quest for new frontiers.50 for him , this meant commitment to global social issues , above all , to the problems of poverty , race and disease . however , this was far from how the image was regarded by others . although in different contexts reactions varied , overall it was greeted with howls of moral indignation and in some places to the sacking of benetton shops .
the germans took the image to court , french billstickers refused to post it and , in britain , the guardian ( the first newspaper to run it as a full - page advertisement ) was inundated with letters of complaint.51 nor was that all . while many in the gay community responded to it favourably , others criticised it as a form of cultural and economic imperialism .
the critique fed a form of political resistance known as culture jamming , or adbusting that hijacks the ads of big brands to talk back to them in order to re - conquer city space.52 toscani s image immediately became the victim of this semiotic robin hoodism : the american aids coalition to unleash power ( act up )
there s only one pullover this photograph should be used to selland pictured a condom.53 here , supposedly , was the subconscious of the benetton campaign x - rayed to uncover not only its opposite meaning , but also ( in the manner of sontag s analysis ) to reveal a deeper truth ostensibly lurking beneath the layers of advertisement euphemism.54 here , then , was yet another field of combat over the meaning of hiv / aids centred on its imagery .
but , again , there was no hint of it at the show in hamburg .
while toscani s image still had the power to shock , it is doubtful if anyone outside of living memory who viewed it at the exhibition ( such as the school children bussed in ) could have guessed what it meant for many visual theorists and aids activists worrying over images of aids since the late 1980s , namely , that it was further testimony to the ongoing crisis over the entire framing of knowledge about the human body,55 with aids , not just a
medical problem but an epidemic of meanings or significations.56 the exhibition in hamburg gave no clue to how images like this had been regarded as testifying to a self - consciously postmodern culture , ethics , aesthetics , disease representation , and politics of identity . nor was the reproduction of toscani s image at the hamburg exhibition intended to illustrate how the lines between the commercial and the medical humanitarian had become so obscured that there was now little to mark the difference between a health poster and an advertisement for the sale of fashion knitwear .
instead , despite the fact that many of the images in the hamburg show had been designed simply to sell condoms , and others , such as that on homophobia , to challenge social injustice , prejudice and exclusion rather than caution against hiv / aids itself,57 the exhibition cohered them all into a would - be historically uniform and medically mediated message against hiv / aids .
thus although not with conscious intent the show flew in the face of the preoccupations of the visual theorists and aids activists of the 1980s and 1990s . where they had seen in representations of aids the postmodern play of signifiers , had argued for a plurality of subjectivities involved in visual engagements , and had construed visual perceptions of the human body in general as involving an onlooker s unconscious construction of their own body through the immediate act of viewing , the curators of the hamburg exhibition saw only medicine and art in modernity .
this was not the only way in which the exhibition displaced the specific and general historical meanings attached to these objects by investing them with others .
the title alone of the exhibition , against aids : posters from around the world did as much .
first and foremost it constructed a particular framework for their perception , one that above all suggested that aesthetic form can travel the world regardless of local geographies and local histories of ethnicity , religion , race , rights , sexuality and gender not to mention alternative aesthetic traditions
. this global aesthetic spin , in effect , harmonised a modernist western transcendent notion of art with the late twentieth - century notion of a spatially transcendent capitalism
an economic system supposedly unfettered by place or national boundary.58 the aesthetic spin and the exhibition as a whole thus further performed for notions of homogeneity and universality attributes long associated with modernity and perceived to be at odds with the pluralities and fragmentations associated with postmodernity during the time of aids in the west.59 the exhibition s title also suggested that the history of aids was about everyone the world over being uniformly against aids .
but that , too , was hardly the case at the point of production of many of these posters .
in the 1980s , christian fundamentalists and other religionists took a rather different line , and gay men and lesbians did not always see government campaigns against aids as being against aids so much as against themselves.60 as noted above , in thatcher s britain the aids campaign was an occasion for the moral high - grounding of heterosexual values.61 to the extent that people ( and international pharmaceutical companies ) were allegedly
against aids in the 1980s and 1990s , their concerns emerged from a multitude of different and often conflicting social and economic interests .
moreover , the relative power of those interests was hierarchically organised , and differently so over time , as virginia berridge has made clear for the history of aids in the uk.62 also , implicit to the entitling of the hamburg exhibition was the idea that nations around the world were homogeneous in their fight against hiv / aids .
this not only collapsed separate national encounters with hiv / aids , such as its very denial by south africa s president thabo mbeki , but effaced the differences between the kinds of media campaigns used in different countries including the often bitter struggles between local , national and international agencies.63 as important , this political gutting of aids posters through their aestheticisation erased national rivalries and pressures involved in medically treating aids victims ( or not , as the case may be ) . through the mixing of posters from different countries , the exhibition dissolved the conventional boundaries between nation - states , while the multitude of images of condoms that it presented served visually to re - unite them around a commercial product .
the images of condoms promoted the idea that campaigns for their use had actually united the countries of the world , a message curiously at odds with the bush administration s contemporaneous funding of medical missionaries advocating sexual abstinence instead of the use of condoms.64 in its own small way , therefore , and for its own particular didactic reasons ( as well as , perhaps , discomfort over germany s nationalistic past ) , the hamburg exhibition effected the same kind of historical effacement and rewrite that the major international media and entertainment companies were also coming to effect by 2006 through their take of up hiv / aids for the purpose of reaping public corporate credibility.65 by then , hiv / aids - funding was a fashionable cause , a benign branding resource for various western philanthropic organisations . at local , national and transnational levels then , against aids : posters from around the world can be seen to have effaced the individual history of the objects on display through a particular universalising and seemingly neutral kind of aestheticisation . at a closer look , however , it both appropriated them into an old script ( a local and fondly held modernist epistemology of viewing and aesthetics ) and a new one globalisation . by collapsing two decades of national histories into a singular and would - be unified world fight against hiv / aids ,
the history of hiv / aids was visually construed in terms of this new global subjectivity .
not only were particular constructions of the recent past left out the local struggles around these objects but also the construction of the present the global media industry s selling of itself through the attack on hiv / aids as a
real or made true through aesthetic representation of an ostensibly international struggle against hiv / aids , but by this same conceit was medically re - appropriated and humanised no matter that over the meaning of
good thing.66 this is not to suggest that hiv / aids was not recognised as a global problem almost from its start . in 1987 , the american aids activist , feminist and visual theorist , paula treichler , for instance , referred to aids as having a potential for global devastation.67 but for her and others in the 1980s the global was only a background problem , and the term was yet without particular epistemic load .
as we have indicated , western nations and their intellectuals and aids activists were gripped more by their own campaigns , interests , and ideologies than by
in fact , the idea in the west that hiv / aids was a global problem was a viewpoint that itself had to be fought for through a world wide media campaign brokered by organisations with internationalist interests .
the campaign can be dated precisely to 27 may 1987 when the world health organisation issued a press release proclaiming that aids is a global epidemic that demands a global attack.68 the who then produced a poster to sell the message ( figure 6)to compete , that is , with other struggles for the meaning of aids.69 the hamburg exhibition abetted that project through a visual rhetoric of shared international struggle against hiv / aids , just as it unwittingly abetted the subsequent take over of aids programs by the media multinationals.70 thus it eclipsed the history that would enable anyone to believe that aids had ever been anything other than global , or for that matter , anything other than mainly a struggle for economic resources for better medical provision for victims of the condition .
images intended for , and often produced by , local sub - groups became artefacts for making up global citizens , with
thereby , the exhibition did far more than merely reinforce what had become the semantic hegemony of the global , as iterated through such credulities as global warming and global terrorism.71 through aesthetics alone it rendered tangible the universalising concept of the global .
although this was not the exhibition s primary intention , it could not help but perform it , and in so doing contribute to a reconstruction of history and consciousness .
far from it ; the globally spun institution - serving celebration of the aesthetics of aids posters merely reflected and reinforced much of what everybody in the west had already come to
feel about hiv / aids by 2006that it was a serious world - wide problem about which everyone needed to be continually reminded.figure 6who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon .
who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon .
we have been concerned with a particular moment in the history of aids posters : not that of their initial
public life on the streets , or in pubs and gay clubs , doctors offices , and so on , but their afterlife in places where they might be displayed or stored.72 through the analysis of an exhibition at one such afterlife location our intention has not been that of negative dismissal , nor critique for the sake of it . nor has it been merely to expose how these often strikingly visual objects that aimed at protecting individual health had their meaning changed through appropriation into a conceptual framework different from that of their initial contexts of production and consumption .
more interesting to us are the intrinsic and unremitting links between these visual objects and wider politics , or how the visual is inherently a part of the latter . while it might have been supposed that aids posters came to political rest once they were retired , categorised , catalogued and stored according to the principles of collecting institutions , the hamburg exhibition proves otherwise .
in fact , as collector s items , they entered a space that was no less political than when they were on the streets in the 1980s and 1990s , and when they were appropriated to western discourses on postmodern identity and on the role of the visual in the cultural negotiation of the self .
retirement home they necessarily became a part of the institutional agenda of the museum fr kunst und gewerbe .
indeed , from the moment such objects become collectors items and are stored and/or displayed as artifacts they become epistemologically loaded through the very process of objectification .
hamburg s museum fr kunst und gewerbe demonstrates that these collecting agendas and the accompanying aesthetic guidelines often have deep historical roots .
but what the analysis of its 2006 exhibition of aids posters also shows is that old and seemingly apolitical agendas ( invented to express specific national political interests ) are neither lost nor rendered innocuous in the contemporary world .
rather , they come to serve new political frameworks linked to the world of today s visitors a world in which aesthetics is the dominant means to a politics constituted on little more than the idea that if it looks good go with it ( an outlook now as pervasive in the practice of science as in the arts of government).73 crucially , this new politics is sustained through , and for , the absenting of critique ; not today the critical outlook entertained by sontag and other pre - postmodern intellectuals , that visual representations ( and popular posters in particular ) covered - up or cloaked lurking ideologies .
postmodernists , unconcerned with that view for the most part , in effect opened the space for the new politics of aesthetics that masks something different : the idea of aesthetics as void of political intention .
the hamburg exhibition of aids posters was , in fact , an early example of the coming - to - reign of these particular politics , with the visual alone being the vehicle for understanding and creating a ( global ) community without distinction .
whereas for brinckmann in the nineteenth century , aesthetics ( in art ) could be consciously used for the political purpose of populist democracy , with aesthetics and politics in clearly separate spheres , for the inheritors of his institution in hamburg aesthetics ( unbeknownst to them ) became the politics , not simple a means to it . ironically , their arrival at these politics their unknowing performance of them through the exhibition was via adherence to brinckmann s legacy . through that , brinckmann s original political agenda was emptied of its original political purpose . installed in its place were the politics of the appearance of political un - intention .
thus did an aesthetic concept born in the nineteenth century to serve nationalistic purposes come to operate for the political work of educating national citizens to global citizenship .
what does this mean for historians working with material objects stored in global - tending museums and archives and who are themselves now operating within a global framework ?
since material objects have no meaning without a framework , and are framed in being collected , the simple answer is that historians have to take into account the afterlives of such objects as much as the object s original lives .
harder is the problem of the historian s own place in the global framework , which , in many respects , is not unlike that of the material object in the global - aspiring museum ( and very much like that of the curators of such exhibitions ) . whether avowed explicitly in
global history , or embraced implicitly in the practice of history writing in contemporary culture , the global operates politically and epistemologically . just as global history s predecessor
world history is now perceived by some of its originators as having been a product of , and agent for , its cold war moment,74 so for our own times , dominated as they are by multi - national corporations and abiding politicians , the take up of global themes in history writing is widely recognised as providing , at the very least , legitimacy to a globalisation discourse , even if , as often the case , the historian s immediate object is the far from reactionary one of provincialising the west and critiquing its hegemony.75 of course , for some historians the global does politically more , overtly serving as a rhetorical strategy for the re - coherence of the discipline of history itself after its pummelling by poststructuralists , deconstructionists and other fragmenting postmodern forces over the past thirty years or more.76 to this end , what could serve better as a reunifying device than the holising connective metaphor of the globe ? thus the global provides a new grand narrative a universalising tool with which to reimpose the meta - narrativity of history .
although seemingly mindless of one of postmodernism s cautions , that totalising worldviews can lead to totalitarianism,77 these historians seek more - or - less intentionally what the curators of the museum fr kunst und gewerbe performed innocently through their exhibition of aids posters . in so doing , they also share company with certain art historians anxious to revive older agendas a means ( as bluntly put by one of them ) to counter the deconstructive criticism of historical culture proposed by self - serving postmodern academics who treat the past as a sour land over which to exercise present concerns and anxieties.78 yet neither global history nor visual culture studies need necessarily lead in this direction . to see the global as a discourse tied politically to institutionally specific agendas ( such as the aesthetics entertained at the museum fr kunst und gewerbe in hamburg in 2006 ) offers the possibility to take an alternative political position , or at least to liberate its historical study from such agendas .
nevertheless , in terms of the less - visible shaping of historical knowledge production , the global framework can never be other than world - making as opposed to being simply historically descriptive.79 no matter how hard we try to stick to the recovery of some truth of how the world came to be globally conceived ( past or present ) , our knowledge productions implicitly reproduce and foster the unifying construct .
just as with material objects ( or words or images ) there is no meaning to historical events outside the conceptual frameworks we wittingly or unwittingly apply to them . in short , there exists no resting place for history writing ; it is always already fashioned and fashioning . like creating a museum exhibition of posters to aestheticise the
global nature of hiv / aids , the business of contributing to a global history of anything entails , by that very act , the politics of constructing a necessarily partial representation of the past. history writing , too , in other words , as a product of its time , can not avoid making up historical consciousness .
this article can not escape the charge that it too contributes to this process merely by discussing an event that was conceptualised in
it might even be seen to compound the problem by drawing attention to spaces where , a priori , the historian is already politically and epistemologically implicated : the museum and the archive .
however , in doing so it has sought to move the discussion beyond the tired call for attending merely to historical contexts , especially of material objects globally attributed .
our purpose has been to encourage historians to an awareness of their own immediate entanglements in history s constructedness the constructedness of the present mediated in history writing as much as through aesthetic assemblages of the global. the historian aruf dirlik , critically inquiring into the point of writing world history , has observed that an awareness of the variety of world histories that have been constructed at different times and in different places [ must cause ] any world historian worthy of the name [ to ] be uncommonly aware of the constructedness of the past.80 similarly , we submit , all historians need be reflective on their contributions to the present that is , to a culture given to re - enchantment through the global. quite how we should historicise material objects of the sort discussed in this paper may be open to debate ; what is not is the necessity to historicise our own historical projects .
otherwise we move perilously close to becoming blind participants in the historically fashioned spaces where memory is increasingly naturalised and neutralised through universalised and universalising concepts mediated aesthetically .
we end up , as it were , nave viewers of the exhibition at hamburg : as blind to the nature of the new post - postmodern politics of aesthetics , as to the modernist would - be universal humanity that the global unwittingly espouses through those politics .
how people interacted with aids posters during the images active life on the streets on buses , billboards , underground trains , and so on and how the power and fear of hiv / aids operated in relation to identity were simply not parts of the 2006 exhibition in hamburg . through the literal framing of the posters ,
their hanging according to the conventions of art galleries , their arrangement ( three or four to a single wall ) , and the choice of them in terms of the quality of their visual language ( bildersprache ) , the organisers denuded them of the local contexts in which they were created and in which they were engaged with politically , intellectually and emotionally .
this absenting can probably be illustrated by paying close attention to the history of any one of the posters in the exhibition .
here , we focus on only a few of them to illustrate the point : semen kit ( figure 4 ) , stand up against homophobia ( figure 3 ) , and toscani s advertisement for benetton ( figure 5 ) .
the first two of these were among the five british examples of posters in the exhibition .
both were issued by hiv / aids charities ( as were the other three british examples in the show ) , and were relatively recent . semen kit ( 1994 ) was produced by the gmfa [ gay men fighting aids ] ( established in 1992 ) , whilst stand up against homophobia ( 1999 ) was distributed by the terrence higgins trust ( established in 1982 and , by the 1990s , britain s leading hiv and sexual health charity ) .
that they were not issued by the state is important , for they were in fact conveying messages alternative to it . in 1986 ,
when thatcher s government announced its intention to spend 20 million over the next twelve months on hiv / aids health information , and commissioned the advertising agency tbwa to undertake it , the resulting iceberg and
tombstone images were crafted to sell a health message to a general audience and to meet the government s inflated interest in family values , heterosexual sex , and nationhood.42 condoms were not then in the frame , nor were gay men , and nor was the global. the story of how the
patriotic heterosexual imaging43 began to be turned around in the 1990s need not concern us here.44 as in most western countries , it was gay men the first victims of the disease who initially confronted the benign , sexually prudish and denying images of the establishment , and then inverted the anti - liberal homophobic rhetoric generated by the gay plague in celebration of their own collective identity ( the gay community ) .
it only needs observing that in the uk this development was significantly different from elsewhere ; bearing upon it were the particularities of social and cultural traditions as well as prevailing political , medical and legal circumstances .
pre - existing affirmative representation in the public media of gays were far less pronounced than in some other places , while the ability of the tabloid press and the conservative government of the day to stir homophobia was greater.45 prime minister margaret thatcher s homophobic legislation of 1988 ( section 28 of the local government act forbidding the promotion of homosexuality ) in particular , did much for heightening alterity , struggle and confrontation .
yet it was precisely these features that the hamburg exhibition eclipsed by having the images hung alongside foreign advertisements and alongside aids posters from china and elsewhere .
the exhibition thereby performed not for the struggle for gay identity within a national context , but for world solidarity .
stand up against homophobia , by virtue of being both a product of 1999 , and by presenting hiv / aids as a part of a wider social issue , served in itself to mask the historical significance of these images .
semen kit performed similarly through its play to a would - be unproblematic history of overt sexual behaviour among men ( inconised in the image of the sailor ) , and by its appropriation from some american aids posters of the by then more - than - a - decade - old image of the gay
body beautiful.46 that gay men in britain , well into the 1990s , were still struggling for a viable visual public identity could not be guessed from the hanging of this poster at hamburg .
nor could it be known from either of these images that the changes in gay identity that occurred in britain in the 1990s were , in part , largely attributable to the uptake of a slick visual language borrowed from the culture of madison avenue - driven international advertising the same visual language , from the same source , that was simultaneously taken up by thatcher and other politicians around the world in electioneering .
but it was not only governments and gays in the 1980s and 1990s who struggled to capture the meaning of aids ; so too did western medicine .
it was its lack of success at turning aids into a meaningful scientific category that , in fact , opened the medical profession to pointed confrontation , and opened up aids to the visual politics of identity . in effect , through aids , and in particular through the early debate over whether hiv caused aids , the medical profession was pulled off its pulpit as the authoritative arbiter of modern secular identity.47 arguably , toscani s poster advertisement for benetton reflects this by blurring the normative boundaries between public art and private anatomy / medicine , as well as by confusing the conventional distinction between commercial marketing and medical humanitarianism.48 for toscani , who regarded the david kirby image as the most significant of those he designed for benetton s shock of reality advertising campaign of 1992,49 it was supposed to show how an international corporation was open to the world s influences and engaged in a continuing quest for new frontiers.50 for him , this meant commitment to global social issues , above all , to the problems of poverty , race and disease .
although in different contexts reactions varied , overall it was greeted with howls of moral indignation and in some places to the sacking of benetton shops .
the germans took the image to court , french billstickers refused to post it and , in britain , the guardian ( the first newspaper to run it as a full - page advertisement ) was inundated with letters of complaint.51 nor was that all . while many in the gay community responded to it favourably , others criticised it as a form of cultural and economic imperialism .
the critique fed a form of political resistance known as culture jamming , or adbusting that hijacks the ads of big brands to talk back to them in order to re - conquer city space.52
toscani s image immediately became the victim of this semiotic robin hoodism : the american aids coalition to unleash power ( act up )
there s only one pullover this photograph should be used to selland pictured a condom.53 here , supposedly , was the subconscious of the benetton campaign x - rayed to uncover not only its opposite meaning , but also ( in the manner of sontag s analysis ) to reveal a deeper truth ostensibly lurking beneath the layers of advertisement euphemism.54 here , then , was yet another field of combat over the meaning of hiv / aids centred on its imagery .
but , again , there was no hint of it at the show in hamburg .
while toscani s image still had the power to shock , it is doubtful if anyone outside of living memory who viewed it at the exhibition ( such as the school children bussed in ) could have guessed what it meant for many visual theorists and aids activists worrying over images of aids since the late 1980s , namely , that it was further testimony to the ongoing crisis over the entire framing of knowledge about the human body,55 with aids , not just a
medical problem but an epidemic of meanings or significations.56 the exhibition in hamburg gave no clue to how images like this had been regarded as testifying to a self - consciously postmodern culture , ethics , aesthetics , disease representation , and politics of identity . nor was the reproduction of toscani s image at the hamburg exhibition intended to illustrate how the lines between the commercial and the medical humanitarian had become so obscured that there was now little to mark the difference between a health poster and an advertisement for the sale of fashion knitwear .
instead , despite the fact that many of the images in the hamburg show had been designed simply to sell condoms , and others , such as that on homophobia , to challenge social injustice , prejudice and exclusion rather than caution against hiv / aids itself,57 the exhibition cohered them all into a would - be historically uniform and medically mediated message against hiv / aids .
thus although not with conscious intent the show flew in the face of the preoccupations of the visual theorists and aids activists of the 1980s and 1990s . where they had seen in representations of aids the postmodern play of signifiers , had argued for a plurality of subjectivities involved in visual engagements , and had construed visual perceptions of the human body in general as involving an onlooker s unconscious construction of their own body through the immediate act of viewing , the curators of the hamburg exhibition saw only medicine and art in modernity .
this was not the only way in which the exhibition displaced the specific and general historical meanings attached to these objects by investing them with others .
the title alone of the exhibition , against aids : posters from around the world did as much .
first and foremost it constructed a particular framework for their perception , one that above all suggested that aesthetic form can travel the world regardless of local geographies and local histories of ethnicity , religion , race , rights , sexuality and gender not to mention alternative aesthetic traditions .
this global aesthetic spin , in effect , harmonised a modernist western transcendent notion of art with the late twentieth - century notion of a spatially transcendent capitalism
an economic system supposedly unfettered by place or national boundary.58 the aesthetic spin and the exhibition as a whole thus further performed for notions of homogeneity and universality attributes long associated with modernity and perceived to be at odds with the pluralities and fragmentations associated with postmodernity during the time of aids in the west.59 the exhibition s title also suggested that the history of aids was about everyone the world over being uniformly against aids .
but that , too , was hardly the case at the point of production of many of these posters . in the 1980s ,
christian fundamentalists and other religionists took a rather different line , and gay men and lesbians did not always see government campaigns against aids as being against aids so much as against themselves.60 as noted above , in thatcher s britain the aids campaign was an occasion for the moral high - grounding of heterosexual values.61 to the extent that people ( and international pharmaceutical companies ) were allegedly against aids in the 1980s and 1990s , their concerns emerged from a multitude of different and often conflicting social and economic interests .
moreover , the relative power of those interests was hierarchically organised , and differently so over time , as virginia berridge has made clear for the history of aids in the uk.62 also , implicit to the entitling of the hamburg exhibition was the idea that nations around the world were homogeneous in their fight against hiv / aids .
this not only collapsed separate national encounters with hiv / aids , such as its very denial by south africa s president thabo mbeki , but effaced the differences between the kinds of media campaigns used in different countries including the often bitter struggles between local , national and international agencies.63 as important , this political gutting of aids posters through their aestheticisation erased national rivalries and pressures involved in medically treating aids victims ( or not , as the case may be ) . through the mixing of posters from different countries , the exhibition dissolved the conventional boundaries between nation - states , while the multitude of images of condoms that it presented served visually to re - unite them around a commercial product .
the images of condoms promoted the idea that campaigns for their use had actually united the countries of the world , a message curiously at odds with the bush administration s contemporaneous funding of medical missionaries advocating sexual abstinence instead of the use of condoms.64 in its own small way , therefore , and for its own particular didactic reasons ( as well as , perhaps , discomfort over germany s nationalistic past ) , the hamburg exhibition effected the same kind of historical effacement and rewrite that the major international media and entertainment companies were also coming to effect by 2006 through their take of up hiv / aids for the purpose of reaping public corporate credibility.65 by then , hiv / aids - funding was a fashionable cause , a benign branding resource for various western philanthropic organisations . at local , national and transnational levels then , against aids : posters from around the world can be seen to have effaced the individual history of the objects on display through a particular universalising and seemingly neutral kind of aestheticisation . at a closer look , however , it both appropriated them into an old script ( a local and fondly held modernist epistemology of viewing and aesthetics ) and a new one globalisation . by collapsing two decades of national histories into a singular and would - be unified world fight against hiv / aids ,
the history of hiv / aids was visually construed in terms of this new global subjectivity . not only were particular constructions of the recent past left out the local struggles around these objects but also the construction of the present the global media industry s selling of itself through the attack on hiv / aids as a
real or made true through aesthetic representation of an ostensibly international struggle against hiv / aids , but by this same conceit was medically re - appropriated and humanised no matter that over the meaning of
good thing.66 this is not to suggest that hiv / aids was not recognised as a global problem almost from its start . in 1987 , the american aids activist , feminist and visual theorist , paula treichler , for instance , referred to aids as having a potential for global devastation.67 but for her and others in the 1980s the global was only a background problem , and the term was yet without particular epistemic load .
as we have indicated , western nations and their intellectuals and aids activists were gripped more by their own campaigns , interests , and ideologies than by
in fact , the idea in the west that hiv / aids was a global problem was a viewpoint that itself had to be fought for through a world wide media campaign brokered by organisations with internationalist interests .
the campaign can be dated precisely to 27 may 1987 when the world health organisation issued a press release proclaiming that aids is a global epidemic that demands a global attack.68 the who then produced a poster to sell the message ( figure 6)to compete , that is , with other struggles for the meaning of aids.69 the hamburg exhibition abetted that project through a visual rhetoric of shared international struggle against hiv / aids , just as it unwittingly abetted the subsequent take over of aids programs by the media multinationals.70 thus it eclipsed the history that would enable anyone to believe that aids had ever been anything other than global , or for that matter , anything other than mainly a struggle for economic resources for better medical provision for victims of the condition .
images intended for , and often produced by , local sub - groups became artefacts for making up global citizens , with
thereby , the exhibition did far more than merely reinforce what had become the semantic hegemony of the global , as iterated through such credulities as global warming and global terrorism.71 through aesthetics alone it rendered tangible the universalising concept of the global .
although this was not the exhibition s primary intention , it could not help but perform it , and in so doing contribute to a reconstruction of history and consciousness .
far from it ; the globally spun institution - serving celebration of the aesthetics of aids posters merely reflected and reinforced much of what everybody in the west had already come to
feel about hiv / aids by 2006that it was a serious world - wide problem about which everyone needed to be continually reminded.figure 6who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon .
who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon .
we have been concerned with a particular moment in the history of aids posters : not that of their initial
public life on the streets , or in pubs and gay clubs , doctors offices , and so on , but their afterlife in places where they might be displayed or stored.72 through the analysis of an exhibition at one such afterlife location our intention has not been that of negative dismissal , nor critique for the sake of it . nor has it been merely to expose how these often strikingly visual objects that aimed at protecting individual health had their meaning changed through appropriation into a conceptual framework different from that of their initial contexts of production and consumption .
more interesting to us are the intrinsic and unremitting links between these visual objects and wider politics , or how the visual is inherently a part of the latter . while it might have been supposed that aids posters came to political rest once they were retired , categorised , catalogued and stored according to the principles of collecting institutions , the hamburg exhibition proves otherwise .
in fact , as collector s items , they entered a space that was no less political than when they were on the streets in the 1980s and 1990s , and when they were appropriated to western discourses on postmodern identity and on the role of the visual in the cultural negotiation of the self .
retirement home they necessarily became a part of the institutional agenda of the museum fr kunst und gewerbe .
indeed , from the moment such objects become collectors items and are stored and/or displayed as artifacts they become epistemologically loaded through the very process of objectification .
hamburg s museum fr kunst und gewerbe demonstrates that these collecting agendas and the accompanying aesthetic guidelines often have deep historical roots .
but what the analysis of its 2006 exhibition of aids posters also shows is that old and seemingly apolitical agendas ( invented to express specific national political interests ) are neither lost nor rendered innocuous in the contemporary world .
rather , they come to serve new political frameworks linked to the world of today s visitors a world in which aesthetics is the dominant means to a politics constituted on little more than the idea that if it looks good go with it ( an outlook now as pervasive in the practice of science as in the arts of government).73 crucially , this new politics is sustained through , and for , the absenting of critique ; not today the critical outlook entertained by sontag and other pre - postmodern intellectuals , that visual representations ( and popular posters in particular ) covered - up or cloaked lurking ideologies .
postmodernists , unconcerned with that view for the most part , in effect opened the space for the new politics of aesthetics that masks something different : the idea of aesthetics as void of political intention .
the hamburg exhibition of aids posters was , in fact , an early example of the coming - to - reign of these particular politics , with the visual alone being the vehicle for understanding and creating a ( global ) community without distinction .
whereas for brinckmann in the nineteenth century , aesthetics ( in art ) could be consciously used for the political purpose of populist democracy , with aesthetics and politics in clearly separate spheres , for the inheritors of his institution in hamburg aesthetics ( unbeknownst to them ) became the politics , not simple a means to it . ironically , their arrival at these politics their unknowing performance of them through the exhibition was via adherence to brinckmann s legacy . through that , brinckmann s original political agenda was emptied of its original political purpose . installed in its place were the politics of the appearance of political un - intention . thus did an aesthetic concept born in the nineteenth century to serve nationalistic purposes come to operate for the political work of educating national citizens to global citizenship .
what does this mean for historians working with material objects stored in global - tending museums and archives and who are themselves now operating within a global framework ?
since material objects have no meaning without a framework , and are framed in being collected , the simple answer is that historians have to take into account the afterlives of such objects as much as the object s original lives .
harder is the problem of the historian s own place in the global framework , which , in many respects , is not unlike that of the material object in the global - aspiring museum ( and very much like that of the curators of such exhibitions ) . whether avowed explicitly in
global history , or embraced implicitly in the practice of history writing in contemporary culture , the global operates politically and epistemologically . just as global history s predecessor
world history is now perceived by some of its originators as having been a product of , and agent for , its cold war moment,74 so for our own times , dominated as they are by multi - national corporations and abiding politicians , the take up of global themes in history writing is widely recognised as providing , at the very least , legitimacy to a globalisation discourse , even if , as often the case , the historian s immediate object is the far from reactionary one of provincialising the west and critiquing its hegemony.75 of course , for some historians the global does politically more , overtly serving as a rhetorical strategy for the re - coherence of the discipline of history itself after its pummelling by poststructuralists , deconstructionists and other fragmenting postmodern forces over the past thirty years or more.76 to this end , what could serve better as a reunifying device than the holising connective metaphor of the globe ? thus the global provides a new grand narrative a universalising tool with which to reimpose the meta - narrativity of history .
although seemingly mindless of one of postmodernism s cautions , that totalising worldviews can lead to totalitarianism,77 these historians seek more - or - less intentionally what the curators of the museum fr kunst und gewerbe performed innocently through their exhibition of aids posters .
in so doing , they also share company with certain art historians anxious to revive older agendas a means ( as bluntly put by one of them ) to counter the deconstructive criticism of historical culture proposed by self - serving postmodern academics who treat the past as a sour land over which to exercise present concerns and anxieties.78 yet neither global history nor visual culture studies need necessarily lead in this direction . to see the global as a discourse tied politically to institutionally specific agendas ( such as the aesthetics entertained at the museum fr kunst und gewerbe in hamburg in 2006 ) offers the possibility to take an alternative political position , or at least to liberate its historical study from such agendas .
nevertheless , in terms of the less - visible shaping of historical knowledge production , the global framework can never be other than world - making as opposed to being simply historically descriptive.79 no matter how hard we try to stick to the recovery of some truth of how the world came to be globally conceived ( past or present ) , our knowledge productions implicitly reproduce and foster the unifying construct .
just as with material objects ( or words or images ) there is no meaning to historical events outside the conceptual frameworks we wittingly or unwittingly apply to them . in short , there exists no resting place for history writing ; it is always already fashioned and fashioning . like creating a museum exhibition of posters to aestheticise the
global nature of hiv / aids , the business of contributing to a global history of anything entails , by that very act , the politics of constructing a necessarily partial representation of the past. history writing , too , in other words , as a product of its time , can not avoid making up historical consciousness .
this article can not escape the charge that it too contributes to this process merely by discussing an event that was conceptualised in
it might even be seen to compound the problem by drawing attention to spaces where , a priori , the historian is already politically and epistemologically implicated : the museum and the archive .
however , in doing so it has sought to move the discussion beyond the tired call for attending merely to historical contexts , especially of material objects globally attributed .
our purpose has been to encourage historians to an awareness of their own immediate entanglements in history s constructedness the constructedness of the present mediated in history writing as much as through aesthetic assemblages of the global. the historian aruf dirlik , critically inquiring into the point of writing world history , has observed that an awareness of the variety of world histories that have been constructed at different times and in different places [ must cause ] any world historian worthy of the name [ to ] be uncommonly aware of the constructedness of the past.80 similarly , we submit , all historians need be reflective on their contributions to the present that is , to a culture given to re - enchantment through the global. quite how we should historicise material objects of the sort discussed in this paper may be open to debate ; what is not is the necessity to historicise our own historical projects .
otherwise we move perilously close to becoming blind participants in the historically fashioned spaces where memory is increasingly naturalised and neutralised through universalised and universalising concepts mediated aesthetically .
we end up , as it were , nave viewers of the exhibition at hamburg : as blind to the nature of the new post - postmodern politics of aesthetics , as to the modernist would - be universal humanity that the global unwittingly espouses through those politics . | drawing on recent visual and spatial turns in history writing , this paper considers aids posters from the perspective of their museum
afterlife as collected material objects .
museum spaces serve changing political and epistemological projects , and the visual objects they house are not immune from them . a recent globally themed exhibition of aids posters at an arts and crafts museum in hamburg is cited in illustration .
the exhibition also serves to draw attention to institutional continuities in collecting agendas . revealed , contrary to postmodernist expectations , is how today s application of aesthetic display for the purpose of making
global connections does not radically break with the virtues and morals attached to the visual at the end of the nineteenth century .
the historicisation of such objects needs to take into account this complicated mix of change and continuity in aesthetic concepts and political inscriptions .
otherwise , historians fall prey to seductive aesthetics without being aware of the politics of them .
this article submits that aesthetics is politics . | Introduction
The Hamburg Exhibition
Aesthetic Framing
Re-picturing AIDS
Conclusion |
single - fiber electromyography ( sfemg ) was introduced by stlberg and ekstedt in the 1960s to study neuromuscular junction transmission failure .
the single - fiber needle ( sfn ) electrodes were designed to study single muscle fiber over small hemispheric recording area with a radius of about 300 m . however ,
sfn electrodes are expensive , and there has been increasing concern about reusing material for invasive medical procedures ; thus , an acceptable , inexpensive alternative to the sfn has been sought .
jitter recorded with concentric needle ( cn ) electrodes has been studied in healthy controls and in myasthenia gravis ( mg ) patients .
it has been shown that cn and sfn electrodes yield comparable jitter results in most studies .
sfemg has been suggested as a quantitative method for supporting chronic partial denervation in amyotrophic lateral sclerosis ( als ) by the revised ei escorial criteria .
due to progressive denervation and reinnervation , immature nerve terminals , and impaired transmission in the endplates , sfemg in als patients showed increased jitter and fiber density ( fd ) , which reflected the neuromuscular junction transmission failure and reinnervation condition due to the progression of als .
sfemg could also provide some information for evaluating the prognosis of als and differentiating als from cervical spondylosis .
conventional cn electromyography is the standard technique for evaluating denervation and reinnervation in neurogenic diseases .
amplitude , duration , area , and phases of motor unit potential ( mup ) are parameters studied conventionally .
instability of the cn - mup is also routinely qualitatively observed , which reflects the dysfunction of neuromuscular junction transmission .
jiggle has been attempted by measuring consecutive amplitude differences and cross - correlational coefficient of consecutive discharges . however ,
jiggle is not commonly used because the special software required is not routinely installed in electromyogram equipment .
although cn electrode has been used to assess jitter in mg patients and healthy controls , no report had used cn electrode to assess jitter in neurogenic diseases to investigate motor unit instability .
signals recorded with cn electrodes , even if visibly indistinguishable from single - fiber action potentials , may actually be composed of synchronized and near - simultaneous action potentials from more than one muscle fiber ; thus , cn electrodes can not be used to measure fd as it is defined in sfemg because of the larger uptake radius .
we are interested in whether we can use the sfemg program and cn electrode rather than sfn electrode to study neuromuscular junction transmission failure and reinnervation in als .
group 2 comprised 23 als patients , including 13 definite and ten probable als patients according to the awaji criteria .
there was no difference in the mean age ( 50.3 , 52.6 years , respectively ) of the two groups at the time of the study .
the study was performed with the sfemg program of the counterpoint emg machine ( dantec electronics , denmark ) . both cn and sfn electrodes were used for the study .
the cn electrodes used were facial needle electrodes with recording surface dimensions of 0.019 mm .
recordings with both electrodes were performed on the extensor digitorum communis ( edc ) muscle under voluntary contraction on the same day . in als
patients , the medical research council scale was grade 5 in the edc muscles studied .
the sfn - jitter and single - fiber needle - fiber density ( sfn - fd ) were defined using sfns and the sfemg program .
action potentials with amplitudes > 200 v and rise times < 300 s were selected for calculating the fd and jitter .
the cn - jitter and spike number were defined using cns and the sfemg program .
the methods and criteria for acquiring and analyzing the parameters were the same for sfn and cn , except that the low - frequency filter was 500 hz when using sfn and 1000 hz when using cn .
the parameter data were collected for each subject as shown in the sfemg program . when studied with cn electrodes ,
the cn - jitter was recorded as the jitter data listed in the sfemg program , and the spike number was recorded as the fd listed in the sfemg program , although the anatomical substrates of the cn - jitter and spike number are different from those of the sfn - jitter and sfn - fd .
differences in parameters between different groups were analyzed by independent sample student 's t - test .
differences in parameters between two needle types in the same groups were analyzed by paired sample student 's t - test .
receiver operating characteristic ( roc ) curve analysis was performed to explore the accuracy of the cn - jitter and spike number in differentiating als patients from the healthy controls .
group 2 comprised 23 als patients , including 13 definite and ten probable als patients according to the awaji criteria .
there was no difference in the mean age ( 50.3 , 52.6 years , respectively ) of the two groups at the time of the study .
the study was performed with the sfemg program of the counterpoint emg machine ( dantec electronics , denmark ) . both cn and sfn electrodes were used for the study .
the cn electrodes used were facial needle electrodes with recording surface dimensions of 0.019 mm .
recordings with both electrodes were performed on the extensor digitorum communis ( edc ) muscle under voluntary contraction on the same day . in als
patients , the medical research council scale was grade 5 in the edc muscles studied .
the sfn - jitter and single - fiber needle - fiber density ( sfn - fd ) were defined using sfns and the sfemg program .
action potentials with amplitudes > 200 v and rise times < 300 s were selected for calculating the fd and jitter .
the cn - jitter and spike number were defined using cns and the sfemg program .
the methods and criteria for acquiring and analyzing the parameters were the same for sfn and cn , except that the low - frequency filter was 500 hz when using sfn and 1000 hz when using cn .
the parameter data were collected for each subject as shown in the sfemg program . when studied with cn electrodes ,
the cn - jitter was recorded as the jitter data listed in the sfemg program , and the spike number was recorded as the fd listed in the sfemg program , although the anatomical substrates of the cn - jitter and spike number are different from those of the sfn - jitter and sfn - fd .
differences in parameters between different groups were analyzed by independent sample student 's t - test .
differences in parameters between two needle types in the same groups were analyzed by paired sample student 's t - test .
receiver operating characteristic ( roc ) curve analysis was performed to explore the accuracy of the cn - jitter and spike number in differentiating als patients from the healthy controls .
the parameters recorded using cn electrodes and sfn electrodes in the two groups are shown in table 1 .
there was no significant difference between the mean cn - jitter and sfn - jitter in als patients ( t = 1.854 , p = 0.072 ) and healthy controls ( t = 0.986 , p = 0.333 ) .
the waveforms recorded from an als patient and a healthy control using sfns and cns in the sfemg program are shown in figure 1 .
parameters recorded using sfn electrode and cn electrode in the edc muscle of als patients and healthy controls data are shown as mean sd . * a significant difference in the parameter between als patients and healthy controls ( p<0.001 ) .
sfn : single - fiber needle ; cn : concentric needle ; edc : extensor digitorum communis ; als : amyotrophic lateral sclerosis ; sd : standard deviation .
waveforms obtained using sfn electrode and cn electrode in the sfemg program in als and healthy controls .
consecutive discharges of the same motor unit were recorded from the extensor digitorum communis muscle during voluntary contraction .
( a ) recorded by sfn electrode in a normal subject , ( b ) recorded by cn electrode in a normal subject , ( c ) recorded by sfn electrode in an als patient , and ( d ) recorded by cn electrode in an als patient .
the low - cut filter setting was 500 hz for ( a ) and ( c ) , 1 khz for ( b ) and ( d ) .
signals recorded with cn were visibly indistinguishable from single - fiber waveforms , although the former might actually comprise synchronized and near - simultaneous action potentials from more than one muscle fiber .
sfn : single - fiber needle ; cn : concentric needle ; sfemg : single - fiber electromyography ; als : amyotrophic lateral sclerosis .
the mean cn - jitter was significantly increased in als patients than in healthy controls ( t = 5.973 , p < 0.001 )
. the mean spike number was significantly increased in als patients ( 2.5 0.5 ) than in healthy controls ( 1.7 0.3 ) ( t = 9.206 , p < 0.001 ) .
roc curve analysis showed that the area under the curve was 0.871 ( 95% confidence interval [ ci ] : 0.753 , 0.989 ) for the mean cn - jitter and 0.966 ( 95% ci : 0.906 , 1.000 ) for the mean spike number [ figure 2 ] .
the sensitivity and specificity in the diagnosis of als were 82.6% and 92.6% for the mean cn - jitter ( cut - off value : 32 s ) and 91.3% and 96.3% for the mean spike number ( cut - off value : 2.0 ) , respectively .
roc curve analysis of spike number and jitter using concentric needles to differentiate als patients from healthy controls .
the dotted line indicated the roc curve of the mean spike number using concentric needles .
both roc curves showed the high area under curve values ( 0.966 for the mean spike number and 0.871 for the mean jitter ) .
sfemg has been studied mainly in patients with neuromuscular transmission disorders . although sfemg has been suggested as one of the quantitative methods for supporting chronic partial denervation in als by the revised ei escorial criteria , few reports have studied sfemg in neurogenic diseases .
we are interested in whether the cn - jitter and spike number can substitute the sfn - jitter and sfn - fd to assess neuromuscular junction transmission and chronic reinnervation in neurogenic diseases . in this study
, we found that both cn and sfn electrodes showed significantly increased jitter in als patients compared with healthy controls . despite differences in the anatomical substrates and physiological meanings between sfn - jitter and cn - jitter , jitter recorded using
cn electrodes has been accepted as a parameter for assessing neuromuscular transmission in mg in most published reports .
the waveform obtained using a cn electrode in the sfemg program represents the mup composed of different numbers of spikes .
both the sfn- and cn - jitters reflect a similar physiological phenomenon ( variability in the arrival time of muscle fiber action potentials to the recording electrode ) and clinical significance .
the jitter of those spikes ( cn - jitter ) reflects the variability of the interval to the recording site between individual components of the mups during firing , whereas the sfn - jitter reflects the variability of the interval to the recording site between individual muscle fibers . unlike in mg , increased jitter in als is the result of an immature endplate due to the progressive degeneration and reinnervation of lower motor neurons .
we suggest that cn - jitter might be used to quantitatively assess mup instability in neurogenic diseases . in als , the variability between mups or mup components results from asynchronous conduction at the motor nerve terminal and neuromuscular junction .
we also found that both the spike number and sfn - fd were increased in als patients compared with the healthy controls .
the spike number , recorded with cns in the sfemg program , is a new term that we first proposed .
we adopted the same criteria for calculating spike number as fd in the sfemg program . although the signals recorded with the cn electrode are visibly indistinguishable from single - fiber action potentials in the sfemg program , the spike number and fd had entirely different anatomical substrates .
the main difference between sfn electrodes and cn electrodes is the areas of their recording surfaces . with a smaller recording area ( 0.0005 mm )
, sfns can identify the action potential from an individual muscle fiber ( i.e. , each spike represents one muscle fiber ) , which allows for the measurement of fd and jitter between two different muscle fibers . for cn electrodes , with a larger recording area ( 0.019 mm ) , each spike of the signal actually comprises synchronized and near - simultaneous action potentials from a group of muscle fiber s. each motor unit may have one or more groups of summated muscle fibers .
the spike number is similar to the number of phases or turns recorded with cn electrodes in conventional emg ; the difference in waveforms between them is due to different filter settings .
each spike mainly reflects the summated action potentials of several muscle fibers nearest to the needle . in healthy controls ,
patients , due to denervation and reinnervation , more muscle fibers are innervated by survival motor neurons ; therefore , the spike number increases .
two pathological and physiological changes may affect the spike number during the denervation - reinnervation process in neurogenic diseases .
first , the actual fd of an individual motor unit increases due to collateral reinnervation , and more muscle fibers in the same area can thus be recorded by cn electrodes .
second , asynchronous conduction increases due to an immature motor nerve terminal and neuromuscular junction following denervation - reinnervation ; thus , the duration of excitatory signal propagation from muscle fibers to the recording needle significantly varied , allowing more waveform spikes to be obtained .
although the spike number obtained with cn electrodes is from groups of muscle fibers and not the actual number of muscle fibers in the recording area , the changes in spike number might have the ability to indirectly reflect changes in fd and asynchronous conduction in the motor unit .
this condition may be similar to calculating the number of motor units using motor unit number estimation , which is also a relative number and not the actual number of motor units .
in fact , many factors might affect the spike number results in neurogenic diseases ; however , all of the theoretical problems are hypotheses or result from computer simulations .
it is not necessary to argue how many exact muscles fibers are involved in composing waveform spikes ; instead , we are interested in whether the method can aid in clinical work or in research .
our data showed that the mean jitter and spike number recorded with cn electrodes in als patients were significantly higher than those in healthy controls .
clinicians often prefer to select an individual point defined by an roc curve to diagnose certain diseases .
our data showed that the mean cn - jitter and spike number had high sensitivity and specificity for differentiating als patients from healthy controls .
however , the sensitivity and specificity of a diagnostic test largely depend on the clinical context in which the test will be used .
although it is seldom necessary to differentiate als patients from healthy controls in practice , the roc curve data still indicated that the cn - jitter and spike number could provide some quantitative information as those recorded with sfns in als patients . in conclusion , theoretically , waveform spikes obtained using cn electrodes with the sfemg program have different anatomical substrates and physiological meaning as those obtained using sfns ; in practice , the parameters recorded with cn and sfn electrodes are highly comparable in als patients .
cn electrodes may be an alternative to sfns in als studies to quantitatively evaluate the changes in jitter and spike number .
the cn - jitter and spike number have high sensitivity and specificity for differentiating als from healthy controls and might be helpful to quantitatively investigate the changes due to denervation - reinnervation in als . | background : single - fiber electromyography ( sfemg ) has been suggested as a quantitative method for supporting chronic partial denervation in amyotrophic lateral sclerosis ( als ) by the revised ei escorial criteria .
although concentric needle ( cn ) electrodes have been used to assess jitter in myasthenia gravis patients and healthy controls , there are few reports using cn electrodes to assess motor unit instability and denervation in neurogenic diseases .
the aim of this study was to determine whether quantitative changes in jitter and spike number using cn electrodes could be used for als studies.methods:twenty-seven healthy controls and 23 als patients were studied using both cn and single - fiber needle ( sfn ) electrodes on the extensor digitorum communis muscle with an sfemg program .
the sfn - jitter and sfn - fiber density data were measured using sfn electrodes . the cn - jitter and spike number were measured using cn electrodes.results:the mean cn - jitter was significantly increased in als patients ( 47.3 17.0 s ) than in healthy controls ( 27.4 3.3 s ) ( p < 0.001 ) .
besides , the mean spike number was significantly increased in als patients ( 2.5 0.5 ) than in healthy controls ( 1.7 0.3 ) ( p < 0.001 ) . the sensitivity and
specificity in the diagnosis of als were 82.6% and 92.6% for cn - jitter ( cut - off value : 32 s ) , and 91.3% and 96.3% for the spike number ( cut - off value : 2.0 ) , respectively .
there was no significant difference between the sfn - jitter and cn - jitter in als patients ; meanwhile , there was no significant difference between the sfn - jitter and cn - jitter in healthy controls.conclusion:cn-jitter and spike number could be used to quantitatively evaluate changes due to denervation - reinnervation in als . | I
M
Subjects
Single-fiber electromyography studies
Statistical analysis
R
D
Financial support and sponsorship
Conflicts of interest |
follicular lymphoma is the most common subtype of non - hodgkin s lymphoma ( nhl ) 1
2 .
gastrointestinal follicular lymphoma mainly comprises scattered small whitish nodules , mass lesions and polyps , while other signs are exceedingly rare .
the recent development of enteroscopy devices , such as double - balloon enteroscopy ( dbe ) and capsule endoscopy , allows gastroenterologists to detect atypical lesions of gastrointestinal follicular lymphoma .
herein , we report an unusual case of gastrointestinal follicular lymphoma presenting with an annular ulcer .
she had no history of abdominal surgery or use of non - steroidal anti - inflammatory drugs ( nsaids ) .
laboratory findings included hemoglobin 5.9 g / dl and mean corpuscular volume of 63.0 fl .
enhanced computed tomography ( ct ) of the abdomen revealed swelling of multiple intraperitoneal lymph nodes and a high density of mesenteric adipose tissue ( fig . 1 ) .
fluorodeoxyglucose ( fdg)-positron emission tomography ( pet ) showed high fdg accumulation at the intraperitoneal lymph nodes .
dbe was performed using a retrograde approach , and severe stenosis with an annular ulcer was detected in the lower ileum ( fig . 2 ) .
enhanced computed tomography ( ct ) of the abdomen revealed swelling of multiple intraperitoneal lymph nodes ( arrow ) and a high density of mesenteric adipose tissue .
double - balloon enteroscopy ( dbe ) was performed using the anal approach , and severe stenosis with an annular ulcer was detected in the lower ileum .
the differential diagnosis of ileal annular ulcers includes intestinal tuberculosis , nsaid - related ulcers , crohn s disease , localized ischemia of the small intestine , and lymphoma .
nsaid - related ulcers were ruled out because she had no history of taking nsaids .
histologic examination of biopsy specimens from the ileal ulcer revealed a concentration of medium - sized atypical lymphocyte - like cells that were immunohistochemically positive for cd10 , cd20 , and bcl-2 antibodies , and negative for cd3 and cd5 antibodies ( fig .
fluorescence in situ hybridization analysis showed igh - bcl2 rearrangement in 67 % of the ileal ulcer cells ( fig .
she was diagnosed with primary ileal follicular lymphoma , stage ii2 based on the lugano classification due to metastasis to distant abdominal lymph nodes .
histologic examination of biopsy specimen from the ileal ulcer ( photomicrograph original magnification 400 ) , and fluorescence in situ hybridization ( fish ) analysis .
chemotherapy with rituximab and bendamustine was selected because of her advanced age and the low grade malignancy of follicular lymphoma .
combination chemotherapy with rituximab and bendamustine has equivalent effects and lower toxicity than polypharmacy with rituximab , cyclophosphamide , hydroxydaunorubicin , oncovin , and prednisone ( r - chop ) in nhl with low grade malignancy such as follicular lymphoma .
moreover , swelling of multiple intraperitoneal lymph nodes observed with enhanced ct , and high fdg accumulation at the intraperitoneal lymph nodes with fdg - pet also disappeared .
most cases of follicular lymphoma are initially detected in the lymph nodes , and widespread nodal involvement such as ann arbor stage iii or iv is a typical presentation of this disease , whereas only 5 10 % of patients present with early stage i or ii 3 . the gastrointestinal tract is the common site of extra - nodal nhl 1
2 .
although extra - nodal involvement usually occurs as a result of disseminated nodal disease , primary gastrointestinal nhl accounts for 30 40 % of all extra - nodal nhl 4 .
most common subtypes of primary gastrointestinal nhl are mucosal - associated lymphoid tissue lymphoma or high grade diffuse large b - cell lymphoma , while primary gastrointestinal follicular lymphoma is a relatively rare disease accounting for only 1 % of gastrointestinal nhl 5 .
gastrointestinal follicular lymphoma lesions comprise mainly scattered small whitish nodules 1 2 mm in diameter and subsequent mass lesions with or without ulceration .
in previous studies of 222 cases with small intestinal follicular lymphoma , scattered small whitish nodules , mass lesions , and polyps were detected as follicular lymphoma lesions in the small intestine in 183 cases ( 82.4 % ) , 29 cases ( 13.1 % ) , and six cases ( 2.7 % ) , respectively , and other morphology was detected in only four cases ( 1.8 % ) 1 .
studies performed before the introduction of dbe or capsule endoscopy revealed that gastrointestinal follicular lymphoma lesions were mostly unifocal , and the proportion of ileal localization was low 6
7
8 .
recent studies , however , reported that dbe detected new jejunoileal follicular lymphoma lesions in 73 85.7 % of cases with duodenal follicular lymphoma 6
7 .
moreover , 24 of 33 patients with gastrointestinal follicular lymphoma ( 72.7 % ) who underwent enteroscopy had multifocal involvement , mostly in the duodenum and ileum 1 .
in addition , fdg - pet was not always effective in detecting gastrointestinal follicular lymphoma lesions , because three - quarters of patients with gastrointestinal follicular lymphoma who had been diagnosed by dbe were negative for fdg - pet 8 .
therefore , investigation of the small intestine is recommended for the diagnosis of gastrointestinal follicular lymphoma . with the increasing use of dbe for observation of small intestinal lesions , the frequency of gastrointestinal follicular lymphoma with atypical changes might increase .
based on the present case , gastrointestinal follicular lymphoma should be considered in the differential diagnosis of annular ulcers in the small intestine . | a 72-year - old woman presented with symptomatic anemia without abdominal symptoms .
she had no history of abdominal surgery or use of non - steroidal anti - inflammatory drugs .
enhanced computed tomography of the abdomen revealed swelling of multiple intraperitoneal lymph nodes and a high density of mesenteric adipose tissue .
fluorodeoxyglucose ( fdg- ) positron emission tomography showed high fdg accumulation at the intraperitoneal lymph nodes .
double - balloon enteroscopy detected severe stenosis with an annular ulcer in the lower ileum .
she was diagnosed with ileal follicular lymphoma based on histologic examination and fluorescence in situ hybridization analysis of the biopsy specimen .
the ileal ulcer was successfully treated by chemotherapy with rituximab and bendamustine for 1 year .
we strongly recommend consideration of gastrointestinal follicular lymphoma in the differential diagnosis of annular ulcers in the small intestine . | Background
Case report
Discussion |
to report on a case of keratoconjunctivitis with periorbital edema after accidental exposure to undiluted juice of ecballium elaterium fruit during the ripening season of this plant .
keratoconjunctivitis with descemet 's membrane folds and superficial upper corneal well - defined edematous areas were noted after an ecballium elaterium fruit burst and its juice splashed into the patient 's left eye .
prompt administration of antibiotic and steroid eye drops along with per os antihistamine therapy , resulted in quick regression of symptoms .
this report demonstrates the toxic effects of this herb to the eye and also that prompt therapy is effective .
a 70-year - old male farmer presented to the emergency department with periorbital pain , periorbital and conjunctival edema , epiphora , hyperemia , irritation , and blurred vision from his left eye after inadvertent exposure to undiluted juice of ecballium elaterium fruit some hours earlier .
the symptoms began within minutes after the herb 's fruit bursted and juice splashed into the eye ( fig .
slit lamp examination revealed corneal epithelial micro - erosions , corneal superficial well - defined edematous areas mainly in the peripheral upper quadrants ( fig .
the patient was treated promptly with eye irrigation , topical steroids for periorbital edema , combination of antibiotic and steroid eye drops along with antihistamine tablets . within four days from the initiation of treatment ,
exposure to ecballium elaterium fruit juice , mainly in its undiluted form , may cause irritation of the mucous membrane .
elaterium is the dried sediment that deposits in the juice of the fruits of ecballium elaterium .
it is a powerful cathartic and contains up to 30% of elaterin , which is the active ingredient .
the toxic dose is 5 mg and the fatal dose 0.6 g. published adverse reactions related to the toxic effect of this remedy include localized swelling of the uvula , the nasal mucosa and others sites of the upper respiratory tract of allergy - prone patients .
also , there is a report of fatal cardiac and renal failure probably related to ecballium elaterium juice intake . regarding the eye , kocak et al .
reported a case of late - onset diffuse lamellar keratitis 11 months after lasik , when an ecballium elaterium herb fruit burst and splashed into the patient 's eye .
also , raikhlin - eisenkraft and bentur report 6 cases with ocular exposure presented with conjunctivitis , corneal edema , and erosion .
this report demonstrates the toxic effect of ecballium elaterium juice to the eye and also , that prompt therapy results in rapid regression of symptoms . | purposeto report on a case of keratoconjunctivitis with periorbital edema after accidental exposure to undiluted juice of ecballium elaterium fruit during the ripening season of this plant.methodscase report.resultskeratoconjunctivitis with descemet 's membrane folds and superficial upper corneal well - defined edematous areas were noted after an ecballium elaterium fruit burst and its juice splashed into the patient 's left eye .
prompt administration of antibiotic and steroid eye drops along with per os antihistamine therapy , resulted in quick regression of symptoms.conclusionsthis report demonstrates the toxic effects of this herb to the eye and also that prompt therapy is effective . | Purpose
Methods
Results
Conclusions
Case Report
Discussion
Conclusion |
cmm is highly characteristic of aggressive invasion , early metastasis , and resistance to chemotherapy or radiotherapy , which results in increased incidence and mortality worldwide . despite improvements in early cmm diagnosis ,
therefore , better understanding of the molecular mechanisms of cmm tumorigenesis and progression will help to explore novel therapeutic agents and prognostic markers in the treatment of patients with cmm .
mirnas are small noncoding rnas , usually 2023 nucleotide long , which regulate the expression of protein - coding genes at the post - transcriptional level .
previous studies have also shown that aberrant mirna expression is involved in the development and progression of cancer [ 510 ] ; thus , mirnas could be used as biomarkers for diagnosis and prognosis of cancer , and as targets for molecular cancer therapy [ 1114 ] .
mir-221 has been reported to be overexpressed in human tumor tissues such as breast cancer , colorectal cancer , and glioblastoma [ 1519 ] .
previous in vitro and in vivo functional studies have confirmed the key role of mir-221 in regulating the progression of human melanoma .
kanemaru et al . found that serum levels of mir-221 were significantly increased in patients with malignant melanoma and that they may be useful for the diagnosis of malignant melanoma .
however , they did not investigate the relationship between serum mir-221 expression level and the prognosis of malignant melanoma .
the present study investigated the feasibility of using serum mir-221 as a noninvasive prognostic biomarker for cmm .
serum samples were obtained from 72 patients with cmm from the department of cosmetic surgery , the third xiangya hospital , from may 2004 to june 2013 .
the histological diagnosis , breslow thickness , and clark level were re - examined in 15 original sections of the primary tumor by the same pathologist , who was unaware of the clinical data .
blood samples were drawn from all patients and healthy control subjects at the beginning of treatment .
a 5-ml sample of peripheral venous blood was drawn from all study participants after an overnight fast and placed at room temperature for 60 min
. then the blood samples were centrifuged at 1000 g for 10 min at 4c to spin down the blood cells .
this project was approved by the clinical research ethics committee of the third xiangya hospital .
the isolation of mirna from serum samples was performed using the mirneasy rna isolation kit ( qiagen , valencia , ca , usa ) according to the manufacturer s instructions , with minor modifications .
briefly , 200 ml of serum was supplemented with 10 ml of 0.05 f 0.0 synthetic nonhuman mirna ( caenorhabditis elegans mir-54 , takara bio , shiga , japan ) as controls , providing an internal reference for normalization of technical variations between samples .
after 1 ml of qiazol lysis reagent ( qiagen ) was added and well mixed ( by gentle vortexing ) , the samples were incubated at room temperature for 5 min .
the aqueous phase was done in an rneasy spin column and rneasy minelute spin column ( takara bio ) .
to carry out quantitative real - time reverse transcription polymerase chain reaction ( rt - pcr ) of serum mir-221 levels , cdna was synthesized from mirna with a mirx mirna first strand synthesis and sybr green quantitative rt - pcr kit ( takara bio ) according to the manufacturer s instructions .
quantitative rt - pcr was carried out using the thermal cycler dice tp800 ( takara bio ) with primers and cdna templates mixed with the sybr premix .
dna was amplified for 50 cycles of denaturation for 5 s at 95c and annealing for 20 s at 60c .
analysis of relative mirna and mrna expression was performed using the ct method , with mir-16 as endogenous controls , following the manufacturer s guidelines .
data analysis was performed using abi prism 7300 sds software ( applied biosystems , foster city , ca , usa ) .
statistical analysis was performed using spss 17.0 ( spss , ibm , chicago , il , usa ) . for continuous variables ,
the mann - whitney u test was used to analyze the relationship between mir-221 expression and clinicopathological variables . the overall survival ( os ) and disease - free survival ( dfs ) were calculated using the kaplan - meier method .
the differences between the survival curves were tested by using the log - rank test .
the cox proportional hazards regression model was used to determine the joint effects of several variables on survival .
serum samples were obtained from 72 patients with cmm from the department of cosmetic surgery , the third xiangya hospital , from may 2004 to june 2013 .
the histological diagnosis , breslow thickness , and clark level were re - examined in 15 original sections of the primary tumor by the same pathologist , who was unaware of the clinical data .
blood samples were drawn from all patients and healthy control subjects at the beginning of treatment .
a 5-ml sample of peripheral venous blood was drawn from all study participants after an overnight fast and placed at room temperature for 60 min
. then the blood samples were centrifuged at 1000 g for 10 min at 4c to spin down the blood cells .
this project was approved by the clinical research ethics committee of the third xiangya hospital .
the isolation of mirna from serum samples was performed using the mirneasy rna isolation kit ( qiagen , valencia , ca , usa ) according to the manufacturer s instructions , with minor modifications .
briefly , 200 ml of serum was supplemented with 10 ml of 0.05 f 0.0 synthetic nonhuman mirna ( caenorhabditis elegans mir-54 , takara bio , shiga , japan ) as controls , providing an internal reference for normalization of technical variations between samples .
after 1 ml of qiazol lysis reagent ( qiagen ) was added and well mixed ( by gentle vortexing ) , the samples were incubated at room temperature for 5 min .
the aqueous phase was done in an rneasy spin column and rneasy minelute spin column ( takara bio ) .
the microrna was eluted from the column with nuclease - free water . to carry out quantitative real - time reverse transcription polymerase chain reaction ( rt - pcr ) of serum mir-221 levels ,
cdna was synthesized from mirna with a mirx mirna first strand synthesis and sybr green quantitative rt - pcr kit ( takara bio ) according to the manufacturer s instructions .
quantitative rt - pcr was carried out using the thermal cycler dice tp800 ( takara bio ) with primers and cdna templates mixed with the sybr premix .
dna was amplified for 50 cycles of denaturation for 5 s at 95c and annealing for 20 s at 60c .
analysis of relative mirna and mrna expression was performed using the ct method , with mir-16 as endogenous controls , following the manufacturer s guidelines .
data analysis was performed using abi prism 7300 sds software ( applied biosystems , foster city , ca , usa ) .
statistical analysis was performed using spss 17.0 ( spss , ibm , chicago , il , usa ) . for continuous variables ,
the mann - whitney u test was used to analyze the relationship between mir-221 expression and clinicopathological variables . the overall survival ( os ) and disease - free survival ( dfs ) were calculated using the kaplan - meier method .
the differences between the survival curves were tested by using the log - rank test .
the cox proportional hazards regression model was used to determine the joint effects of several variables on survival .
expression levels of mir-221 were detectable in circulation in our study population . using mir-16 to normalize samples
, we observed that the serum mir-221 levels were significantly higher in patients with cmm than in healthy controls ( p<0.0001 , figure 1 ) . in this study , patients with values less than the median expression level of 2.95 were assigned to the low expression group ( n=36 ) , whereas those with values 2.95 were assigned to the high expression group ( n=36 ) .
as shown in table 1 , high mir-221 expression was correlated with tumor thickness ( p=0.03 ) , poor differentiation ( p=0.01 ) , higher t classification ( p=0.04 ) , higher n classification ( p=0.009 ) , metastasis ( p<0.001 ) , and advanced clinical stage ( p<0.001 ) .
however , high mir-221 expression was not associated with other clinicopathological factors of patients , including age , sex , ulceration , histologic type , or tumor site ( all p>0.05 ) . as shown in figure 2 ,
patients with high serum mir-221 level had a significantly lower 5-year os rate ( 22.1% vs. 54.6% ; p=0.018 ; figure 2a ) and rfs rate ( 12.5% vs. 45.2% ; p=0.008 ; figure 2b ) than those with low serum mir-221 level .
table 2 shows multivariate analyses of clinical variables and serum mir-221 levels for their prognostic influence on os and rfs . in a multivariate cox model
, we found that mir-221 expression was an independent poor prognostic factor for both 5-year os ( hazards ratio [ hr]=3.189 , 95% confidence interval [ ci]=1.7826.777 , p=0.007 ) and 5-year dfs ( hr=2.119 , ci=1.9628.552 , p=0.01 ) in cmm .
cmm is an aggressive and chemoresistant form of skin cancer characterized by malignant growth and poor patient prognosis .
although many novel strategies have been used recently for the diagnosis and treatment of cmm , the prognosis is still very poor .
improvement in cmm outcomes largely depends on increasing our understanding of the molecular biology and regulatory mechanisms underlying the growth of cmm .
furthermore , there is still a need to improve early detection screening methods and to identify new prognostic biomarkers .
ideal biomarkers should be easy to measure and have a strong association with clinical outcome .
mirnas may match these proposed criteria and could be used as biomarkers for diagnosis and prognosis of cancer . among all the cancer - related mirnas , mir-221 , which is considered a micro - oncogene , was reported to be increasingly expressed in various carcinomas , including liver cancer , breast cancer , prostate cancer , colorectal carcinoma , melanoma , and acute myeloid leukemia [ 1519 ] .
furthermore , high level of mir-221 expression is correlated with metastasis , tumor capsular infiltration , tumor stage , and poor prognosis .
mir-221 plays an important role in epithelial - to - mesenchymal transition ( emt ) .
it has been identified as a basal - like subtype - specific mirna that downregulates the expression of epithelial - specific genes and enhances the expression of mesenchymal - specific genes .
the basal - like transcription factor , fosl1 , can directly stimulate the transcription of mir-221 . the abundance of mir-221 declines with the suppression of mitogen - activated or extracellular signal - regulated protein kinase ( mek ) .
the mir-221-mediated reduction in e - cadherin is dependent on the targeting of the 3-utr of trichorhinophalangeal syndrome type 1 ( trps1 ) .
trps1 inhibits emt by directly repressing the expression of zinc finger e - box - binding homeobox 2 ( zeb2 ) .
thus , mir-221 could contribute to the aggressive clinical behavior of various types of cancers .
kanemaru et al . found that serum levels of mir-221 were significantly increased in patients with malignant melanoma and that mir-221 levels may be useful for the diagnosis of malignant melanoma .
however , they did not investigate the relationship between serum mir-221 expression level and the prognosis of malignant melanoma .
our study aimed to investigate the feasibility of using serum mir-221 as a noninvasive prognostic biomarker for cmm . in the present study , we showed that individuals with cmm had significantly elevated levels of tissue mir-221 expression , and that high level of mir-221 expression was significantly correlated with tumor thickness , poor differentiation , t classification , n classification , metastasis , and advanced clinical stage , suggesting that the overexpression of mir-221 might be able to play a critical role in the development of cmm .
in addition , we also found that the 5-year os and dfs rates of patients with high mir-221 expression were significantly lower than those of patients with low mir-221 expression , suggesting that mir-221 has potential for use as a molecular marker for predicting the prognosis of cmm patients . to further confirm this ,
our data indicate that mir-221 status might be an independent prognostic factor for cmm patients . to the best of our knowledge ,
we are the first to report that patients with higher serum mir-221 levels have a significantly lower survival rate than those with lower expression levels , revealing the usefulness of serum mir-221 expression as an independent prognosis factor in cmm .
expression levels of mir-221 were detectable in circulation in our study population . using mir-16 to normalize samples
, we observed that the serum mir-221 levels were significantly higher in patients with cmm than in healthy controls ( p<0.0001 , figure 1 ) . in this study , patients with values less than the median expression level of 2.95 were assigned to the low expression group ( n=36 ) , whereas those with values 2.95 were assigned to the high expression group ( n=36 ) .
as shown in table 1 , high mir-221 expression was correlated with tumor thickness ( p=0.03 ) , poor differentiation ( p=0.01 ) , higher t classification ( p=0.04 ) , higher n classification ( p=0.009 ) , metastasis ( p<0.001 ) , and advanced clinical stage ( p<0.001 ) .
however , high mir-221 expression was not associated with other clinicopathological factors of patients , including age , sex , ulceration , histologic type , or tumor site ( all p>0.05 ) .
as shown in figure 2 , patients with high serum mir-221 level had a significantly lower 5-year os rate ( 22.1% vs. 54.6% ; p=0.018 ; figure 2a ) and rfs rate ( 12.5% vs. 45.2% ; p=0.008 ; figure 2b ) than those with low serum mir-221 level .
table 2 shows multivariate analyses of clinical variables and serum mir-221 levels for their prognostic influence on os and rfs . in a multivariate cox model
, we found that mir-221 expression was an independent poor prognostic factor for both 5-year os ( hazards ratio [ hr]=3.189 , 95% confidence interval [ ci]=1.7826.777 , p=0.007 ) and 5-year dfs ( hr=2.119 , ci=1.9628.552 , p=0.01 ) in cmm .
cmm is an aggressive and chemoresistant form of skin cancer characterized by malignant growth and poor patient prognosis .
although many novel strategies have been used recently for the diagnosis and treatment of cmm , the prognosis is still very poor .
improvement in cmm outcomes largely depends on increasing our understanding of the molecular biology and regulatory mechanisms underlying the growth of cmm .
furthermore , there is still a need to improve early detection screening methods and to identify new prognostic biomarkers .
ideal biomarkers should be easy to measure and have a strong association with clinical outcome .
mirnas may match these proposed criteria and could be used as biomarkers for diagnosis and prognosis of cancer . among all the cancer - related mirnas ,
mir-221 , which is considered a micro - oncogene , was reported to be increasingly expressed in various carcinomas , including liver cancer , breast cancer , prostate cancer , colorectal carcinoma , melanoma , and acute myeloid leukemia [ 1519 ] .
furthermore , high level of mir-221 expression is correlated with metastasis , tumor capsular infiltration , tumor stage , and poor prognosis .
mir-221 plays an important role in epithelial - to - mesenchymal transition ( emt ) .
it has been identified as a basal - like subtype - specific mirna that downregulates the expression of epithelial - specific genes and enhances the expression of mesenchymal - specific genes .
the basal - like transcription factor , fosl1 , can directly stimulate the transcription of mir-221 . the abundance of mir-221 declines with the suppression of mitogen - activated or extracellular signal - regulated protein kinase ( mek ) .
the mir-221-mediated reduction in e - cadherin is dependent on the targeting of the 3-utr of trichorhinophalangeal syndrome type 1 ( trps1 ) .
trps1 inhibits emt by directly repressing the expression of zinc finger e - box - binding homeobox 2 ( zeb2 ) .
thus , mir-221 could contribute to the aggressive clinical behavior of various types of cancers .
kanemaru et al . found that serum levels of mir-221 were significantly increased in patients with malignant melanoma and that mir-221 levels may be useful for the diagnosis of malignant melanoma .
however , they did not investigate the relationship between serum mir-221 expression level and the prognosis of malignant melanoma .
our study aimed to investigate the feasibility of using serum mir-221 as a noninvasive prognostic biomarker for cmm . in the present study , we showed that individuals with cmm had significantly elevated levels of tissue mir-221 expression , and that high level of mir-221 expression was significantly correlated with tumor thickness , poor differentiation , t classification , n classification , metastasis , and advanced clinical stage , suggesting that the overexpression of mir-221 might be able to play a critical role in the development of cmm .
in addition , we also found that the 5-year os and dfs rates of patients with high mir-221 expression were significantly lower than those of patients with low mir-221 expression , suggesting that mir-221 has potential for use as a molecular marker for predicting the prognosis of cmm patients . to further confirm this ,
our data indicate that mir-221 status might be an independent prognostic factor for cmm patients . to the best of our knowledge ,
we are the first to report that patients with higher serum mir-221 levels have a significantly lower survival rate than those with lower expression levels , revealing the usefulness of serum mir-221 expression as an independent prognosis factor in cmm .
in summary , our analysis shows that level of mir-221 is elevated in the circulation of cmm patients , and it is independently associated with poor survival . based on these results | backgroundthe aim of this study was to investigate the feasibility of using serum mir-221 as a noninvasive prognostic biomarker for cutaneous malignant melanoma ( cmm).material / methodswe measured the expression levels of mir-221 in serum samples from 72 cmm patients and 54 healthy controls by real - time quantitative polymerase chain reaction ( rt - pcr ) .
the overall survival ( os ) and disease - free survival ( dfs ) were calculated using the kaplan - meier method .
the differences between the survival curves were tested by using the log - rank test .
the cox proportional hazards regression model was used to determine the joint effects of several variables on survival.resultsthe serum mir-221 levels were significantly higher in patients with cmm than in healthy controls ( p<0.0001 ) .
patients with high serum mir-221 levels had a significantly lower 5-year os rate ( 22.1% vs. 54.6% ; p=0.018 ) and rfs rate ( 12.5% vs. 45.2% ; p=0.008 ) than those with low serum mir-221 level . in a multivariate cox model , we found that mir-221 expression was an independent predictor of poor 5-year os ( hazards ratio [ hr]=3.189 , 95% confidence interval [ ci]=1.7826.777 , p=0.007 ) and 5-year dfs ( hr=2.119 , ci=1.9628.552 , p=0.01 ) in cmm patients.conclusionsour data indicate that serum mir-221expression level has prognostic value in patients with cmm . | Background
Material and Methods
Patients and serum samples
miRNA extraction and quantitative RT-PCR analysis
Statistical analysis
Results
Increased miR-221 expression level in CMM patients and its relationship with clinicopathological variables
Expression of miR-221 in serum samples in relation to prognosis of CMM patients
Discussion
Conclusions |
acute upper gastrointestinal bleeding ( ugib ) is a common and potentially life - threatening emergency facing clinicians with an overall annual incidence of approximately 100 hospital admissions per 100,000 population .
peptic ulcer bleeding is a common medical emergency accounting for 50%70% of cases of acute nonvariceal ugib and is significantly associated with morbidity , mortality , and health costs .
initial hemostatic rates of 80% to 95% can be achieved with effective endoscopic therapies such as local injection , contact thermal coagulation , and hemoclips . despite remarkable advances in diagnosis and treatment of peptic ulcer bleeding , bleeding recurs in 10%30% of patients .
therefore , recurrence of bleeding after initial endoscopic hemostasis has been described as the single most adverse independent prognostic factor for this group of patients .
endoscopic hemostasis including mechanical , thermal , and by injection of various agents , whether epinephrine , distilled water , cyanoacrylate , ethanol , or polidocanol , has been found to decrease the risk of rebleeding , need for surgery , and the mortality .
although vigorous efforts have been made to delineate the ideal endoscopic method for achieving hemostasis , there is still a great deal of uncertainty about a highly effective and simple hemostatic technique to reduce the risk of rebleeding in patients with nonvariceal ugib .
endoscopic injection of dilute epinephrine is considered a highly effective and popular endoscopic scheme for ugib , owing to its low cost , wide availability , lack of severe local or systemic injury , and easy to administer . however
, epinephrine injection may induce cardiovascular complications and mild histologic changes such as hematoma and perforation . because mechanical and chemical effects of epinephrine are short - lived
, the addition of a second agent , such as polidocanol or ethanol , may be beneficial for preventing recurrent bleeding by prolonging hemostatic mechanisms of epinephrine .
therefore , wide variation in the rate of rebleeding can be explained by difference in the mechanisms of the action of the injected agents and differences in injection volume .
however , clinical trials in patients with bleeding ulcers disclosed that no single solution is superior to another for hemostasis .
furthermore , results from several studies have revealed that injection of normal saline solution or distilled water has a primary role in hemostasis similar to those of epinephrine in the endoscopic therapy of patients with bleeding ulcers . in this regard , there has been no clinical study that evaluates the effects of fresh frozen plasma ( ffp ) in the endoscopic therapy of patients with ugib .
ffp , the most commonly prescribed hemostatic agent , is prepared either from a single whole blood donation or obtained by apheresis , frozen within a specific period and stored at a proper temperature up to 1 year that will maintain all of the coagulation factors , electrolytes , and plasma proteins .
it has been shown that ffp is a good source of coagulation factors which can correct mild to moderate coagulopathy .
therefore , if the injection of ffp has similar effects epinephrine , it might be an alternative solution for the treatment of patients with bleeding ulcers because of its low cost , wide availability , rapidity , ease of application , and lack of severe complication .
this study was designed to shed light on the possible role of ffp as a hemostatic agent in patients with high - risk bleeding peptic ulcers .
a prospective , randomized , single - blind , parallel group trial was conducted at al - zahra hospital from august 23 , 2015 , to april 21 , 2016 .
all cases were informed about the procedure of the survey and written informed consent was obtained from all them .
all patients with hematemesis , melena , or hematochezia underwent for emergency endoscopy within 24 h of admission . only men and women over 18 years of age with ulcers indicative of a high - risk for spontaneous recurrent bleeding including bleeding visible vessel ( spurting or oozing ) , adherent clot , and nonbleeding visible vessel were selected .
patients were excluded from the study if they had another possible bleeding site or an acute significant illness , were pregnant , had a severe bleeding tendency , and unable , or unwilling to give written consent .
after an explanation of the nature of the study and patient consultation with their gastroenterologist , informed written consents were attained from all patients .
the ethical institutional committee of isfahan university of medical sciences approved the protocol of study .
overall , a total of 100 patients with high - risk bleeding peptic ulcers were enrolled in the study , and completed interventions without interruption .
after the enrollment , all patients were randomly divided into one of the two groups using random allocation method .
patients were stratified according to their forrest classification and then randomized 1:1 into two intervention groups : group a who received injection of epinephrine diluted 1:20,000 in saline solution alone in volumes of 8 ml aliquots by multiple punctures into and around the bleeding point and group b who received injection of epinephrine diluted 1:20,000 in saline solution plus thawed ffp in volumes of 8 ml around the ulcer base and beneath the bleeding source . during the period of endoscopic treatment ,
, endoscopic biopsy specimens from the gastric antrum and one from the gastric body were obtained for a rapid urease test for helicobacter pylori . in our study ,
initial hemostasis was defined as endoscopically verified cessation of hemorrhage for at least 5 min after the first endoscopic treatment and maximal water irrigation for 10 s. recurrent bleeding was suspected clinically as one or more signs of ongoing or new bleeding , including vomiting of fresh blood , melena or bloody stool , instability of vital signs or hemodynamic , and a reduction of hemoglobin concentration by more than 20
patients with rebleeding were confirmed by endoscopy immediately and retreated with endoscopic treatments such as epinephrine injection plus argon plasma coagulation ( apc ) , transcatheter arterial embolization , and emergent surgery .
after endoscopy treatment , all patients were closely monitored in an intensive care unit until the first follow - up and blood transfusion was given to maintain the hemoglobin level at > 10
the outcomes assessed in the study were the in - hospital recurrent bleeding , duration of hospitalization , surgery , and 30-day mortality .
analysis of the current study was performed using the spss for hardware ( version 20.0 ; spss , chicago , il , usa ) .
t - test , chi - square , and tests were applied to ensure homogeneity between the two groups in terms of age and gender .
results of endpoints parameter at baseline and 24 h after treatment were compared between the two groups using paired student 's t - tests . for quantitative data ,
results of the analysis were summarized by mean standard deviation or number ( percent ) .
all tests were two - tailed , and p < 0.05 was considered as a significant .
a total of 108 patients with ugib were recruited during the study period . of these , eight patients were excluded due to endoscopically uncontrollable bleeding , gastric malignancy , and multiple sites of bleeding at endoscopy .
the two treatment groups well matched for demographic and clinical characteristics including age , gender , nsaid ingestion , comorbid disease , positive h. pylori , hemodynamic status at entrance , bleeding stigma , ulcer size , and transfusion requirements during the first 24 h [ table 1 ] .
there were one hundred patients who were included in the study , fifty in each group .
other clinical and endoscopic data for the patients at study entry are summarized in table 1 .
initial hemostasis was achieved in 47 of 50 patients ( 94% ) in the group a and 49 of 50 patients ( 98% ) in the group b. clinical and endoscopic characteristics of the patients at study entry we found no statistically significant differences in the rate of initial hemostasis between group a and group b ( p = 0.61 ) .
there were no significant differences in the rate of recurrent bleeding between group a ( 14% ) and group b ( 8% ) ( p = 0.52 ) .
seven patients in group a had recurrent bleeding but were controlled in three patients with additional apc , and in three by surgical intervention .
recurrent bleeding was also controlled in three patients of group b. recurrent bleeding was controlled in one patient by apc , and in two patients by surgical intervention .
the endoscopic findings indicated that duodenal ulcer was the source of bleeding in 54 patients .
there were no significant differences between two groups in terms of ulcer type , ulcer size , volume of epinephrine used during endoscopic hemostasis , and forrest class .
major complications from endoscopic treatment including severe abdominal pain following hemostasis , perforation , and endoscopic therapy induced bleeding were observed more frequently in the group b than in the group a and the differences were not statistically significant .
we did not find procedure - related cardiovascular or respiratory complication such as cardiac arrhythmia , considerable change in oxygen saturation , significant changes in systolic pressure , and abrupt change in pulse rates after endoscopic hemostasis in groups a and b. furthermore , there were no significant differences between group a and group b with respect to the surgery rate , bleeding death , procedure related death , and duration of hospitalization [ table 2 ] .
bleeding peptic ulcer is a potentially life - threatening disease with a considerable mortality and morbidity .
the effectiveness of endoscopic treatment for ugib is well established ; endoscopic therapy is commonly used as the first - line treatment in patients with nonvariceal upper gi , and epinephrine alone is among the most widely used injection solutions . in an effort for more effective hemostasis and to reduce the risk of rebleeding in patients with high - risk of ugib , a number of endoscopic treatment modalities are firmly established including injection , thermal , and mechanical . although the combination of epinephrine injection with a thermal method shows a consistent trend in favor of combined treatment , there is not yet strong evidence that any modality is superior to injection of epinephrine alone for treatment of patients with ugib .
one of the reasons might be that combined therapy is not available in all hospitals , and it is more technically demanding .
it has been shown that unsatisfactory visualization of the bleeding site , unsuccessful application of epinephrine injection , and recurrent bleeding in actively bleeding ulcers may be associated with the age , gender , comorbid disease , positive h. pylori , hemodynamic status at entrance , ulcer size , ulcer location , and transfusion requirements during the first 24 h , which were similar between two groups in our study , indicating that the outcomes of the study are unlikely influenced by these factors .
a number of agents with different mechanisms of action such as vessel compression , vasoconstriction , and platelet aggregation have been investigated .
therefore , the addition of ffp , the most commonly prescribed hemostatic agent , would be advantageous in theory . despite the theoretical advantages of ffp
, previous studies revealed no significant differences between injection of epinephrine alone or in combination with other solutions such as distilled water and normal saline solution .
although it is difficult to sort out these inconsistent outcomes of previous studies , the difference may in part be clarified by a small number of patients , different subtype of peptic ulcer bleeding , combined with the fact that epinephrine injection is adequate in most cases with acute ugib .
these possible reasons are explained in previous meta - analyses that compared efficacy of injection of epinephrine alone with epinephrine and a second injected solution in the patients with peptic ulcer bleeding .
meta - analysis of the recent controlled trials revealed that combined therapy seems to be more effective than epinephrine injection alone , but a particular form of treatment is not equal or superior to another .
although the absolute improvement in the outcomes of our study is relatively small , we failed to detect a significant reduction in the need for surgery , overall rate of mortality , duration of hospitalization , and rate of rebleeding . in a similar study ,
140 patients with ulcers and endoscopic features indicative of a high - risk for spontaneous recurrent bleeding were assigned to endoscopic injection with epinephrine ( group a ) and epinephrine plus human thrombin ( group b ) .
they reported that endoscopic injection of epinephrine alone results in rebleeding rates of 20% , an overall mortality of 10% , and administration of 297 units of blood .
the combination of endoscopic injection of epinephrine with an injection of human thrombin might be superior , resulting in rebleeding rates of 4.5% , an overall mortality of 0% , and administration of 219 units of blood .
our findings are inconsistent with the results of their study that highlighting advantages of the combination of epinephrine plus a second solution
. a possible explanation for the discrepancy may lie in the fact that high - dose continuous intravenous ppis greatly lowers the rate of rebleeding , resulting in the conflicting findings but can not serve as a specific explanation for the failure of ffp because the dose of ppis reported for both studies appears identical . in another study ,
415 patients with high - risk bleeding ulcers were randomized to injection of epinephrine alone or epinephrine plus ethanolamine .
they found no significant differences in the rate of mortality or in the proportions of the patients who required surgery .
in addition , they showed a trend toward less recurrent bleeding after injection of the epinephrine plus ethanolamine .
therefore , the findings of their study suggested that adding ethanolamine to epinephrine can effectively prevent recurrent bleeding and might be some of the value in patients with peptic ulcer bleeding .
however , pescatore et al . found that injection of epinephrine alone and epinephrine plus fibrin glue does not have comparable recurrent bleeding rates , indicating that adding fibrin glue to epinephrine is not superior to epinephrine injection alone in the treatment of high - risk bleeding peptic ulcers . due to
increasing number of reports describing differential and conflicting results , more extensive studies in larger groups of patients should be undertaken to analyze the putative role of adding a second agent to epinephrine for injection treatment of patients with high - risk bleeding ulcers .
in summary , this single - center , prospective , randomized , controlled trial of patients with ugib has shown that injection of epinephrine alone was equally effective as injection of epinephrine plus ffp to endoscopic hemostasis .
epinephrine alone and epinephrine plus ffp were not different in recurrent bleeding , surgery , blood transfusion , or mortality . financial support of isfahan university of medical sciences , isfahan ( research project number : 395100 ) .
financial support of isfahan university of medical sciences , isfahan ( research project number : 395100 ) .
mkh contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , agreed for all aspect of the work .
mk contributed in the conception of the work , conducting the study , revising the draft , agreed for all aspects of work .
ash contributed in the conception of the work , conducting the study , agreed for all aspects of the work .
vs contributed in the conception of the work , revising the draft , agreed all aspects of the work . | background : acute upper gastrointestinal bleeding is a common and potentially life - threatening emergency with substantial mortality . fresh frozen plasma ( ffp ) , a good source of coagulation factors , might be an ideal injection agent based on its physiologic properties . therefore , we evaluated the role of ffp as a hemostatic agent in patients with high - risk bleeding peptic ulcers.materials and methods : from august 2015 to april 2016 , 108 consecutive patients with high - risk bleeding ulcers were admitted to our university hospital .
they were randomly assigned to undergo injection of epinephrine alone ( a ) or epinephrine plus ffp ( b ) .
the primary outcomes assessed were the initial hemostasis , recurrent bleeding , hospital stay , blood transfusion , surgery rate , and 14-day mortality.results:initial hemostasis was achieved in 47 of 50 patients ( 94% ) in the group a and 49 of 50 patients ( 98% ) in the group b ( p = 0.61 ) .
there were no significant differences in the rate of recurrent bleeding between group a ( 14% ) and group b ( 8% ) ( p = 0.52 ) .
we found no significant differences between group a and group b with respect to the surgery rate , bleeding death , procedure - related death , and duration of hospitalization ( p > 0.05).conclusion : it is concluded the injection of epinephrine alone was equally effective as injection of epinephrine plus ffp to endoscopic hemostasis .
epinephrine alone and epinephrine plus ffp were not different in recurrent bleeding , rate of surgery , blood transfusion , or mortality . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
Financial support and sponsorship
Conflicts of interest
AUTHORS CONTRIBUTION |
the gastrointestinal tract ( gi ) is a common site of presentation for extranodal lymphomas , which comprise between 5 and 10% of all primary gi neoplasms .
the commonest type of primary gi lymphoma is diffuse large b - cell lymphoma , which is a type of non - hodgkin 's lymphoma and accounts for about 45% of all cases of primary gi lymphoma .
mucosa - associated lymphoid tissue lymphoma ( malt lymphoma or maltoma ) , also known as extranodal marginal zone b - cell lymphoma , is a subtype of b - cell non - hodgkin 's lymphoma and comprises approximately 17% of all gi tract lymphomas .
peyer 's patches , found in the small intestine , are examples of prominent , normal malt .
antigenic stimulus in the gi tract , e.g. helicobacter pylori ( hp ) infection in the stomach , leads to the activation of the gi immune response and the proliferation of malt . in the presence of sustained antigenic stimulus , lymphocytes within the malt
maltomas are not limited to the gi tract and can occur at any site in which malt is able to proliferate .
they can arise not only in response to the chronic inflammation seen in infections such as hp gastritis , but also in response to the chronic inflammation seen in autoimmune conditions such as sjogren 's syndrome , where they can occur in the salivary glands and in the thyroid in response to hashimoto 's thyroiditis .
the commonest gi site of maltoma is the stomach , with approximately 50% of lesions arising here , most commonly in response to chronic hp infection .
less common sites include the small bowel , which accounts for 2030% , and the colon , with colonic maltomas being relatively rare and only accounting for approximately 10% of all gi maltomas .
although maltomas themselves are not uncommon , the presence of synchronous upper and lower gi maltomas appears to be a more rare occurrence , with only a few cases reported [ 2 , 3 , 4 ] .
here we present a case of synchronous upper and lower gi maltomas and the potential management difficulties of maltomas .
a 73-year - old man with type 2 diabetes mellitus was referred from primary care with a 4-month history of alternating bowel habit associated with excessive mucus .
there was no rectal bleeding and he did not have any abdominal pain , nor had he lost any weight .
his abdomen was soft and non - tender and there were no palpable masses or organomegaly .
g / l , a white blood cell count of 3.1 10/l , a neutrophil count of 1.7 10/l , platelets 203 10/l , sodium 140 mmol / l , potassium 5.1 mmol / l , urea 7.8 mmol / l , creatinine 94 mol / l , albumin 43 g / l , bilirubin 4 mol / l , alanine aminotransferase 15 iu / l and alkaline phosphatase 36 iu / l .
he underwent a colonoscopy , which revealed a large 5-cm , ulcerated , polypoid sigmoid lesion ( fig . 1 ) .
potential diagnoses at this point included colonic cancer , gastrointestinal stromal tumour ( gist ) or maltoma . the biopsies from this lesion showed an intense lymphoid infiltrate , but no overt evidence of adenocarcinoma .
subsequent immunohistochemical ( ihc ) analysis showed a lymphoid infiltrate with numerous cd20 + b cells .
bcl-2 was positive , whilst cyclin d1 , cd10 , cd43 and bcl6 were negative .
the patient underwent a staging ct scan , which confirmed the sigmoid lesion but also revealed a gastric cardia mass , but showed no evidence of other metastases .
an oesophagogastroduodenoscopy revealed nodular gastritis throughout the corpus and a 30-mm spherical mass in the cardia with some proximal ulceration ( fig . 2 ) .
he also underwent a linear endoscopic ultrasound which confirmed a 30-mm smooth heterogenous hypoechoic lesion that involved mucosa , submucosa and the muscularis propria ( fig .
fine needle aspiration samples from the cardia mass revealed that there were no spindle cells present , which excluded a gist .
the biopsies from the gastric lesion , similarly to the sigmoid lesion , showed a dense lymphoid infiltrate and similar immunohistochemistry ( fig .
4 ) , with the exception that the gastric biopsies were cd43 positive on immunohistochemistry , whilst the colonic lesion was negative .
this resulted in a diagnosis of synchronous upper and lower gi tract maltomas , with differing immunohistochemistry patterns multi - focal malt lymphoma .
fluorescence in - situ hybridisation ( fish ) studies of both sets of biopsies were conducted to assess for the presence of the malt1 translocation these were negative .
the patient underwent a sigmoid resection for the lesion which confirmed that the sigmoid lesion was a maltoma .
the gastric maltoma did not respond to either successful hp cure or a course of rituximab ; however , after 3 cycles of bendamustine and rituximab chemotherapy , a repeat ct scan showed near complete resolution of the gastric lesion .
the diagnosis of maltoma usually relies on the careful application of histological and ihc analyses of the biopsy samples obtained during endoscopy .
histology will show the presence of lymphoepithelial lesions , defined by the infiltration and distortion of epithelial structures by aggregates of ( usually three or more ) neoplastic lymphoid cells . despite there being no specific ihc marker for maltoma ,
the ihc pattern is virtually identical to that of nonneoplastic marginalzone b cells : cd20 + , igd , igm ( > iga > igg)+ , cd5 , cd10 , cyclin d1 .
attempts have been made to determine the key endoscopic features which would suggest a lesion is a maltoma .
gastric maltomas have had a 6-point classification proposed : ( 1 ) exophytic : a single mass with irregular or polypoid aspect ; ( 2 ) ulcerative : single to multiple ulcerations / erosions ; ( 3 ) hypertrophic : enlarged folds and nodular pattern ; ( 4 ) petechial haemorrhage : several petechiae at mucosal level ; ( 5 ) normal / hyperemic mucosa : normal to reddish mucosa , and ( 6 ) mixed : more of the above - mentioned pattern present .
the role of endoscopic ultrasound was also covered and the authors concluded that 4 tumour patterns can be present : ( 1 ) superficial : where there is thickening of the second and third layers without involvement of either the first or fourth layer ; ( 2 ) diffuse - infiltrative : diffuse trans - mural involvement of the gastric wall with irregular layer border and absence of uniform echogenicity ; ( 3 ) mass - forming : localized hypoechoic mass at the third / fourth layer , with good demarcation from the surrounding tissue , and ( 4 ) mixed - type : a combination of the previous three patterns .
colonic maltomas are much rarer and make a consensus harder to arrive at , but the review suggested the usual endoscopic appearance is of a whitish polypoid lesion , usually not ulcerated and 12 cm in diameter , preserving a smooth aspect at endoscopy .
gastric maltoma is regarded as an indolent disease , and the conventional treatment for low - grade gastric maltoma is hp cure .
approximately 20% of patients do not respond to hp cure and will require treatment with rituximab with or without chemotherapy .
interestingly , the absence of malt1 translocation is usually an indicator of probable successful response to hp cure , although this was not the case in our patient .
the treatment for colonic maltoma depends on the patients age , co - morbidities , disease stage , etc . , but usually involves a combination of surgery and/or rituximab + / chemotherapy [ conventionally chop ( cyclophosphamide , doxorubicin , vincristine and prednisolone , although bendamustine with rituximab is also used ] .
in this case , a 72-year - old man presented with a few months of altered bowel habit . he was found to have a sigmoid lesion , which later proved to be maltoma .
a staging ct scan showed a synchronous gastric lesion , which was also found to be a maltoma after biopsy .
interestingly , the two lesions were shown to have a slightly different ihc profile , thus leading to a diagnosis of multifocal malt lymphoma .
although rare in the literature , the presence of cases such as this does raise the question of whether all patients with confirmed maltoma in the gi tract , especially gastric maltoma , should undergo staging ct and colonoscopy to exclude synchronous lesions .
complete staging has been suggested as mandatory by fischbach in his review on current maltoma diagnosis and management , and the classification should be based on either the ann - arbor system or the tmn system , although the latter has not been validated .
currently , patients with confirmed gastric maltoma are unlikely to have a ct scan due to the belief that it is an indolent disease .
the detection of synchronous lesions will obviously affect their long - term prognosis and care , as synchronous lesions will likely require far more aggressive therapy than low - grade isolated gastric lesions . | mucosa - associated lymphoid tissue lymphoma ( maltoma ) is a subtype of b - cell non - hodgkin 's lymphoma , comprising 17% of all gastrointestinal ( gi ) tract lymphomas .
it is associated with chronic inflammation and autoimmunity , for example helicobacter pylori gastritis and sjogren 's syndrome , respectively .
approximately 50% of gi maltomas occur in the stomach , with small bowel and colonic lesions being less frequent .
synchronous upper and lower gi maltomas occur rarely , with few cases reported .
we present the case of a 73-year - old patient who presented with change in bowel habit and was found to have synchronous multifocal upper and lower gi maltomas , which did not respond to h. pylori cure or to rituximab therapy , but did respond to a combination of surgery and chemotherapy with rituximab and bendamustine . | Introduction
Case Report
Discussion
Conclusion
Statement of Ethics
Disclosure Statement |
epidemic keratoconjunctivitis ( ekc ) is an acute infection of the eye caused by adenovirus , of which several different types have been implicated .
common manifestations of this infection include inflammation of the conjunctivae , edema of the eyelid , pain , photophobia , and blurred vision .
transmission may occur through direct contact with eye secretions of an infected person , or indirectly through contact with contaminated surfaces , instruments or solutions .
ekc is highly contagious and frequently occurs in epidemic fashion , as its name implies .
the incubation period can range from 2 days to 2 weeks , with an average of 8 - 10 days , and infected people remain contagious for up to 2 weeks .
viral particles can remain infectious on surfaces for up to a month . in march 2009
, the florida department of health ( fldoh ) was contacted by a regional academic ophthalmology referral center , regarding a local outpatient ophthalmology practice ( practice a ) that had requested assistance with an outbreak of ekc at the practice .
practice a was contacted and a site visit was arranged . at the same time , anecdotal reports were noted of increased ekc activity throughout the county . to assess whether the problems experienced at practice a were isolated or part of a more wide - spread increase in ekc activity , a brief survey was developed of outpatient ophthalmology clinics in the county .
this report describes the investigation of the ekc outbreak at practice a and the survey results of other out - patient ophthalmology clinics in the county .
the practice consists of a main office ( office a1 ) and an auxiliary office ( office a2 ) , located in different parts of the county , and only office a1 was visited by investigators .
office staff assembled a line list from patient charts of all persons diagnosed with ekc at practice a during the previous 5 months .
confirmed cases were defined as those with compatible clinical symptoms of ekc and laboratory confirmed adenovirus infection through viral culture of ocular swab specimens .
suspect cases were defined as those with a clinical diagnosis of ekc , without laboratory confirmation .
the line list included the onset date of symptoms , dates previously seen in the practice prior to symptoms , and which office visited .
based on the estimated outside incubation period for ekc of 2 weeks , cases were further classified as possibly healthcare associated if they were seen at practice a for an unrelated condition within 2-weeks of the onset of ekc symptoms .
specific information regarding medical procedures performed during previous visits or past visits to other providers was not obtained . during the site visit ,
a rapid test was performed on 2 patients with compatible symptoms , using the adeno detector test kit [ www.rpt-tests.com/products_ad.html ] .
specimens for viral culture were obtained from the same 2 patients and from 2 additional patients seen the next day .
viral cultures were performed at the bascom palmer eye institute , microbiology laboratory , in miami , fl .
viral culture was conducted by inoculation of a549 and mrc cells ( viromed laboratories , minnetonka , mn , usa ) and incubation for up to 21 days , followed by immunoflourescent staining with monoclonal antibody ( pathodx , remel , lenexa , ks , usa ) .
viral culture isolates positive for adenovirus were forwarded to the centers for disease control and prevention ( cdc ) for viral serotyping by polymerase chain reaction , using primers specific for adenovirus serotypes .
fldoh developed a list of all licensed ophthalmologists in the county from medical licensing records .
a cover letter and brief 1-page questionnaire were faxed to all ophthalmology practices in the county alerting them to the possible ekc problem , and requesting the completion and return of the questionnaire .
the questionnaire included items regarding their perceptions of recent ekc incidence , number of ekc cases diagnosed in their clinic during the last three months , similar illness among office staff , and any laboratory test results available .
analyses of data collected from both practice a and the county wide survey were descriptive and no statistical tests of significance were performed .
the practice consists of a main office ( office a1 ) and an auxiliary office ( office a2 ) , located in different parts of the county , and only office a1 was visited by investigators .
office staff assembled a line list from patient charts of all persons diagnosed with ekc at practice a during the previous 5 months .
confirmed cases were defined as those with compatible clinical symptoms of ekc and laboratory confirmed adenovirus infection through viral culture of ocular swab specimens .
suspect cases were defined as those with a clinical diagnosis of ekc , without laboratory confirmation .
the line list included the onset date of symptoms , dates previously seen in the practice prior to symptoms , and which office visited .
based on the estimated outside incubation period for ekc of 2 weeks , cases were further classified as possibly healthcare associated if they were seen at practice a for an unrelated condition within 2-weeks of the onset of ekc symptoms .
specific information regarding medical procedures performed during previous visits or past visits to other providers was not obtained . during the site visit ,
a rapid test was performed on 2 patients with compatible symptoms , using the adeno detector test kit [ www.rpt-tests.com/products_ad.html ] .
specimens for viral culture were obtained from the same 2 patients and from 2 additional patients seen the next day .
viral cultures were performed at the bascom palmer eye institute , microbiology laboratory , in miami , fl .
viral culture was conducted by inoculation of a549 and mrc cells ( viromed laboratories , minnetonka , mn , usa ) and incubation for up to 21 days , followed by immunoflourescent staining with monoclonal antibody ( pathodx , remel , lenexa , ks , usa ) .
viral culture isolates positive for adenovirus were forwarded to the centers for disease control and prevention ( cdc ) for viral serotyping by polymerase chain reaction , using primers specific for adenovirus serotypes .
fldoh developed a list of all licensed ophthalmologists in the county from medical licensing records .
a cover letter and brief 1-page questionnaire were faxed to all ophthalmology practices in the county alerting them to the possible ekc problem , and requesting the completion and return of the questionnaire .
the questionnaire included items regarding their perceptions of recent ekc incidence , number of ekc cases diagnosed in their clinic during the last three months , similar illness among office staff , and any laboratory test results available .
analyses of data collected from both practice a and the county wide survey were descriptive and no statistical tests of significance were performed .
practice a is a busy , outpatient ophthalmology practice that serves a predominantly elderly patient population and sees approximately 150 - 200 patients per week .
the practice has 1 general ophthalmologist ( physician a ) , specializing in cataract and corneal surgery , who founded the practice and has worked for several years in the community . at office a1 , there are 4 exam rooms and 2 ophthalmology technicians who assist with patient care ; 2 other staff assist with administrative duties .
office a2 is a smaller satellite facility where physician a and the technicians work 2 half - days per week .
approximately two - thirds of patient visits occur at office a1 , where all patient records are maintained for the practice .
interviews with staff revealed that the first ekc patient believed to be part of this outbreak was seen in office a1 in late november 2008 .
a total of 37 patients were clinically diagnosed with ekc at practice a from november 2008 through march 2009 .
twenty - three ( 62% ) ekc patients were seen at office a1 , 12 ( 32% ) at office a2 , and 2 patients were seen at both offices ( figure 1 ) . of the 4 patients seen at office a1 for whom
specimens were collected for viral culture , all 4 were positive for adenovirus , serotype 8 .
two of the four laboratory confirmed patients had rapid tests performed during the clinical visit , and both of these patients were negative for adenovirus by rapid test .
all of the laboratory confirmed patients had a previous visit at office a1 within the previous 2 weeks , prior to their symptom onset .
interviews revealed that physician a had symptoms compatible with ekc , with onset of these symptoms on approximately february 5 , 2009 and lasting approximately 4 - 5 weeks before resolution of symptoms .
no other staff members had similar symptoms and no diagnostic specimens were collected from any staff members . among the other 37 confirmed or suspected ekc patients , the median age was 79 years ( table 1 ) .
nineteen ekc patients ( 51% ) had been seen for an unrelated condition by physician a , within 2 weeks of the onset of their symptoms ( figure 2 ) .
four additional ekc cases were seen at practice a between 15 and 17 days prior to symptom onset .
of the 19 ekc patients seen at practice a within 2 weeks of symptom onset , 13 had been seen at office a1 , 5 at office a2 , and 1 at both offices . on march 18 , 2009 ,
the day the health department was notified of the outbreak , practice a was closed for terminal cleaning .
all surfaces were wiped by office staff with a bleach solution , reusable vials of drops were discarded , and tonometers and other medical equipment were thoroughly cleaned .
when practice a opened the following day , one exam room in office a1 was dedicated exclusively for ekc patients , and ekc and non - ekc patients were separated on different sides of the waiting room . for patient encounters ,
office staff implemented aggressive use of gloves and wiping of surfaces with germicidal wipes following each patient encounter .
these control measures were initiated independently by practice a , prior to the health department becoming involved in the investigation . following implementation of these control measures ,
2 additional cases were diagnosed with ekc at practice a , 2 and 6 days respectively following the terminal cleaning . the patient with onset 2 days after implementation of control measures had a previous office visit 15 days earlier ; the other patient had not been seen in practice a during the past year . since prevention measures had already been implemented and the outbreak had begun to dissipate by the time the epidemiologic investigation was under way , no environmental samples were collected and extensive analyses were not conducted to identify additional risk factors associated with infection .
no additional ekc patients were seen at practice a in the two months following this outbreak , however , physician a reported seeing 4 additional ekc patients within 6 months following the outbreak .
approximately 8 ekc case patients from the outbreak , including physician a , developed post - viral keratitis , requiring long - term topical steroid treatment to control corneal infiltrates .
medical licensing records revealed 29 ophthalmologists in general practice in the county , including physician a. in april 2009 , questionnaires were faxed to all 28 providers , other than physician a , and responses were received from 20 ( 71% ) . of those who responded , 4 ( 20% ) reported an increase in conjunctivitis during the previous 6 months above normal seasonal patterns .
eight of twenty ( 40% ) respondents believed they had seen patients from january through march 2009 with symptoms compatible with ekc or acute hemorrhagic conjunctivitis ( ahc ) . among these providers ,
the median number of ekc / ahc patients seen per clinic during this period was 10 ( range 1 to 25 ) with a total of 84 patients with compatible symptoms seen at these 8 clinics . none of these 84 suspect case - patients were laboratory confirmed . for comparison , during the same 3-month period , physician a saw 28 ekc patients , making 112 total ekc patients for all 21 providers . because denominator data were not collected on the total number of patients seen at each clinic , attack rates at each clinic
two of the eight clinics ( 25% ) reported seeing symptoms compatible with ekc in patients who had visited their clinic in the previous 2 weeks for an unrelated problem .
of these 2 clinics , one reported seeing ekc symptoms in 10 return patients , and the other clinic reported 1 ekc case in a return patient .
none of the 20 clinics who responded to the survey reported similar symptoms among clinic staff .
finally , 7 of 18 ( 39% ) clinics that responded indicated future interest in submitting specimens to reference laboratories for diagnostic testing .
practice a is a busy , outpatient ophthalmology practice that serves a predominantly elderly patient population and sees approximately 150 - 200 patients per week .
the practice has 1 general ophthalmologist ( physician a ) , specializing in cataract and corneal surgery , who founded the practice and has worked for several years in the community . at office a1 , there are 4 exam rooms and 2 ophthalmology technicians who assist with patient care ; 2 other staff assist with administrative duties .
office a2 is a smaller satellite facility where physician a and the technicians work 2 half - days per week .
approximately two - thirds of patient visits occur at office a1 , where all patient records are maintained for the practice .
interviews with staff revealed that the first ekc patient believed to be part of this outbreak was seen in office a1 in late november 2008 .
a total of 37 patients were clinically diagnosed with ekc at practice a from november 2008 through march 2009 .
twenty - three ( 62% ) ekc patients were seen at office a1 , 12 ( 32% ) at office a2 , and 2 patients were seen at both offices ( figure 1 ) . of the 4 patients seen at office a1 for whom
specimens were collected for viral culture , all 4 were positive for adenovirus , serotype 8 .
two of the four laboratory confirmed patients had rapid tests performed during the clinical visit , and both of these patients were negative for adenovirus by rapid test .
all of the laboratory confirmed patients had a previous visit at office a1 within the previous 2 weeks , prior to their symptom onset .
interviews revealed that physician a had symptoms compatible with ekc , with onset of these symptoms on approximately february 5 , 2009 and lasting approximately 4 - 5 weeks before resolution of symptoms .
no other staff members had similar symptoms and no diagnostic specimens were collected from any staff members . among the other 37 confirmed or suspected ekc patients , the median age was 79 years ( table 1 ) .
nineteen ekc patients ( 51% ) had been seen for an unrelated condition by physician a , within 2 weeks of the onset of their symptoms ( figure 2 ) .
four additional ekc cases were seen at practice a between 15 and 17 days prior to symptom onset .
of the 19 ekc patients seen at practice a within 2 weeks of symptom onset , 13 had been seen at office a1 , 5 at office a2 , and 1 at both offices . on march 18 , 2009 ,
the day the health department was notified of the outbreak , practice a was closed for terminal cleaning .
all surfaces were wiped by office staff with a bleach solution , reusable vials of drops were discarded , and tonometers and other medical equipment were thoroughly cleaned .
when practice a opened the following day , one exam room in office a1 was dedicated exclusively for ekc patients , and ekc and non - ekc patients were separated on different sides of the waiting room . for patient encounters ,
office staff implemented aggressive use of gloves and wiping of surfaces with germicidal wipes following each patient encounter .
these control measures were initiated independently by practice a , prior to the health department becoming involved in the investigation . following implementation of these control measures ,
2 additional cases were diagnosed with ekc at practice a , 2 and 6 days respectively following the terminal cleaning . the patient with onset 2 days after implementation of control measures had a previous office visit 15 days earlier ; the other patient had not been seen in practice a during the past year . since prevention measures had already been implemented and the outbreak had begun to dissipate by the time the epidemiologic investigation was under way , no environmental samples were collected and extensive analyses were not conducted to identify additional risk factors associated with infection .
no additional ekc patients were seen at practice a in the two months following this outbreak , however , physician a reported seeing 4 additional ekc patients within 6 months following the outbreak .
approximately 8 ekc case patients from the outbreak , including physician a , developed post - viral keratitis , requiring long - term topical steroid treatment to control corneal infiltrates .
medical licensing records revealed 29 ophthalmologists in general practice in the county , including physician a. in april 2009 , questionnaires were faxed to all 28 providers , other than physician a , and responses were received from 20 ( 71% ) . of those who responded , 4 ( 20% ) reported an increase in conjunctivitis during the previous 6 months above normal seasonal patterns .
eight of twenty ( 40% ) respondents believed they had seen patients from january through march 2009 with symptoms compatible with ekc or acute hemorrhagic conjunctivitis ( ahc ) . among these providers ,
the median number of ekc / ahc patients seen per clinic during this period was 10 ( range 1 to 25 ) with a total of 84 patients with compatible symptoms seen at these 8 clinics . none of these 84 suspect case - patients were laboratory confirmed . for comparison , during the same 3-month period , physician a saw 28 ekc patients , making 112 total ekc patients for all 21 providers . because denominator data were not collected on the total number of patients seen at each clinic , attack rates at each clinic
two of the eight clinics ( 25% ) reported seeing symptoms compatible with ekc in patients who had visited their clinic in the previous 2 weeks for an unrelated problem .
of these 2 clinics , one reported seeing ekc symptoms in 10 return patients , and the other clinic reported 1 ekc case in a return patient .
none of the 20 clinics who responded to the survey reported similar symptoms among clinic staff .
finally , 7 of 18 ( 39% ) clinics that responded indicated future interest in submitting specimens to reference laboratories for diagnostic testing .
the outbreak at practice a described in this report involved 37 patients diagnosed with ekc over a 5 month period .
approximately one - half of case - patients had recently been seen by physician a for an unrelated condition , and previous visits occurred at both offices of the practice .
one additional suspect case involved the ophthalmologist , raising the strong possibility of iatrogenic transmission within the practice .
at least 2 other ophthalmology clinics in the county reported a similar number of case - patients during the same time period and at least 1 of these clinics may also have had nosocomial transmission , based on the large number of ekc patients who were recently seen for unrelated conditions .
patients diagnosed with ekc were also seen in several other clinics throughout the county during this time frame . taken together , these observations suggest broad distribution of ekc - like illness in the community with focal amplification in practice a , likely due to nosocomial transmission .
the etiologic agent identified in 4 ekc patients at practice a was adenovirus , serotype 8 ( ad8 ) .
ad8 is a common cause of severe ekc , along with adenoviral serotypes 19 and 37 .
other serotypes , such as ad3 , ad4 , ad7 , and ad11 , generally cause milder conjunctivitis with systemic involvement .
diagnosis of severe ekc is usually only clinical , but milder forms of the illness due to these other serotypes may require laboratory confirmation . among those infected with ad8 , eyelid edema may be significantly more common , and serotype ad8 may persist longer in ocular tissue and secretions than other serotypes . the fact that ad8 was found to be the etiologic agent in this outbreak suggests that those patients diagnosed clinically at practice a , without laboratory confirmation , were also likely infected with ad8 .
there is no population based disease surveillance for ekc in florida or other states , making estimates of disease incidence difficult in the usa .
this episode was classified as an outbreak based on the experienced clinician reporting it as such , rather than by comparing disease incidence in practice a to background rates , which are not reliably available for florida . in japan , where nationwide surveillance has occurred over several years , an estimated 1 million cases of adenoviral conjunctivitis occur each year . in the usa and elsewhere ,
outbreaks of ekc are believed to be common , and several outbreak investigations have been described in the literature .
risk factors for ekc transmission identified in healthcare settings include use of inadequately disinfected tonometers to measure intraocular pressure , use of multi - dose drops , or exposure to specific care givers .
adenoviruses have been shown to persist in the environment and remain viable in a desiccated state for lengthy periods and to be recoverable from the hands of infected individuals , even following hand washing .
guidelines for the disinfection and sterilization of ophthalmology equipment that may contribute to ekc transmission have been published by the healthcare infection control practices advisory committee .
firstly , we only became aware of the outbreak in practice a late in its course , after strict control measures had been implemented .
thus , sampling to identify an environmental source of infection was unlikely to yield useful information and no environmental sampling was conducted .
in addition , more than one month had elapsed since physician a became symptomatic , making diagnostic testing of physician a impractical .
since the outbreak began to come under control at the time the health department investigation was beginning , fewer patients with active symptoms were available for laboratory confirmation to support an epidemiologic investigation of risk factors .
since physician a was the only ophthalmologist in the practice , all patients were exposed to physician a , making measures of association with this exposure difficult to estimate .
therefore , a specific common source of transmission could not be conclusively identified . when practitioners are confronted with a suspected outbreak of ekc at their facility , there are some recognized best practices , many of which were followed by practice a. they sought laboratory assistance to identify the pathogen , conducted thorough cleaning of the facility and equipment with appropriate disinfectants , segregated symptomatic and non - symptomatic patients , and cooperated fully with the health department in the investigation .
however , healthcare providers with known or suspected ekc should also avoid direct patient contact for 14 days following onset of symptoms in the most recently involved eye , which practice a did not follow . under florida administrative code 64d-3 , any disease outbreak in a community , hospital or other institution is reportable to public health authorities .
our survey of ophthalmologists in the county found that at least 2 other outpatient facilities witnessed a similar number of ekc patients during the same time period , yet neither facility reported these as outbreaks . without laboratory confirmation and serotyping ,
it is difficult to determine if the patients diagnosed with ekc seen at these other facilities were somehow linked to patients at practice a. our findings suggest that passive surveillance based on administrative rule results in poor surveillance sensitivity to detect ekc outbreaks in florida .
the same is likely true in many other states where disease outbreaks are reportable , but individual cases of the same disease are not .
ophthalmologists and other providers experiencing a perceived increase in ekc activity among patients in their facility are encouraged to contact their local health department .
laboratory diagnostic support to confirm certain infectious causes may be available through state health department laboratories . | epidemic keratoconjunctivitis ( ekc ) is an acute eye infection caused by adenovirus .
we investigated an outbreak of ekc at an outpatient ophthalmology practice in the context of a suspected community wide increase in ekc activity .
a site visit was made to the facility reporting the outbreak .
a line list was created of patients clinically diagnosed with ekc at the practice during the previous 5 months .
a questionnaire was faxed to all other licensed ophthalmologists in the county regarding recent ekc activity in their facility .
descriptive data analyses were conducted .
the outbreak facility reported 37 patients clinically diagnosed with ekc during the previous 5 months .
in addition , the single ophthalmologist at the practice also had symptoms compatible with ekc during the outbreak period .
specimens were collected on 4 patients and all were positive for adenovirus serotype 8 .
forty percent of ophthalmologists surveyed in the county saw at least one ekc patient in the previous 3 months , and 20% reported a perceived increase in ekc activity in recent months over normal seasonal patterns .
the outbreak at the facility likely began as part of a widespread community increase in ekc that may have been amplified at the facility through nosocomial transmission .
medical providers experiencing increases in ekc activity above seasonally expected norms should contact their public health department for assistance with etiologic diagnoses and outbreak control . | Introduction
Materials and Methods
Investigation of practice A
Survey of outpatient ophthalmology clinics
Results
Investigation of practice A
Survey of outpatient clinics in the community
Discussion |
prone positioning is widely used to improve oxygenation of patients with acute respiratory distress syndrome ( ards ) . in the previous issue , robak and colleagues report the short - term impact of combining two positioning strategies , prone and upright positioning . although randomized controlled trials failed to demonstrate an overall mortality benefit with the prone position , a trend toward better survival was observed for patients with severe ards [ 2 - 5 ] .
the use of the prone position is supported by a strong physiological rationale and a significant improvement of the oxygenation status is observed in roughly 70% of ards patients .
several mechanisms have been advocated to explain this effect , but the most determinant probably involves better aeration and recruitment of the vertebra - diaphragmatic lung regions , as a consequence of the reduction in ventral chest wall ( cw ) compliance due to direct compression on the bed .
interestingly , either high baseline cw compliance or a greater reduction in its magnitude with the prone position were associated with oxygenation improvement .
upright positioning has also been associated with oxygenation improvement in ards patients and was proposed as an alternative to the prone position .
we previously demonstrated , in a group of 16 ards patients submitted to upright positioning , a significant increase in the pao2/fio2 ratio ( 94 33 versus 142 49 mmhg , p < 0.0032 ) in comparison to the baseline value obtained with the supine position . with respect to the responders , the significant increase in the measured end - expiratory lung volume , coupled with the change in chord compliance after switching to upright positioning , suggested a time - dependent lung recruitment phenomenon .
based on physiological studies , one can postulate that the oxygenation improvement is mainly due to a lung volume redistribution induced by reduced ventral cw compliance during prone positioning , while an increase of the lung volume ( recruitment ) appears more significant during upright positioning . because of their complementary mechanisms of action , combining prone and upright positioning can make sense in severe ards patients .
likewise , it has already been shown that the combination of prone positioning with an adjunctive technique to increase lung volume , the recruitment maneuvers , has additive positive effects on oxygenation . in the physiological study of roback and colleagues ,
the upright - prone position was achieved by raising the head of the bed and lowering the foot end to obtain an angle of at least 20 ( reverse trendelenburg ) .
this was associated with a significant improvement in the pao2/fio2 ratio , without any short - term adverse events .
the proportion of responders increased by combining the prone with the upright position , which is particularly relevant since the prone position is usually discontinued in the absence of oxygenation improvement .
this study was the first to specifically investigate the effect on gas exchange of combining prone and upright positioning .
unfortunately , the study design precluded conclusions about the precise physiological mechanisms involved . measurements of the lung volumes would have been useful to better document the alteration of respiratory mechanics related to both prone and upright positioning .
regarding the redistribution of aerated lung regions , directly assessing the regional changes could have added to our current understanding . since computed tomography ( ct ) scanning
can not be done during upright positioning , alternative techniques , such as electrical impedance tomography , may have permitted a better delineation of the importance of ventilation redistribution from non - dependent toward dependent parts of the lung with the upright - prone position . beyond the impact on oxygenation , other beneficial effects
although the level of evidence is weak , both prone and upright positions have been associated with a decrease of ventilator - associated pneumonia .
an additive effect of these two strategies is plausible , but the pathophysiology of ventilator - associated pneumonia is complex and one should be prudent before making such an assumption .
furthermore , head elevation of patients ventilated in the prone position was shown to improve the tolerance of enteral feeding .
in summary , the use of prone and upright positioning should not be considered as opposing and mutually exclusive strategies , but more as complementary ones .
even if the level of evidence is debatable , combining prone and upright positioning seems to enhance the response rate in terms of oxygenation . whenever the prone position is used , combining it with the upright position
this strategy is easy to implement at no additional cost and with a low risk of major complications .
specific policies should be implemented to encourage caregivers to avoid as much as possible a strictly supine position .
future studies should aim at determining to what extent the addition of the upright position during , and perhaps also between , prone sessions can promote the maintenance of beneficial effects . | positional strategies have been proposed for mechanically ventilated patients with acute respiratory distress syndrome . despite different physiological mechanisms involved , oxygenation improvement has been demonstrated with both prone and upright positions . in the previous issue of critical care , robak and
colleagues reported the first study evaluating the short - term effects of combining prone and upright positioning .
the combined positioning enhanced the response rate in terms of oxygenation .
other benefits , such as a reduction in ventilator - associated pneumonia and better enteral feeding tolerance , can potentially be expected . | None
Abbreviations
Competing interests |
protein - energy malnutrition ( pem ) is highly prevalent in chronic kidney disease ( ckd ) patients on hemodialysis ( hd ) [ 14 ] .
low serum albumin and transferrin are surrogate markers of pem , with low serum albumin being one of the most commonly used in ckd .
the progressive deterioration of kidney function in ckd leads to retention of many substances that are normally excreted by the kidney .
the retention of uremic toxins and other comorbid conditions can lead to a lowered appetite and a decrease in protein and energy intake , which is often compounded by ill - advised imposition of various dietary restrictions . among the causes of malnutrition in hd patients
, inadequate dietary protein intake appears to be one of the most common and important .
low protein intake and low serum albumin have been shown to be independently associated with increased risk of mortality in patients on dialysis [ 9 , 10 ] .
mortality increases significantly as albumin levels fall below 39 g / l . in the united states
g / l in 50% of hypoalbuminemic dialysis patients could save 1,400 lives and decrease hospitalizations by 6,300 per year , although a causal relationship has not been established .
the national kidney foundation kidney disease outcomes quality initiative ( nkf kdoqi ) has proposed a minimum intake of 1.2 g protein per kilogram of body weight per day to ensure a neutral protein balance in the hemodialysis population . despite this recommendation ,
inadequate protein intake persists in many chronic hd patients [ 8 , 14 , 15 ] .
moreover , the persistent marked reduction of daily nutrient intake , even if limited to a single day of the week , has been shown to be an independent determinant of reversible impairment of nutritional status .
studies have suggested that individual dietetic counseling has significant potential to decrease the incidence of pem in hd patients [ 16 , 17 ] .
however , standardized approaches to counseling have not been well documented . complicating the problem , increasing dietary protein intake brings about the associated concerns of hyperphosphatemia and hyperkalemia , as potential consequences of increased dietary phosphorus and potassium burden [ 7 , 18 , 19 ] .
the ep-10 protein counter , a protein exchange list developed for the quantification and education of dietary protein intake during dietetic counseling , has recently been validated in the asian setting .
the purpose of this study was to investigate the effectiveness of using the ep-10 in dietetic assessment and counseling to improve the albumin and transferrin levels of ckd patients on hd .
forty - five patients on stable hemodialysis with serum albumin of less than 38 g / l were enrolled in the study .
forty of these patients were on phosphate binders prior to enrollment into the study .
data on age , sex , race , other relevant biochemical results , cause of renal disease , medications , years of dialysis , and comorbid conditions were collected at baseline from the patients ' medical and clinical charts .
patients ' dry weight and body mass index ( bmi ) were obtained from the same sources at baseline and at three months after intervention .
serum albumin , serum transferrin , kinetic modeling ( kt / v ) , potassium , and phosphate levels were measured at baseline before nutritional intervention and at three months after intervention .
predialysis serum albumin levels were tested in the laboratory using bromocresol green colorimetric assay at wavelength of 630 nm .
kt / v was calculated using the following urea kinetic modeling formula [ 21 , 22 ] :
( 1)ktv=ln(r0.008t)+((43.5r)ufw ) ,
where r is the ratio of postdialysis to predialysis serum urea nitrogen , t is time of dialysis in hours , uf is the amount of ultrafiltration ( in liters ) , and w is the postdialysis weight ( in kg ) .
nutritional intervention was provided to patients by the same clinical dietitian ( sll ) trained in renal nutrition .
areas covered included , but were not limited to , dietary recall , dietary protein intake quantification and assessment , and individualized dietary recommendations based on the eating habits of the patient .
the latter two were done using the ep-10 method as described by lim et al .
patients were educated on the importance of adequate protein intake and the number of protein exchanges required using the ep-10 method . recommended protein intakes
were based on the nkf kdoqi recommendation of 1.2 g protein per kilogram , body weight per day .
these were then explained to the patient using food models and pictures of protein portions based on ep-10 protein exchanges . the ep-10 food exchange list and a written individualized meal plan that included protein distribution were also given to each patient .
patients whose usual dietary intake provided insufficient calories were educated on ways to increase their caloric intake .
patients assessed as being unable to meet their nutritional requirement through diet alone were prescribed suitable nutritional supplements .
in addition to education on protein and caloric requirements , dietary implications of metabolic disorders such as hyperphosphatemia , hyperkalemia , hyperlipidemia , and diabetes mellitus were addressed during the counseling session when necessary .
all statistical analyses were performed using statistical package for social science ( pasw statistics , rel . 18.0.1 . 2009 .
the mean differences between the biochemical parameters at baseline and at three months postintervention were evaluated using the paired t - test when the normality assumption was satisfied . where the latter was not satisfied , the wilcoxon signed - rank test was used .
table 1 shows the mean age , age range , gender ratio , diabetic status , and mean duration of hd of the patients . using the ep-10 method ,
the mean daily dietary protein intake of the study subjects was 45 10 g. this was significantly lower than their mean protein requirement , which was 68 9 g per day based on the 1.2 g protein / kg body weight per day as recommended by nkf kdoqi .
of the forty - five patients , 31 ( 69% ) reported no loss of appetite .
eight patients were underweight ( bmi < 20 kg / m ) , 15 patients were of normal weight ( bmi 2024.9 kg / m ) , and 22 patients were overweight or obese ( bmi > 25
six showed an increase in dry weight of between 0.4 to 4.4 kg three months after intervention while the two remaining underweight patients maintained their dry weight .
table 2 shows the mean differences between the biochemical parameters at baseline and three months postnutritional intervention .
thirty - eight ( 84% ) patients showed a rise in albumin levels , while two ( 4% ) patients maintained their albumin level .
all eight underweight patients showed an increase in albumin levels between 1.0 to 13.0 g / l ( mean increase of 6.6 4.8
thirty - six ( 80% ) patients had low transferrin levels ( < 200 mg / dl ) initially .
after nutritional intervention , 28 out of these 36 ( 78% ) patients had an increase in transferrin levels and two maintained their levels .
there was no significant rise in the mean levels of potassium , phosphate , and kt / v ( table 2 ) .
none of the five patients who were not prescribed phosphate binders had a significant increase in serum phosphorus .
hemodialysis , amongst other renal replacement therapy , is an accepted and successful treatment modality for supporting patients with chronic renal failure .
however , factors like malnutrition , age , and other comorbidities , with emphasis on cardiovascular disease , still cause serious complications and adversely affect quality of life and risk of mortality in this population [ 14 , 20 , 24 ] .
food exchange lists , primarily carbohydrate exchange lists , have been widely used for many years to facilitate meal planning in diabetes and weight reduction .
however , there is a lack of literature showing that exchange lists improve understanding and dietary compliance .
the results of this study show that the ep-10 protein counter , when incorporated into individualized dietetic counseling , can potentially improve the nutritional parameters of hd patients .
both albumin and transferrin are hepatic transport proteins with transferrin having a shorter half - life than albumin . both reflect protein status and are used extensively as markers of nutritional status . in a study by neyra et al .
( 2000 ) , it was found that changes in albumin can be reliably predicted by earlier changes in serum transferrin .
decreases in serum albumin and/or transferrin can be caused by insufficient protein and/or caloric intake .
these levels are also decreased in inflammation , as both albumin and transferrin are negative acute phase proteins .
however , research has shown that albumin levels in hd patients can respond to nutritional interventions despite high levels of inflammatory markers .
several studies have reported a strong association between hypoalbuminemia and morbidity and mortality rates in hd patients , suggesting that nutritional interventions that maintain or increase serum albumin to normal levels may be associated with improved long - term survival [ 1 , 2631 ] .
( 1993 ) found serum albumin to be the single most powerful predictor of patient survival , with a 2 g / l decrease in serum albumin levels associated with a 25% decrease in survival .
given this strong association between hypoalbuminemia and patient outcomes , an intervention that successfully improves serum albumin levels may exert substantial effects on overall patient health and survival .
there are many barriers to adequate nutritional intake in hd patients , including poor nutrition knowledge , poor appetite , and other functional or lifestyle issues .
targeting nutritional barriers has been shown to increase albumin levels , even in the presence of elevated inflammatory markers . in our study ,
insufficient protein and/or energy intake did not appear to be a consequence of poor appetite , as the majority of our hypoalbuminemic patients reported having a normal appetite .
the significant improvement in albumin and transferrin levels of patients in this study after nutritional intervention suggests that the ep-10 protein counter can be easily understood and used by patients to increase their protein intake and thus overcomes at least one barrier to nutritional adequacy .
the kidney plays an important role in maintaining the homeostasis of phosphorus and potassium in the body .
however , the renal mechanisms that ensure a balance of phosphorus and potassium are impaired in patients with kidney disease , leading to increased risk of hyperphosphatemia and hyperkalemia , both of which are associated with higher mortality in patients on hd [ 32 , 33 ] . while limited evidence is available on the correlation between dietary protein intake and serum potassium , dietary phosphorus intake is strongly and positively correlated with dietary protein intake , and a high protein intake is often associated with the development of hyperphosphatemia .
hyperphosphatemia may worsen hyperparathyroidism and renal osteodystrophy , promote vascular calcification and cardiovascular events , and increase mortality [ 3437 ] . in vivo phosphorus
hence it is naturally found in foods that are rich in protein , and imposing dietary phosphorus restriction is often associated with a reduction in dietary protein intake , leading to an increased risk of pem and mortality .
the results of this study show that using the ep-10 as a dietary assessment and educational tool to increase dietary protein intake to recommended levels did not cause an increase in serum phosphorous , potassium , or kt / v .
this suggests that patients are able to understand and utilize the ep-10 to meet their recommended protein intake and achieve an improvement in their albumin level yet avoid the associated complications of hyperphosphatemia and hyperkalemia .
it also suggests that restricting dietary protein below the current nkf kdoqi guidelines in an attempt to manage serum phosphorous is not necessary with prescription of and compliance to phosphate binders .
more research needs to be conducted to understand the reasons for insufficient energy and/or protein intake in ckd patients on hd . amongst many possible reasons , it is important to determine if a relationship exists between a patient 's degree of knowledge and understanding and their dietary compliance , as successful individual dietetic counseling relies on this relationship . as this study does not include
a control group , future research should focus on a randomized control trial to assess the effectiveness of ep-10 versus conventional methods in the clinical setting .
the results of this study show that using the ep-10 as an assessment and educational tool during nutritional counseling of hemodialysis patients has the potential to significantly improve albumin and transferrin levels . |
objective .
the expedited 10 g protein counter ( ep-10 ) is a quick and valid clinical tool for dietary protein quantification .
this study aims to assess the clinical effectiveness of the ep-10 in improving serum albumin and transferrin in chronic hemodialysis patients .
methods .
forty - five patients with low serum albumin ( < 38
g / l ) were enrolled in this study .
parameters measured included dry weight , height , dietary intake , and levels of serum albumin , transferrin , potassium , phosphate , and kinetic modeling ( kt / v ) .
the nutritional intervention incorporated the ep-10 in two ways ( 1 ) to quantify protein intake of patients and ( 2 ) to educate patients to meet their protein requirements .
mean values of the nutritional parameters before and after intervention were compared using paired t - test . results .
three months after nutritional intervention , mean albumin levels increased significantly from 32.2 4.8 g /
l to 37.0 3.2 g / l ( p < 0.001 ) .
thirty - eight ( 84% ) patients showed an increase in albumin levels , while two ( 4% ) maintained their levels .
of the thirty - six ( 80% ) patients with low transferrin levels ( < 200 mg / dl ) , 28 ( 78% ) had an increase and two maintained their levels after intervention .
mean transferrin levels increased significantly from 169.4 39.9 mg / dl to 180.9 38.1 mg / dl ( p < 0.05 ) . conclusion .
nutritional intervention incorporating the ep-10 method is able to make significant improvements to albumin and transferrin levels of chronic hemodialysis patients . | 1. Introduction
2. Methods
3. Statistical Analysis
4. Results
5. Discussion |
cytomegalovirus ( cmv ) , part of herpesviridae genus , is a common virus , with positive serology found in more than two - thirds of the population .
however , the virus remains latent in immunocompetent individuals , while reactivation may occur in the setting of immunosuppression .
cmv is known to be opportunistic in immunocompromised individuals with hiv infection , organ transplantation , immunosuppressive chemotherapy , and corticosteroid therapy . in immunocompromised patient
, cmv infections can have an effect on the retina , respiratory system , central nervous system and gastrointestinal ( gi ) tract .
however , over the past decade , there have been emerging cases of severe cmv infections in immunocompetent individuals , with the gi tract found to be the most frequently affected site . in both immunocompromised and immunocompetent individuals ,
the majority of cmv enteritis cases have been reported in the ileum , including severe complications leading to perforation and ischemia , , .
presented is a case report of cmv duodenitis that manifested with life - threatening hemorrhage in an immunocompetent patient .
past medical history included alcohol abuse and hypertension , treated with hydrochlorothiazide . upon admission ,
the patient was found to have methicillin - sensitive staphylococcus aureus ( mssa ) bacteremia with aortic valvular endocarditis and disseminated septic emboli .
investigations revealed wide spread infection , including mssa meningitis , c6 - 7 osteomyelitis , and spinal and sternoclavicular joint abscesses .
multiple septic emboli infarcts were identified in the brain , liver , spleen and kidneys .
the patient ultimately required an aortic valve replacement . while in hospital , the patient developed significant upper gi bleeding , requiring aggressive blood transfusions .
general surgery was initially consulted , but less - invasive therapies were favored at the time due to the patient s severe comorbidities .
the duodenal ulcers were found to be actively bleeding on five subsequent endoscopies , with attempts to halt the bleeding through epinephrine injection , bicap cauterization , endoclipping of vessels , hemospray with procoagulant , and argon plasma coagulation .
ir embolization of the gastroduodenal artery and multiple branches of the superior and inferior pancreaticoduodenal arteries also failed to control bleeding ( fig . 1 , fig
3 ) . the patient continued to hemorrhage , requiring daily transfusions to maintain hemodynamic stability , totaling more than 95 units of packed red cells during the admission in the intensive care unit .
the last endoscopy revealed a generalized mucosal bleed throughout the duodenum . with no further options available within gi or ir s scopes of practice ,
duodenal resection , which included a whipple procedure , was recommended as treatment for the unrelenting duodenal hemorrhage .
however , given the patient 's critical condition and ongoing hemorrhage , it was felt that the patient would be at high risk ( > 30% ) for pancreaticojejunostomy anastomotic leak and significant mortality .
therefore , duodenal resection with total pancreatectomy and splenectomy was performed to control the hemorrhage and remove the risk of pancreaticojejunostomy leak .
multiple aberrant blood vessels extending into the duodenum were successfully controlled at the time of surgery .
the patient was critically ill throughout the procedure , and was transferred to the icu after the resection of the duodenum and pancreas .
the patient returned to the operating room in stages to complete the gastrojejunostomy and hepaticojejunostomy .
gross examination of the resected specimen revealed a flattened , congested , and focally ulcerated duodenal mucosa .
histologic sections from an ulcer revealed viral cytopathic changes in keeping with cmv ( fig .
the surrounding surface duodenal mucosa had extensive autolysis ; no vasculitis or intravascular thrombi were identified .
notably , ductal epithelial cells had viral cytopathic changes , which were also in keeping with cmv ( fig .
immunohistochemical stains for cmv were positive in both the duodenum and pancreas ( see insets of figs .
post - duodenal resection , the patient remained hemodynamically stable , requiring no additional blood transfusions .
infectious disease initiated intravenous ganciclovir , 5 mg / kg twice daily then transitioned to oral valganciclovir 900 mg bid once tolerating an oral diet for a total course of three weeks . while on antiviral medications , the completed blood count and creatinine were closely monitored for evidence of pancytopenia and renal dysfunction .
cmv infections commonly occur in the gi tract , while localized duodenal infection is very rare .
a 2011 study of cmv infection in the upper gi tract , found the stomach to be the most common site of involvement and only one out of thirty cases involved the duodenum .
literature review identified four case reports of cmv duodenitis , , , , where all four cases involved immunocompromised patients .
presented here is an atypical case of cmv duodenitis causing life - threatening hemorrhage in an immunocompetent individual , with no history of immunosuppressive treatments or infections . however , this patient did present with critical illness from his disseminated mssa bacteremia and infective endocarditis , which could have caused a transient depression in immunity .
icu admission , use of antibiotics , and use of antacids including h2 blockers and ppis , are found to be risk factors for cmv reactivation in immunocompetent patients . a recent review of reactivation of cmv infection in immunocompetent critically - ill patients found that cmv colitis was the most prevalent manifestation , carrying a 71% in - hospital mortality rate despite specific treatment with ganciclovir .
although severe gi cmv is uncommon in immunocompetent hosts , there can be a significant mortality rate associated and thus cmv reactivation should be considered in the differential diagnosis of uncontrolled gi bleeding , especially in the setting of a critically - ill patient .
the diagnostic gold standard for cmv infection is histopathological examination of endoscopic biopsy or surgical specimen .
hematoxylin - eosin stains typically show intranuclear owl - eye or cowdry type a inclusion bodies in stromal and endothelial cells , which are hypertrophic cells containing eosinophilic intranuclear viral inclusions .
serologic detection of cmv igm and igg are often not helpful in identifying active disease but may determine recent or past exposure .
virologic testing options include cmv dna pcr and cmv blood antigenemia assays . however , it s not uncommon to have isolated gastrointestinal cmv disease without detectable viremia , as in the case of this patient
. therefore , the roles of serologic and virologic testing appears to be limited in the diagnosis of gi - restricted cmv disease .
first - line treatment for gastrointestinal cmv disease is intravenous ganciclovir 5 mg / kg bid or oral valganciclovir 900 mg bid .
there is significant toxicity with ganciclovir , including headaches , transaminitis , fever , rash , and myelosuppression in the form of pancytopenia .
an alternative is foscarnet , which can be used as initial treatment or in ganciclovir failure .
the role of treatment is unclear in immunocompetent hosts as some will resolve disease without any treatment .
there are limited data on duration of therapy for immunocompetent hosts , but studies have documented response in two to three weeks , .
unfortunately , gastrointestinal cmv disease is not always readily diagnosed , especially in an immunocompetent patient as suspicion may be low . in the case of cmv duodenitis , there does appear to be a significant potential for mortality .
here we presented an immunocompetent patient with cmv duodenitis that manifested with ongoing hemorrhage , failing multiple attempts at non - surgical treatments , and ultimately requiring surgical intervention .
this is the first known case report of life - threatening hemorrhage from cmv duodenitis managed successfully with surgery . among the four cmv duodenitis case reports found during literature review ,
three manifested with life - threatening bleeding , only one patient survived , , , . in the surviving case , cmv was identified emergently on histopathologic examination of endoscopic biopsy .
the patient was started on ganciclovir immediately and stabilized after 4 days of therapy .
interestingly , acute necrotizing pancreatitis secondary to arterial embolizations , was found in our patient .
this likely exacerbated the duodenal bleed and would have lead to further complications if surgical intervention was delayed .
gi cmv infections should be on the differential diagnosis of immunocompetent patients presenting with recurrent gi bleeding , especially in critically ill patients due to transiently suppressed immunity .
cmv duodenitis has significant potential to be life - threatening . if failing noninvasive treatments , surgical intervention may be indicated .
written informed consent was obtained from the patient for publication of this case report and the accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
( 1 ) lucy shen : drafting of the article , critical revision of the article for important intellectual and clinical content ; ( 2 ) david youssef : drafting of the article , critical revision of the article for important intellectual and clinical content ; ( 3 ) suzan abu - abed : image creation , critical revision of the article for important intellectual and clinical content ; ( 4 ) sangita k. malhotra : critical revision of the article for important intellectual and clinical content ; ( 5 ) kenneth atkinson : critical revision of the article for important intellectual and clinical content ; ( 6 ) elena vikis : critical revision of the article for important intellectual and clinical content ; ( 7 ) george melich : critical revision of the article for important intellectual and clinical content , final approval of the version to be submitted ; ( 8) shawn mackenzie : drafting of the article , critical revision of the article for important intellectual and clinical content , final approval of the version to be submitted . | highlightsgastrointestinal cytomegalovirus infections can occur in immunocompetent patients.diagnosis relies on histopathologic examination of endoscopic biopsy specimen.early recognition and antiviral treatment are important to patient outcome.cytomegalovirus duodenitis has significant potential to be life - threatening . | Introduction
Presentation of case
Outcome and follow-up
Discussion
Conclusion
Conflicts of interest
Funding sources
Ethical approval
Consent
Author contributions
Registration of research studies
Guarantor |
there are different types of pediatric renal tumors , like wilms tumor ( wt ) , mesoblastic nephroma , multicystic nephroma , clear cell sarcoma , rhabdoid tumor , nephroblastomatosis , intrarenal neuroblastoma , etc . among these ,
wt or nephroblastoma is the most common one ( 85% ) . it is seen primarily in infants , with 50% of the cases occurring below the age of 3 years .
wt is usually sporadic , although 1 - 2% of the patients have a positive family history . grossly , most cases of wt are large , solitary , gray - white and fleshy , well - circumscribed , rounded and soft in consistency .
cut - sections show predominantly solid pale areas with cystic change , necrosis and hemorrhage .
microscopically , it comprises three major components , undifferentiated blastema , mesenchymal ( stromal ) tissue and epithelial tissue .
when all the three components present together , then it is called a triphasic or classic wt , but it may be bi- or monophasic also .
additional morphologic features that can be encountered in wt include ciliated mucinous , endocrine cells of various types , renin - producing cells , neuroepithelium , neuroblast , rhabdomyoblast and mature ganglion cells , neuroglia , adipose tissue and cartilage , bone and hematopoietic cells . among the different prognostic factors , stage and histologic type play important roles in assessing the prognosis and survival of patients with wt .
capsular invasion , rupture at surgery , renal sinus involvement , renal vein invasion , tumor implants , lymph node metastasis and bilaterality are the main criteria used .
other important prognostic factors are age , extensive tubular differentiation , skeletal muscle differentiation , mucin production , deoxyribonucleic acid ( dna ) ploidy , p53 mutation , proliferation marker ( mib - i or ki67 ) , proliferating cell nuclear antigen ( pcna ) , p27 protein expression , vascular endothelial growth factor receptor ( vegfr ) , platelet derived growth factor receptor ( pdgfr ) and type i insulin - like growth factor receptor expression have prognostic significance , but their roles are still not yet well established .
this study was carried out to analyze the histopathological spectrum of pediatric renal tumors and to study the expression of proliferation marker ( ki-67 ) and p53 in wt and correlate its expression in epithelial and blastemal components in different stages .
cases were collected and records were documented as per the following proforma : ( a ) identification : name , age , sex , ( b ) clinical findings , ( c ) radiological findings and ( d ) peroperative findings . after receiving the specimen containing tumor in buffered formalin , macroscopical examination of the tumor was carried out .
a graphic record of the site of each block was indicated on a photograph of the bisected specimen using a digital camera . besides the adrenal glands , lymph nodes , renal vessels , renal sinus , capsule , pelvis and ureter wherever removed along with the kidney tumor , had been examined and sections were taken to make a pathological staging according to the national wilms tumor study ( nwts-5 ) staging . hematoxylin - eosin stain was performed and used for tumor diagnosis , histologic type and staging .
further sections were taken on poly - l - lysine - coated slides from tumor - containing blocks for immunohistochemistry for ki-67 and p53 using a standard immunohistochemistry protocol .
we examined a total of 27 cases of pediatric renal tumors , of which 20 cases were wt ( 74.1% ) , three cases were mesoblastic nephroma ( 11.1% ) , three cases were clear cell sarcoma ( 11.1% ) and one case was rhabdoid tumor ( 3.7% ) [ figure 1a ] .
photomicrograph of a case of triphasic wilm 's tumor ( wt ) [ hematoxylin - eosin ( h / e ) ; lp ] ( a ) a case of triphasic wt ( h / e ) ( b ) low - power view of a case of epithelial predominant wt ( h / e ; lp ) ( c ) high - power view of a case of blastema predominant wt ( h / e ; lp ) ( d ) high - power view of unfavorable histology showing abnormal mitosis , nuclear enlargement and hyperchromasia ( h / e ; hp ) regarding the age distribution of wt cases , it was seen that all the cases were in between 8 and 74 months , with a peak age incidence of 1 - 3 years of age [ table 1 ] .
distribution of different histologic types of pediatric renal tumors according to age on analyzing the sex distribution of wt , it was observed that 13 cases ( 65% ) were males and seven cases ( 35% ) were females , with a male : female ratio of 1.9:1 .
of the 18 cases of wt , 10 cases presented in stage i , six cases presented in stage ii , one case presented in stage iii and one case presented in stage iv .
most cases of wt were in stage i ( 55.55% ) and in stage ii ( 33.33% ) [ table 2 ] . in two cases , based on the computed tomography scan findings and fine needle aspiration cytology ( fnac )
reports suggestive of wt , pre - operative chemotherapy was administered because of the large size . as these specimens showed extensive necrosis and fibrosis , nwts-5 staging
distribution of pediatric renal tumors according to stages epithelial predominance [ figure 1b ] and blastema predominance [ figure 1c ] were equally distributed and did not show any correlation with the staging .
unfavorable histology was found in one [ figure 1d ] out of 18 cases and , therefore , the percentage of unfavorable histology was 5.6% .
ki-67 labeling index was determined for blastema and epithelial components separately . of the 18 cases of wt ,
the range of ki-67-positive cells in the epithelial component in stage i was 20 - 68% ( mean 44.8% ) and in blastema this was 12 - 48% ( mean 31.7% ) ; in stage ii , the epithelial component was 65 - 80% ( mean 74.3% ) [ figure 2a and b ] and in blastema it was 40 - 58% ( mean 47.8% ) ; in stage iii , in the epithelial component , this was 78% and in blastema it was 68% [ figure 3a and b ] .
immunostaining of the single case of stage iv could not be done as blocks were not available .
combined epithelial and blastemal ki-67 expression in different stages were : stage i-38.25% , stage ii-61.08% and stage iii-73% .
total proliferation index ( pi ) of the epithelial component was 57.2% , and in the blastema component it was 39.53% [ table 3 ] .
( a ) low - power view of very high ki 67 labeling index in the epithelial component ( ihc ; lp ) ( b ) high - power view of case 2a ( ihc ; hp ) ( a ) low - power view showing low ki 67 labeling index in the stromal component than in the blastema component ( ihc ; lp ) ( b ) high - power view showing case 3a ( ihc ; hp ) range of ki-67 proliferation index in wt and stage of advancement on analyzing the data , it was found that the epithelial component had a significantly higher pi compared with the blastema component in stage i ( p=0.05 ) [ figure 4a ] , in stage ii ( p=0.02 ) and in all stages together ( p=0.002 ) . again , comparing the pi between the stages , it was found that stage i showed a significantly lower pi compared with stage ii for the epithelial component ( p=0.001 ) [ figure 4b ] , blastema component ( p=0.03 ) and the epithelial and blastema components together ( p=0.002 ) . ( a )
photomicrograph showing increased labeling index of ki-67 in the epithelial component than that of blastema in stage i ( ihc ; lp ) ( b ) photomicrograph showing stage ii wilm 's tumor with higher labeling index of ki-67 than stage i ( ihc ; lp ) expression of p53 was performed in a semiquantitative manner as mild , moderate and marked .
only one case of stage i , two cases of stage ii and one case of stage iii showed moderate expression of p53 , and all the others showed a low level of expression .
p53 expression did not show any significant difference of expression between the epithelial and the blastema components .
however , it was observed that the single case of stage iii had a higher intensity of staining than the other cases in the lower stages .
we have examined different histologic subtypes of cases of pediatric renal tumors in our study . among a total of 27 cases of pediatric renal tumors , the incidence of wt was
74.1% , of mesoblastic nephroma was 11.1% , of clear cell sarcoma was 11.1% and of rhabdoid tumor was 3.7% .
our percentage of wt was less than that of the world literature , which is approximately 85% .
because the world literature is based on western data , this difference may partly be due to the small number of cases present in this study and regional variation due to unknown causes .
regarding sex distribution , in our study , there is a definite male predominance in wt ( male : female=1.9:1 ) , which is similar to the study by mishra , et al . and other asian studies , but the ratio is somewhat greater than that of american / european data .
this may be because of the social practices favoring the male child , who is more frequently brought to the hospital than the female child . analyzing the distribution of 18 wt cases in different stages according to clinicopathological staging , we had 10 cases ( 55.55% ) in stage i , six cases ( 33.33% ) in stage ii and one case each ( 5.5% ) in stage iii and stage iv .
patient numbers in stage i and ii is greater than that in stage iii and iv , which correlates with other studies . of the 18 cases ,
17 cases ( 94.4% ) showed a favorable histology and only one case ( 5.6% ) had an unfavorable histology .
this is similar to the percentage of unfavorable histology of 5% quoted in the sternberg 's diagnostic surgical pathology .
we have further analyzed the distribution of proliferation marker ( ki-67 ) and p53 in different stages of wt , with special reference to various histological components . in our study , it was observed that the pi of the epithelial component was significantly higher than that of blastema in all stages .
others , i.e. khine , ghanem and juszkiewicz , have also made a similar observation ; however , none of these studies give any plausible explanation for these observations .
this is difficult to understand if the epithelial component is considered as a differentiation product from other components of the tumor .
nephroblastoma is a heterogenous tumor composed of epithelial , blastema and stromal components , and the epithelial component is expected to have the lowest proliferative activity .
the dense blastema component , which is considered to be less - differentiated tissue from the epithelium , had a lower proliferation rate than the epithelial component .
khine et al . in a study similar to ours , with eight cases of pediatric nephroblastoma , noted that the pi of ki-67 and pcna of the epithelial component was higher than that of the other components .
although we have not got survival data in our study , we found that the pi was higher in higher stages . in a study by juszkiewicz et al .
, the pi of neoplastic cells was determined in epithelial , blastema and stromal components of the tumors . in tumors after chemotherapy , a higher pi in epithelial component and a lower index in the blastema component was found .
found that increased pcna index in the epithelial component was associated with a higher stage of clinical advancement , as seen in our study .
it was also observed that pi in stage ii is significantly higher than stage i for the individual component as well as overall .
statistical analysis could not be performed in other stages due to the small number of cases .
sredni et al . found that overall survival was higher for patients with p53 negative than with p53-positive cases , and they concluded that increased p53 expression in wt seems to be associated with advanced disease and relapse .
huang et al . showed that p53 accumulation in favorable histology wt is associated with angiogenesis and clinically aggressive disease .
although this study performed on a small number of cases did not show any statistical significance between p53 expression and staging , higher intensity was noted in stage ii and iii .
literature on the significance of p53 in wt is more conflicting . in a study by malkin et al .
however , a later study by lahoti et al . had shown that p53 mutation correlated with recurrence and metastasis .
difference in staining pattern , staining protocol , antibody used and detection system used may be responsible for this discrepancy . from this study
, we found that in our set - up , wt forms 74.1% of all pediatric renal tumors , with a definite male predominance .
majority of the cases of wt presented in stage i ( 55.55% ) and stage ii ( 33.33% ) . | context : the spectrum of pediatric renal tumors is different from adult renal tumors , and wilms tumor ( wt ) forms the majority . the histological type and clinicopathological staging are the two important prognostic parameters .
the role of newer prognostic factors is not clear.aims:this study was performed to analyze the histopathological spectrum of pediatric renal tumors and to study the expression of proliferation markers ( ki-67 and p53 ) in wt and correlate its expression in epithelial and blastema components in different stages.materials and methods : twenty - seven cases of pediatric renal tumors were collected over 2 years .
hematoxylin - eosin staining was used for diagnosis .
immunostaining was performed for ki-67 and p53 .
ki-67 proliferation index ( pi ) and p53 expression were determined in each case and for the epithelial and blastema components separately.statistical analysis and results : we had 20 cases of wt ( 74.1% ) , three cases of mesoblastic nephroma ( 11.1% ) , three cases of clear cell sarcoma ( 11.1% ) and one case of rhabdoid tumor ( 3.7% ) .
it was observed that the pi of the epithelial component ( 57.2% ) was significantly higher than that of blastema ( 39.53% ) in all stages .
the pi in stage ii is significantly higher than that in stage i. statistical analysis could not be performed in stages iii and iv due to the small number of cases .
p53 expression did not show any significant difference in the epithelial and blastema components .
there was also no significant difference between the stages.conclusion:in this study , we found the differences between pi of different tissue components of wt , with the epithelial component having a higher pi , which correlated with the stage of advancement of the disease . | INTRODUCTION
MATERIALS AND METHODS
RESULTS AND ANALYSIS
DISCUSSION |
significant pulmonary disease occurs in the majority of cf patients , manifested as a progressive cough , increased dyspnea and decline in lung function caused by acute and chronic infection and inflammation in the airways . with advances in therapy and a multidisciplinary approach ,
life expectancy has increased dramatically from a median of several years to a projected median survival of over 50 years of age for individuals born in 2000 .
several factors may contribute to the increase in survival of cf patients , including early diagnosis of cf followed by early preventive treatment and close clinical follow - up , aggressive treatment of malnutrition and the enhanced use of antibiotics and physiotherapy both as intermittent routine treatment and as treatment for respiratory exacerbations , the vast majority of which are caused by bacterial pathogens .
furthermore , frequent intravenous ( iv ) antibiotics treatment at the hospital and vigorous eradication treatment for first isolation of pseudomonas aeruginosa were also shown by the scandinavian centers to be beneficial [ 37 ] .
intravenous antibiotics are usually the treatment of choice in such exacerbations and may be given either as home treatment or in a hospital setting .
several studies have compared both treatment settings , but to date no conclusion as to which is superior has been established in the literature [ 811 ] . in most studies ,
inspection of improvement in pulmonary function test ( pfts ) indices during the interventions had shown large variation , indicating that some of the patients vastly improved or deteriorated , while other patients did not .
furthermore , the effect of the treatment settings in different clinical preconditions such as gender , chronic colonization of pseudomonas aeruginosa and cf - related complications and the probability of a patient to improve his lung function at a specific treatment setting were not fully explored .
moreover , it has been shown in previous studies by sanders et al that about 25% of the patients treated for pulmonary exacerbations fail to recover to baseline pfts , especially female patients , undernourished patients , and patients with persistent infection with pseudomonas aeruginosa , burkholderia capacia complex or methicillin - resistant staphylococcus aureus .
the aim of this study was to investigate the likelihood of a cf patient to improve in a specific treatment setting and to investigate whether home or hospital setting results were influenced by clinical preconditions .
the study population consisted of patients with proven cf by clinical characteristics and identified cftr alteration mutation and/or pathological sweat chloride test ( above 60 mmol / l ) .
patients were aged 12 to 47 years , and all were part of the national cf center , edmond and lily safra children s hospital , tel hashomer , israel .
we retrospectively obtained all data of hospital and home treatments between the years 20072009 from records of the cf patients .
inclusion criteria were the necessity of home or hospital treatment due to acute respiratory exacerbation .
patients who needed supplemental oxygen , iv fluid resuscitation and supplemental caloric intake such as total parental nutrition ( tpn ) or patients who were unlikely to manage home treatment were hospitalized .
all patients , whether treated at home or in the hospital , received at least 2 anti - pseudomonal antibiotics based on the sensitivities of the most recent sputum culture . where aminoglycosides were prescribed , doses were adjusted according to blood levels and kidney function test . nebulized antipseudomonal antibiotics such as tobi or colistin were continued .
patients colonized with staphylococcus aureus were also treated with anti - staphylococcal antibiotics during the acute episode .
patients treated at the hospital routinely received physiotherapy twice a day , were put on a high - calorie diet , were treated by a multi - disciplinary team according to need , and performed forced spirometry twice a week .
patients treated at home usually performed physiotherapy 3 to 5 times a week provided by community physiotherapists or family members , and performed forced spirometry and had a physician follow - up once or twice weekly .
patients completed their treatment when clinical and pfts improvement was achieved as decided by the treating physician , and treatment length ranged from 10 days to 5 weeks .
the study was approved by the appropriate ethical committees related to the institution in which it was performed .
forced spirometry was performed according to the report working party standardization of lung function tests ers / ats guidelines prior to start of treatment , during treatment and within a week post - treatment regardless of the treatment site .
all curves were stored , and the best curve with the maximal forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) were analyzed for the study .
the effects of the treatment site were primarily judged by changes in fev1 pre- and post - treatment .
in addition , the effects of treatment site were related to other clinical preconditions such as cf alteration mutations , gender , body mass index ( bmi ) , baseline fev1 ( % predicted ) , chronic colonization of pseudomonas aeruginosa , mycobacterium abscessus and methicillin - resistant staphylococcus aureus ( mrsa ) , cf - related complications such as pancreatic insufficiency ( pi ) , cf - related diabetes mellitus ( cfrdm ) , distal intestinal obstruction syndrome ( dios ) , liver and kidney disease , pancreatitis and allergic bronchopulmonary aspergillosis ( abpa ) .
the effect of treatment site on weight gain , duration of treatment , time to next exacerbation and the cost of the treatment were also inspected . the change in spirometry indices in relation to treatment
setting ( groups ) and the effects of the clinical preconditions were compared using paired and unpaired t - tests , as appropriate .
the odds ratio of a patient to improve his fev1 in relation to home or hospital setting and in relation to clinical preconditions was analyzed using chi - square or fisher test .
the study population consisted of patients with proven cf by clinical characteristics and identified cftr alteration mutation and/or pathological sweat chloride test ( above 60 mmol / l ) .
patients were aged 12 to 47 years , and all were part of the national cf center , edmond and lily safra children s hospital , tel hashomer , israel .
we retrospectively obtained all data of hospital and home treatments between the years 20072009 from records of the cf patients .
inclusion criteria were the necessity of home or hospital treatment due to acute respiratory exacerbation .
patients who needed supplemental oxygen , iv fluid resuscitation and supplemental caloric intake such as total parental nutrition ( tpn ) or patients who were unlikely to manage home treatment were hospitalized .
all patients , whether treated at home or in the hospital , received at least 2 anti - pseudomonal antibiotics based on the sensitivities of the most recent sputum culture . where aminoglycosides were prescribed , doses were adjusted according to blood levels and kidney function test . nebulized antipseudomonal antibiotics such as tobi or colistin were continued .
patients colonized with staphylococcus aureus were also treated with anti - staphylococcal antibiotics during the acute episode .
patients treated at the hospital routinely received physiotherapy twice a day , were put on a high - calorie diet , were treated by a multi - disciplinary team according to need , and performed forced spirometry twice a week .
patients treated at home usually performed physiotherapy 3 to 5 times a week provided by community physiotherapists or family members , and performed forced spirometry and had a physician follow - up once or twice weekly .
patients completed their treatment when clinical and pfts improvement was achieved as decided by the treating physician , and treatment length ranged from 10 days to 5 weeks .
the study was approved by the appropriate ethical committees related to the institution in which it was performed .
forced spirometry was performed according to the report working party standardization of lung function tests ers / ats guidelines prior to start of treatment , during treatment and within a week post - treatment regardless of the treatment site .
all curves were stored , and the best curve with the maximal forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) were analyzed for the study .
the effects of the treatment site were primarily judged by changes in fev1 pre- and post - treatment . in addition , the effects of treatment site were related to other clinical preconditions such as cf alteration mutations , gender , body mass index ( bmi ) , baseline fev1 ( % predicted ) , chronic colonization of pseudomonas aeruginosa , mycobacterium abscessus and methicillin - resistant staphylococcus aureus ( mrsa ) , cf - related complications such as pancreatic insufficiency ( pi ) , cf - related diabetes mellitus ( cfrdm ) , distal intestinal obstruction syndrome ( dios ) , liver and kidney disease , pancreatitis and allergic bronchopulmonary aspergillosis ( abpa ) .
the effect of treatment site on weight gain , duration of treatment , time to next exacerbation and the cost of the treatment were also inspected .
the change in spirometry indices in relation to treatment setting ( groups ) and the effects of the clinical preconditions were compared using paired and unpaired t - tests , as appropriate .
the odds ratio of a patient to improve his fev1 in relation to home or hospital setting and in relation to clinical preconditions was analyzed using chi - square or fisher test .
data were analyzed from 54 patients , 12 to 47 years of age who underwent 1 or up to 3 treatments at home or at the hospital .
twenty - four ( 44% ) patients received only hospital treatments , 12 ( 22% ) received only home treatments , and 18 ( 33% ) received both treatments during the enrolment period
. a total of 139 treatments were conducted , of which 84 ( 60% ) were at the hospital and 55 ( 40% ) were at home .
the anthropometric data , bacterial colonization , cf - related complications and lung function indices prior to intervention are presented in table 1 .
there were significant differences between the home and hospital groups concerning height and weight , but no difference in calculated body mass index ( bmi ) . in the hospital group
more patients had cf - related complications compared to the home group , in particular , pancreatic insufficiency ( pi ) ( 75 ( 89% ) vs. 38 ( 69% ) , respectively ; p<0.01 and for pi 64 ( 76% ) vs. 15 ( 27% ) , respectively ; p<0.01 ) .
the percent of patients suffering from severe lung disease differed between settings : n=44 ( 52% ) at the hospital and n=13 ( 24% ) at home , as reflected by the different spirometry indices outlined in table 1 mean duration of treatment was 9.76.7 days for the hospital site and 16.39.1 days for the home site ( p<0.02 ) .
specifically , treatment duration in patients colonized with pseudomonas aeruginosa was significantly shorter at the hospital than at home ( 11.15.5 vs. 18.011.0 days , respectively ; p<0.01 ) , as well as in patients without cf - related complications ( 11.96.6 vs. 18.512 days , respectively ; p<0.01 ) . in
in that respect , the cost of stay at the hospital was $ 540 usd / day , with an approximate treatment cost of $ 5200 usd / admission .
cost of home treatment was $ 140 usd / day with an approximate total treatment cost of $ 2300 usd .
the effect of treatment site on lung function for the entire group is presented in table 2 .
there was no significant difference in mean percent change in spirometry indices in relation to treatment site .
the effect of treatment site on changes in fev1 in relation to the main clinical preconditions is presented in table 3 .
looking at hospital versus home treatments , patients having cf - related complications improved their fev1 significantly more at the hospital compared to at home ( 13.1%19.4 vs. 1.9%14.9 , respectively ; p<0.01 ) , especially patients suffering from cfrdm ( 11.4%18.7 vs. 1.7%14.6 , respectively ; p<0.05 ) and patients with pi ( 14.419.6 vs. 4.122.5 , respectively ; p<0.01 ) . at the hospital
, patients colonized with pseudomonas aeruginosa improved their fev1 significantly more compared to patients not colonized with pseudomonas aeruginosa ( 21.4%21 vs. 7.4%29 , respectively ; p<0.02 ) .
when looking at the home site only , those having cf - related complications improved fev1 less than those free of complications ( 1.9%14.9 vs. 24.6%42.1 , respectively ; p<0.02 ) , especially patients suffering from cfrdm , who improved their fev1 significantly less than patients without cfdrm ( 1.7%14.6 vs. 17%30.8 , respectively ; p<0.02 ) .
changes in fev1 were not influenced by the treatment site in relation to sex , cftr alteration mutation , chronic colonization of mrsa or mycobacterium abscessus .
the odds ratio of a patient to improve or deteriorate their fev1 in different treatment sites in relation to clinical preconditions is presented in table 4 .
the odds to improve fev1 ( more than 10% baseline ) at the hospital for patients with cf - related complications , especially cfrdm , were 3 times higher than the odds ratio to improve at home ( 0.96 vs. 0.32 and 0.79 vs. 0.25 , respectively ) .
the odds to deteriorate fev1 ( more than 10% baseline ) at home compared to at the hospital were higher for the entire study group ( 0.18 vs. 0.09 ) and for male patients ( 0.26 vs. 0.06 ) .
patients not colonized with pseudomonas aeruginosa deteriorated at the hospital significantly more than those colonized with pseudomonas aeruginosa ( 0.32 vs. 0.05 ) .
age , baseline pfts prior to treatment , cftr alteration mutation and pancreatic insufficiency did not affect the changes in fev1 in either of the sites .
other findings : patients treated at the hospital improved their weight by 1.364.6 kg compared to patients treated at home who improved their weight by 0.493.6 kg ( p=0.06 ) .
in the present study we investigated the preferable treatment site , hospital versus home , during respiratory exacerbations in our cf population in relation to hospitalization duration , change in pfts as a representative for clinical improvement , and the odds ratio of a patient to improve or deteriorate pfts in either of the settings .
we found that while for the entire group mean improvement in pfts was similar in both sites , the improvement was achieved within a shorter duration of treatment when carried out at the hospital .
we further found that the hospital site was most beneficial to the specific subgroup of patients with cf - related complications , especially cfrdm .
the improvement in pft was accompanied by a trend toward better improvement in weight gain at the hospital .
although all patients improved their lung functions in both treatment settings , we noticed that there was a greater risk for deterioration of lung functions at home for all patients and especially for males . on the contrary ,
pulmonary exacerbations are a major source of morbidity for cystic fibrosis patients and are likely to contribute to lung function decline .
therefore , determining the optimal approach to exacerbation therapy is an important issue for which there has been little objective data .
hospital site : hospital treatment provides the patient a comprehensive supportive multidisciplinary care with physiotherapy twice a day physiotherapy and diet monitoring in our center .
this may explain why patients with cf - related complications , cfrdm in particular , significantly improve pfts more at the hospital and were significantly more likely to do so . a significant portion of our adult patients have cf - related complications .
naturally these patients are no longer living with their parents who used to support them during exacerbations at an earlier age . in consequence
, their ability to treat themselves , especially to monitor their glucose blood level due to cfrdm , may be compromised compared to monitoring and treatment given at the hospital .
showed that even non - diabetic cf patients tend to lose their glucose blood level balance during acute pulmonary exacerbations .
in their pilot study they showed that these patients may benefit from glucose blood level monitoring and may improve pfts after being treated with insulin when required .
indeed , the efforts of a cf endocrinologist and a specialized dietitian and customized food may explain both the better weight gain and the better control of cfrdm , and in consequence the better pfts achieved in our hospitalized patients .
patients colonized with pseudomonas aeruginosa also showed a trend of better improvement in pfts at the hospital as compared to those treated at home .
this significantly shorter duration yielded similar improvement in pfts compared to the longer duration of home treatment .
cochrane review by balaguer and gonzalez de dios found that maximal recovery of lung function occurs within the first 8 days of therapy with intravenous antibiotics at the hospital .
nazer et al . demonstrated that even patients with different disease severity levels improved more and in a shorter time when treated at the hospital .
the disadvantage of hospital treatment is the possibility of cross - infections , stopping one s daily routine and the cost of admission , which is significantly higher than the cost of home treatment .
thornton et al in the united kingdom showed hospital treatment to be more expensive than home treatment .
we believe that the difference in costs is similar in most parts of the world , but should not be a factor when evaluating the best option for the patient .
home site : there is a large worldwide drive for treating exacerbation in cf patients at home .
the cystic fibrosis pulmonary guidelines from 2009 concluded against delivery of intravenous antibiotic treatment in a non - hospital setting unless resources and equipment are equivalent to those in the hospital setting .
home treatment is characterized by self - care management and is considered to enable the patient to maintain daily routine and quality of life ( qol ) .
wolter et al showed no significant improvement of emotional well - being after home treatment compared to hospital treatment , and even better qol after hospital treatment .
more recently , esmond et al . showed the same results with no differences in qol between the 2 treatment sites , which might be explained by the anxiety accompanying an unfamiliar self - administered home treatment .
we have shown that for the entire study group and especially for male patients the odds ratio of deterioration of more the 10% from baseline fev1 at home were much higher compared to the odds ratio to deteriorate after treatment at the hospital .
we suggest that both the aggressive comprehensive treatment at the hospital as well as a poorer adherence during home treatment may contribute to this finding .
we found that at home our patients often ( but this has not been thoroughly investigated ) encounter technical problems managing venous access , forget treatments , are unable to fit them into a busy schedule or are under pressure to return to daily routine before treatment is finished and improvement is accomplished .
phillips showed that up to 84% of adult patients had drugs left over post - treatment and bosworth and nielson found decreased frequency and probably quality of chest physiotherapy when patients were treated at home .
indeed , adherence during home treatment without medical staff supervision is probably harder to maintain and should be considered when prescribing home treatment .
in conclusion , our results suggest that hospital treatment for acute respiratory exacerbations in cf may be superior to home treatment , as indicated by a shorter duration of hospitalization , better improvement in fev1 in patients with cf - related complications ( cfrdm in particular ) and a trend toward better weight gain .
our study is limited as a single center retrospective study and reflects the national center s experience and should promote further studies on this issue .
this study provides further insights into the advantages or disadvantages of treating subgroups of patients at the hospital versus home treatment for respiratory exacerbation . before the widespread use of home treatment , | summarybackgroundtreatment of respiratory exacerbations in cystic fibrosis ( cf ) is important in slowing disease progression .
the treatment may be given either at home or at the hospital .
the aim of our study was to compare both treatment settings.material/methodswe retrospectively analyzed data of 139 treatments in 54 cf patients ( age range1247 y ) treated for respiratory exacerbations at the hospital ( n=84 ) and/or at home ( n=55 ) .
primary outcomes were improvement in pulmonary function tests ( pfts ) , weight gain and duration of treatment in relation to treatment setting .
secondary outcomes were these same parameters , but in relation to different clinical preconditions and cf - related complications.resultsmean improvement in fev1 ( % predicted ) was similar between the hospital and home treatments ( 14.334.4% vs. 14.320.2% , respectively ; ns ) , yet treatment duration was significantly shorter at the hospital ( 9.76.7 vs. 16.39.1 days , respectively ; p<0.02 ) , especially for patients colonized with pseudomonas aeruginosa ( 11.15.5 vs. 18.011.0 days , respectively ; p<0.01 ) . at the hospital ,
a subgroup of patients with cf - related complications improved their fev1 significantly more than those at home ( 13.119.4% vs. 1.914.9% , respectively ; p<0.02 ) , particularly patients with cf - related diabetes mellitus ( cfrdm ) ( 11.418.7% vs. 1.714.6% , respectively ; p<0.05 ) .
patients tended to gain more weight at the hospital compared to home treatment ( 1.364.6 kg and 0.493.6 kg respectively ; p=0.06).conclusionshospital treatment for acute respiratory exacerbations in cf may be superior to home treatment , as indicated by a shorter duration of hospitalization , better improvement in fev1 in patients with cf - related complications , cfrdm in particular and a trend toward better weight gain . | Background
Material and Methods
Subjects
Study design
Spirometry
Analysis of data
Statistical analysis
Results
Discussion
Conclusions |
diabetic kidney disease is the leading cause of end - stage renal disease ( esrd ) , and the number of individuals requiring chronic dialysis or renal transplantation is still increasing in most countries .
both urinary albumin excretion and estimated glomerular filtration rate ( egfr ) are independently associated with the worsening of kidney function [ 2 , 3 ] .
we previously reported that a very high proportion of japanese subjects with type 2 diabetes mellitus with stage 3 chronic kidney disease ( ckd ) progressed to stage 4 ckd within 3 years , and that the existence of albuminuria further accelerated the clinical course .
although the clinical course of the deterioration of kidney function might differ among study populations , another study from the united kingdom reconfirmed a similar relation between albuminuria and egfr .
we also found that hemoglobin concentration was a significant predictor of deterioration to ckd stage 4 .
anemia is a common complication of chronic kidney disease ( ckd ) . in diabetes mellitus
, anemia develops earlier and is more severe than in patients with renal impairment with other causes [ 5 , 6 ] .
anemia in diabetes mellitus is known to be a risk factor for the development and progression of micro- and macrovascular complications of diabetes mellitus as well as increased mortality independent of the presence or severity of diabetic nephropathy .
a reduced hemoglobin concentration , even within the normal range , is associated with an increased risk of esrd and death in subjects with chronic renal failure .
it was also reported that lower hemoglobin level was related to subsequent decline of kidney function in subjects with persistent macroalbuminuria and also in those without albuminuria . however , there is no report on the risk factors for decline of kidney function according to clinical stage , divided by egfr .
especially , data on the gfr decline in subjects with type 2 diabetes mellitus whose gfr was less than 60 ml / min/1.73 m are scarce despite the clinical importance of this stage in relation to further deterioration of kidney function .
we prospectively evaluated the significance of hemoglobin concentration and other clinical factors in the three - year decline of kidney function in a hospital - based study cohort .
the details of the study participants were described previously . all of the subjects admitted to saiseikai central hospital for a 2-week diabetic education program from january 1999 to december 2004 were study candidates .
those subjects whose egfr was less than 30 ml / min/1.73 m and those with a strong suspicion of renal disease with causes other than diabetic nephropathy ( e.g. , glomerulonephritis ) were excluded . of the 1901 subjects , 7 died ( 5 due to cancer , 1 sudden death , and 1 acute myocardial infarction ) , 38 moved to other medical facilities for medical or personal reasons , and 553 withdrew for unknown reasons . as a result
, we analyzed 1303 japanese subjects with type 2 diabetes mellitus whose egfr was 30 ml / min/1.73 m. the subjects were instructed on a standard life - style modification according to the recommendations of the japan diabetes society ( jds ) .
blood collection after an overnight fast was performed in the early morning of day 2 of hospitalization .
serum fasting glucose , total cholesterol , hdl cholesterol , triglycerides , and serum creatinine were determined by enzymatic methods , and several other biochemical assays were performed with autoanalyzers .
almost all subjects were evaluated within four months after the end of the three - year period .
hba1c level on admission was determined by high - performance liquid chromatography ( hplc : arkray inc . ,
kyoto , japan ) according to the recommended method by ( jds ) at that time and converted to the national glycohemoglobin standardization program ( ngsp ) value .
twenty - four - hour urine collection was performed from day 2 to day 3 of hospitalization .
microalbuminuria was defined as albumin excretion rate ( aer ) in 24-hour urine collection of 20 to 200 g / min and macroalbuminuria as more than 200 g / min .
egfr ( ml / min/1.73 m ) was calculated as 194 cr age ( with further multiplication by 0.739 for female subjects ) using the equation provided by the japanese society of nephrology .
the study subjects were subdivided by egfr level , as follows : stratum 1 : egfrbaseline90 ml / min/1.73 m , stratum 2 : 60 egfrbaseline < 90 ml / min/1.73 m , and stratum 3 : 30 egfrbaseline < 60 ml / min/1.73 m. annual gfr decline was calculated by the formula : ( egfrbaseline egfrafter 3 years)/3
. annual percent reduction of egfr from the baseline value was calculated by the formula : annual gfr decline / egfrbaseline 100 .
data are expressed as mean standard deviation ( sd ) or number ( % ) except for triglyceride and aer .
differences in continuous variables among the three groups were tested by ad hoc fisher 's least significant difference ( lsd ) multiple comparison method after the analysis of variance ( anova ) .
multiple linear regression analysis with forced entry method was performed to detect independent significant predictors of decline of kidney function .
stratified analysis of risk factors for gfr reduction by egfr strata was needed , as the annual gfr reductions were different among egfr strata .
the mean age of this study cohort was around 60 years , two - thirds were male , and mean duration of diabetes was about ten years .
mean systolic blood pressure was 132 mmhg and mean diastolic blood pressure 75 mmhg .
about 30% of study participants had diabetic retinopathy . over 70% of hypertensive subjects and around 90% of hypertensive subjects with macroalbuminuria
briefly , subjects with advanced egfr stratum were older , predominantly male , and had longer disease duration , higher systolic blood pressure , better glycemic control , higher aer , and lower hemoglobin level compared to those with less advanced egfr strata .
the annual gfr decline was 2.3 5.4 ml / min/1.73 m in overall subjects .
that in male subjects was 2.5 5.4 and that in female subjects was 2.0 5.4 ml / min/1.73 m , with no significant sex difference .
annual gfr decline was 1.5 4.9 , 2.9 5.0 , and 7.1 7.5 ml / min/1.73 m in subjects with normoalbuminuria , microalbuminuria , and macroalbuminuria , respectively
. annual percent reduction of egfr was 1.4 5.5 , 3.2 5.7 , and 9.8 8.8% in subjects with normoalbuminuria , microalbuminuria , and macroalbuminuria , respectively .
annual gfr decline was significantly greater in stratum 1 compared to the other strata , and when we analyzed the annual percent reductions of egfr , the differences between stratum 1 and the other strata and also between stratum 2 and stratum 3 were significant ( table 1 ) .
the results of multiple linear regression analysis of annual egfr decline are shown in table 2 .
age , male sex , systolic blood pressure , logarithmically transformed aer , egfr strata , and hemoglobin concentration were significantly correlated with annual egfr decline .
the results of multiple linear regression analysis of the annual egfr decline stratified by egfr strata are shown in table 3 .
it revealed that factors that were significantly correlated with the annual egfr decline were different among the egfr strata .
aer at baseline was significantly correlated with the annual egfr decline in all egfr strata .
systolic blood pressure showed a significant correlation with the annual egfr decline only in stratum 2 .
the existence of diabetic retinopathy was not correlated with the annual egfr decline in all egfr strata .
hba1c at baseline was significantly associated with the annual egfr decline in stratum 1 and 2 but not in stratum 3 . on the other hand ,
the hemoglobin concentration showed a significant correlation with the annual egfr decline only in stratum 3 , but this correlation was relatively strong ( partial regression coefficient : 0.249 , p < 0.01 ) .
it has been confirmed that both the amount of urinary albumin and egfr value are useful to predict the outcome of kidney function in subjects with type 2 diabetes [ 24 ] .
also , as the decline of gfr is finally the most important clinical marker for the development of esrd , several risk factors for the decline of gfr have been advocated .
albuminuria / proteinuria , glycemic control , blood pressure control , existence of diabetic retinopathy , and the existence of anemia are proposed representative risk factors [ 24 , 9 , 10 ] .
we demonstrated that annual egfr decline rate and the factors correlated with the egfr reduction varied according to the baseline egfr status in this study .
annual percent reduction of egfr from the baseline value showed a u shape pattern , being higher in stratum 1 and stratum 3 than in stratum 2 .
the steeper decline of egfr in stratum 1 might reflect the hyperfiltration state in type 2 diabetes [ 13 , 14 ] .
the degree of albuminuria was correlated with the annual egfr decline in all egfr strata in our study . on the other hand ,
the fact that over 70% of hypertensive patients were treated with antihypertensive agents that were predominantly ras blockers in our study might have influenced the study results .
hba1c at baseline was significantly associated with the annual egfr decline in the less advanced egfr strata but not in stratum 3 . as multiple linear regression analysis of stratum 3
subjects revealed that hba1c was not correlated with the annual egfr decline , the effect of reduced hemoglobin concentration on hba1c value in accordance with the deterioration of kidney function was negligible .
we consider that these results suggest the importance of earlier blood glucose control in preventing diabetic nephropathy , although this observational study can not provide a definite conclusion about therapy .
as logarithmically transformed aer was significantly higher in subjects with retinopathy compared to those without , the relation of retinopathy to worsening of kidney function might be confounded by albuminuria .
on the other hand , although there is a common mechanism of diabetic microvascular disease in retinopathy and nephropathy , diabetic kidney disease in type 2 diabetes mellitus might be influenced by various factors other than hyperglycemia compared to retinopathy .
at least , from the fact that over half of subjects with stratum 3 in this study did not have any sign of diabetic retinopathy , the absence of diabetic retinopathy did not imply a benign renal outcome in subjects with type 2 diabetes with an advanced egfr stratum . on the contrary
, hemoglobin concentration did not show an association with the annual egfr decline in less advanced egfr strata .
however , the correlation between hemoglobin concentration and the annual egfr decline in stratum 3 has a strong clinical significance , because egfr decline has a greater clinical impact in patients with a more advanced egfr stratum .
it has been reported that anemia develops earlier in diabetes [ 5 , 6 ] , and anemia in diabetes mellitus is related to a worse outcome of vascular complications as well as increased mortality [ 710 ] .
a previous study showed that hemoglobin concentration was correlated with the worsening of kidney function with or without albuminuria [ 9 , 10 ] .
however , in our study , a correlation of hemoglobin concentration with egfr decline was observed only in the reduced egfr group , independent of albuminuria .
this may imply that the etiology of the hemoglobin reduction is linked to the pathophysiology of the egfr reduction , rather than that of urinary albumin excretion
. the main cause of anemia in diabetes is suspected to be reduced erythropoietin production relative to the degree of anemia , and some researchers observed the microscopic injury of the renal tubulointerstitium , where erythropoietin is produced , in diabetic subjects [ 15 , 16 ] .
it has been revealed that the decline of kidney function in ckd was more strongly correlated with tubular and interstitial changes than with glomerular changes , and our results corresponded well with this finding if the reduced hemoglobin concentration in the advanced egfr stratum reflected renal tubulointerstitial damage .
although it was reported that pathologic damage of the tubulointerstitium was found relatively early in the clinical course , hemoglobin concentration was not correlated with the egfr decline in the less advanced stratum in our study , but was significantly correlated only in stratum 3 .
however , we consider it reasonable that advanced tubulointerstitial damage , which gives rise to organ dysfunction such as reduced hemoglobin concentration , corresponds to advanced egfr stratum to some extent .
recently reported the risk factors for egfr decline in association with the progression of albuminuria in japanese type 2 diabetes .
the significance of albuminuria in egfr decline and that of hba1c in earlier stages corresponded to our results , but the effects of the existence of retinopathy and anemia on egfr decline were different .
we do not know how the differences arose , but the fact that our study population included subjects of a more advanced stage compared to their study might have influenced the results .
a limitation of our study is that it was an observational study performed at a single hospital in japan .
the drop - out rate was considerably high . as we demonstrated in our prior publication , the dropout subjects were dominantly male , younger with a shorter duration of diabetes mellitus , with a higher ratio of smokers , lower serum creatinine level , and less frequent history of cvd .
so the higher dropout rate could have made the disease progression rate in this study higher than the real incidence .
baseline data obtained just after admission might reflect poor glycemic control and high blood pressure prior to admission , which might have affected the gfr and aer levels in our study .
aer determined in this study was a single measurement , although it is usually recommended to evaluate it on three different days .
moreover , as is customary with observational studies , we could not distinguish an observed association as a cause or effect .
similar previous reports in terms of the progression of kidney dysfunction have been performed in various situations .
one study was performed in caucasian subjects with macroalbuminuria , in which subjects with retinopathy comprised 70% of the total .
the degree and prevalence of possible risk factors as well as other diabetic complications in the study populations might influence the study results , so we should be cautious in comparing and interpreting the results of different study populations .
we reported that the factors associated with the annual egfr decline might be different among egfr strata in subjects with type 2 diabetes mellitus , and that hemoglobin concentration as well as aer was a very important clinical factor to predict the worsening of kidney function , especially in stratum 3 .
further understanding of how each clinical marker such as albuminuria , egfr decline , and the reduction of hemoglobin concentration reflects the renal pathology is important . |
introduction . there is no report about risk factors for renal deterioration according to the clinical stage , divided by the estimated glomerular filtration rate ( egfr ) in type 2 diabetes .
materials and methods .
we evaluated the factors correlated with the annual egfr decline in 1303 subjects with type 2 diabetes whose egfr was 30 ml / min/1.73 m2 .
egfr strata were defined by baseline egfr value as follows : stratum 1 : 90 , stratum 2 : 60 , < 90 , and stratum 3 : 30 , < 60 . results . the annual egfr decline was 2.3 5.4 ml / min/1.73 m2 in overall subjects .
multiple linear regression analysis demonstrated that age , male sex , systolic blood pressure , logarithmically transformed albumin excretion rate ( aer ) , egfr strata , and hemoglobin concentration were significantly correlated with the annual egfr decline .
when stratified by egfr , the factors that showed a significant correlation were different among egfr strata .
aer was significantly correlated with annual egfr decline in all egfr strata .
hemoglobin concentration showed a significant correlation only in the advanced egfr stratum .
conclusion .
the factors correlated with the annual egfr decline were different among egfr strata in type 2 diabetes mellitus , and hemoglobin concentration and aer were important factors for renal deterioration , especially in the advanced egfr stratum . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
patients < 5 years of age who were admitted for lrti to the pediatric intensive care unit ( picu ) of wilhelmina children s hospital , utrecht , the netherlands , were enrolled from october through may during 20052008 .
patients were excluded if they had any of the following : asthma exacerbation , immunocompromised state , indication for antimicrobial drugs other than for lrti , and repeated picu admission for lrti during the study period .
control group participants were children < 18 years of age ( median 2.2 years ) who were admitted to the picu from october 2005 through march 2006 for reasons other than lrti .
underlying illnesses were defined as chronic pulmonary disease , congenital heart disease , immunodeficiency , malignancy , neurologic disease , or gastrointestinal disease ( 8) . to assess the severity of illness , we used the lowest ratio during the first 24 hours of the partial pressure of oxygen in arterial blood ( pao2 ) to the inspired oxygen fraction ( fio2 ) .
these ratios were acquired from the pediatric intensive care evaluation database , which contains validated clinical data for all dutch picu admissions .
nasopharyngeal aspirates were collected from all patients in the lrti group as part of the investigation of their illnesses . in the control group , nasopharyngeal aspirates were taken from intubated patients , and throat swabs were taken from extubated children as part of routine surveillance to identify transmission of respiratory syncytial virus ( rsv ) . because rsv surveillance was conducted as part of normal patient care , patient consent / ethical approval was not needed , according to the medical ethical research council of our institution .
specimens from patients in the lrti group were initially examined for rsv , influenza viruses , parainfluenza viruses , adenoviruses , rhinoviruses , coronaviruses , human metapneumovirus , and mycoplasma pneumoniae by using real - time pcr as previously described ( 1,9,10 ) .
all samples were retrospectively tested for hbov , kipyv , and wupyv also by using real - time pcr as previously described ( 11,12 ) .
after nucleic acid extraction using the magna pure lc 1.0 nucleic acid isolation system ( roche diagnostics , rotkreuz , switzerland ) , amplification was carried out in a 25-l reaction mixture on a 7500 fast real - time pcr system ( applied biosystems , foster city , ca , usa ) .
positive controls for the kipyv and wupyv pcr were provided by s. bialasiewicz and t.p .
sloots , university of queensland , queensland , australia , and the positive control for hbov was provided by t. allander , karolinska institute , stockholm , sweden .
ct values are inversely correlated with viral load ; i.e. , low ct values indicate high viral loads .
of 90 lrti patients enrolled , 78 ( 86.7% ) had sufficient material stored for hbov , kipyv , and wupyv testing .
eighty - eight control patients were enrolled , of which 83 ( 94.3% ) had sufficient material stored to be included .
the main clinical conditions of control patients were cardiac disease requiring surgery ( 33.7% ) , trauma ( 8.4% ) , sepsis ( 8.4% ) , and upper respiratory tract infection ( 8.4% ) .
a total of 57 ( 68.7% ) nasopharyngeal aspirates and 26 ( 31.3% ) throat swabs from the 83 patients who had sufficient samples were tested .
* picu , pediatric intensive care unit , wilhelmina children s hospital , utrecht , the netherlands ; lrti , lower respiratory tract infection ; iqr , interquartile range ; pao2/fio2 , ratio of the partial pressure of oxygen in arterial blood to the inspired oxygen fraction . in lrti patients ,
hbov was found in 4 ( 5.1% ) and wupyv in 2 ( 2.6% ) of the 78 patients .
table 2 shows ct values and clinical characteristics for lrti patients whose samples were positive as well as for controls whose samples were positive for these viruses .
rsv was found in 52 ( 66.7% ) , influenza viruses in 3 ( 3.8% ) , parainfluenza viruses in 2 ( 2.6% ) , adenoviruses in 4 ( 5.1% ) , rhinoviruses in 20 ( 25.6% ) , coronaviruses in 6 ( 7.7% ) , human metapneumovirus in 5 ( 6.4% ) , and mycoplasma pneumoniae in 1 ( 1.3% ) of the patients .
multiple respiratory viruses were found in 3 of the 4 lrti patients with hbov infection and in both patients with wupyv infection ( table 2 ) .
one patient had a single infection with hbov ( i.e. , no other virus was detected ) .
this patient was born at 31 weeks of gestational age and had a history of a grade iv idiopathic respiratory distress syndrome .
she was admitted to the picu at 19 months of age with a severe lrti . bacterial throat and
* picu , pediatric intensive care unit , wilhelmina children s hospital , university medical center utrecht , the netherlands ; lrti , lower respiratory tract infection ; hbov , human bocavirus ; kipyv , ki polyomavirus ; wupyv , wu polyomavirus ; ct , cycle threshold ; npa , nasopharyngeal aspirate ; adeno , adenovirus ; hmpv , human metapneumovirus ; rsv , respiratory syncytial virus ; irds , idiopathic respiratory distress syndrome ; pvl , periventricular leukomalacia ; na , not assessed ; urti , upper respiratory tract infection ; aml , acute myeloid leukemia ; bmt , bone marrow transplant ; vsd , ventricular septal defect ; alte , apparent life - threatening event . in the control group ,
hbov was found in samples from 4 ( 4.8% ) patients , kipyv was found in 4 ( 4.8% ) , and wupyv was present in 2 ( 2.4% ) samples from the 83 patients whose samples could be tested .
median ct values for hbov , kipyv , and wupyv combined were 18.1 ( interquartile range [ iqr ] 20.4 ) for the lrti group and 34.4 ( iqr 5.1 ) for the control group ( p = 0.09 ; figure ) .
the ct values indicate that , on average , the viral load in the lrti group might be higher than in the control group .
cycle threshold ( ct ) values of lower respiratory tract infection ( lrti ) and control patients with human bocavirus ( hbov ) , ki polyomavirus ( kipyv ) , and wu polyomavirus ( wupyv ) infections .
lrti patients are those admitted to the pediatric intensive care unit , wilhelmina children s hospital , university medical center utrecht , the netherlands ; control patients are patients admitted to the pediatric intensive care unit with other diagnoses .
in the present study , the prevalences of hbov , kipyv , and wupyv in picu patients with lrti ( n = 78 ) or without lrti ( n = 83 ) were similar ( 5.1% , 0% , 2.6% ; and 4.8% , 4.8% , 2.4% , respectively ) .
most hbov- and kipyw - positive lrti patients were co - infected with other viruses .
first , lrti patients were younger than controls ( lrti , 100% < 5 years ; controls , 40% > 5 years ) .
however , the positivity rates for hbov , kipyv , and wupyv in control children < 5 years were similar to rates of control children > 5 years of age ( 6/49 vs. 3/34 , respectively ) .
studies have also shown that the highest incidence of kipyv / wupyv infection occurs in children 1 year of age , slightly older than the children in our lrti group ( 1315 ) .
the young age of the lrti group may have resulted in a lower than expected positivity rate for this group .
second , all lrti patients had nasopharyngeal aspirates taken ; however , 68.7% of controls had provided nasopharyngeal aspirates .
hbov and kipyv / wupyv infections were more common in controls who had nasopharyngeal aspirate samples taken than in those who had throat swab samples taken ( 8/57 vs. 1/26 ) .
this difference in positivity for nasopharyngeal aspirates strengthens our conclusion that these viruses are not found more frequently in picu children with lrti .
sampling errors make precise quantification of viral loads difficult . nevertheless , in the lrti group , low ct values , which indicate high viral loads , were found in nasopharyngeal samples taken from 3 patients infected with hbov ( ct 15 ) and from 1 patient infected with wupyv ( ct = 19 ) .
ct values found in nasopharyngeal samples from patients in the control group were much higher , 3239 .
a possible explanation for this difference is that high viral loads in the young lrti population represent symptomatic primary infection , whereas the low viral load in the older controls might represent asymptomatic long - term shedding .
prevalences of hbov , kipyv , and wupyv infections in children in the picu is low ( <5% for lrti patients and controls ) , and these agents are unlikely to be a major cause of lrti at the picu .
however , hbov might be pathogenic in some picu patients because 1 person with a hbov single infection in a high quantity was identified .
further studies using quantitative viral detection are needed to investigate the probability that hbov , kipyv , and wupyv represent etiologic agents of lrti . | we evaluated the prevalence of human bocavirus and ki and wu polyomaviruses in pediatric intensive care patients with and without lower respiratory tract infection ( lrti ) .
the prevalence of these viruses was 5.1% , 0% , and 2.6% , respectively , in children with lrti and 4.8% , 4.8% , and 2.4% , respectively , in those without lrti . | The Study
Conclusions |
in mammalian germ cells , reactive oxygen species ( ros ) production , including hydrogen peroxide ( h2o2 ) , superoxide anion ( o2
) , and/or hydroxyl radical ( oh ) , has been shown to be a physiological event required for maturation , capacitation , acrosomal reaction of spermatozoa , binding with the zona pellucida , and oocyte fusion ( 1 )
. however , excessive generation of ros damages the structural and functional parameters of sperm ( 2 ) .
antioxidants , including superoxide dismutase , glutathione peroxidase , and catalase play an important defensive role in neutralizing ros ( 3 ) . the balance between ros generation and antioxidants
all cellular components , including polyunsaturated fatty acids , proteins , and nucleic acids , are potential targets of ros . however , sperm 's membrane polyunsaturated fatty acids have a critical role in sperm function because lipid peroxidation leads to a loss of integrity in the sperm plasma membrane ( 4 ) .
a lot of studies support the role of nitric oxide as a messenger in a wide array of biological processes such as neurotransmission ( 5 ) , regulation of vascular wall tone ( 6 ) , and immune system activity as an effector molecule with bactericidal and antiviral properties ( 7 ) .
no is a free radical which is generated from the oxidation of l - arginin to l - citrulline by nitric oxide synthase ( nos ) isozymes ( 9 ) .
nos activity has been detected in human and rat testis , epididymis , prostate , and seminal vescicles ( 10 ) .
several studies have shown that this enzyme is associated with acrosome and tails of mouse and human sperm and it appears to be involved in sperm motility and acrosomal reaction that are the main factors in fertilization process ( 1113 ) .
several studies have demonstrated that certain chemicals present in cigarette smoke can adversely affect male fertility ( 14 , 15 ) . some of these compounds , especially nicotine and its metabolite cotenine , and cadmium have been found in seminal plasma at a concentration proportional to those in serum ; therefore , these substances seem to be able to pass from blood - testis barrier ( 16 ) . consequently , seminal plasma of male smokers can be considered as a toxic environment for sperm .
a relationship has also been found between cigarette smoking and alteration of sperm quality ( 14 , 17 , 18 ) .
indeed , cigarette smoking is linked to increased levels of ros in seminal plasma ( 15 ) .
although cigarette smoking is a hazardous habit but more people seem to consume cigarettes on a regular basis .
the highest prevalence of smoking is observed in young males , aged 2039 years , during their reproductive age ( 19 ) .
although a lot of studies have been done on the effects of smoking on male reproductive function ( 14 , 15 , 19 , 21 ) , but the role of cigarette smoking on nitric oxide and lipid peroxidation levels in sperm seem to be limited in the literature .
therefore , the objective of the present study was to evaluate the effects of cigarette smoking on the amount of sperm nitric oxide and sperm oxidation by measuring the levels of nitrite ( no2
) , nitrate ( no3
) , and malondialdehyde ( mda ) in the sperm of normozoospermic men with the habit .
the study was approved by the institutional review board of the institute research committee of ahwaz jundishapour university of medical sciences .
semen samples were obtained from male partner of couples attending razi laboratory in ahwaz , iran for a routine semen analysis .
subsequently , a questionnaire was distributed to obtain demographic data , smoking habits , alcohol use , and use of other substances and drugs .
individuals who had consumed alcohol and/or narcotic drugs in the past three months were not eligible for the study .
other exclusion criteria included recent fever or exposure to gonadotoxins ( e.g. chemotherapy or radiotherapy ) , to pesticides or heavy metals ( professionally ) .
none of the participants were receiving or had received any kind of vitamin treatment in the past three months .
a total of 147 men with a mean age of 30 years ( ranging from 1741 years ) provided 147 semen sample for analysis .
the 147 men categorized according to the number of cigarettes they usually smoked per day over three months before providing the sample , were divided into two groups of nonsmokers ( n=83 ) and smokers ( n=64 ) .
semen samples were collected by masturbation into a sterile container after 23 days of sexual abstinence .
following a ten - minute liquefaction period of semen at 37 c and 5% co2 , samples were examined for volume , sperm motility and count according to world health organization guidelines ( 22 ) .
all the collected samples had a volume of 3.0 ml and sperm concentration/ ml of 2010 .
sperm were separated from seminal plasma by centrifugation at 1000 g for 10 minutes at room temperature .
the sperm were washed twice by adding phosphate buffer ( 20 mm , ph=7.4 ) and centrifuged at 2500 g for 5 minutes .
finally , sperm samples were resolved in phosphate buffer and were aliquoted as a homogenous mixture with 110 sperm .
the aliquots were stored at 80 c for nitrite , nitrate , and malon dialdehyde assays .
lipid peroxides , derived from polyunsaturated fatty acids , are unstable molecules which decompose to form a complex series of compounds , most abundantly malondialdehyde ( mda ) ( 23 ) . therefore , content of sperm lipid peroxidation was determined by measuring mda as described by rao et al .
sperm samples were lysed using rapid freeze and thaw method , respectively at 80 c and 35 c , for least three times .
the samples were centrifuged at 4000 g for 10 minutes , and the supernatant was used for the assays .
the mda level of each sample was measured by its color reaction with thiobarbituric acid reagent at 100 c for one hour , which its maximum absorbance is at 534 nm
( 23 ) .
the concentration of mda was calculated using the extinction coefficient of 1.5610
mol.l.cm
and expressed as nmol of mda equivalents per 10 sperm .
no assay is difficult because it decomposes rapidly into nitrite ( no2
) and nitrate ( no3
) in biological solutions .
therefore , no2
and no3
assay is often used as a measure of no radical production .
sperm specimens lysed through rapid freeze - thawing at 80 c and 35 c , for three times respectively .
the sperm no2
levels were measured using the griess reagent , as previously described ( 24 , 25 ) .
the griess reagent consists of sulfanilamide ( 58 mm in 3 m hcl ) and n-1-naphtylethylenediamine ( 722 m ) .
it is necessary to use protein - free samples for no2
and no3
assays ; therefore , somogyi method is used to eliminate protein interference ( 26 ) .
briefly , 8 ml distilled water , 0.5 ml zinc sulphate ( 10% ) , and 0.5 ml naoh ( 0.5 n ) , were added to 1 ml of semen sample .
the mixture was later centrifuged for 10 minutes at 4000 g , and finally the supernatant was collected .
no2
concentration of sperm samples was determined by spectrophotometry at 540 nm with the addition of griess reagent to deproteinized samples converting no2
into a deep purple azo compound . in this study , 0.7850 m concentration of sodium nitrite was used for plotting the standard curve , and the results were reported as nmol/10 sperm .
the first step is the reduction of no3
to no2
using copper coated cadmium granules , as previously reported ( 25 ) .
t - test was employed for comparisons between sperm nitrite , nitrate , and malondialdehyde levels in smoker and nonsmoker men .
the coefficients of correlation were calculated analyzed by linear ( pearson ) analysis . according to
lipid peroxides , derived from polyunsaturated fatty acids , are unstable molecules which decompose to form a complex series of compounds , most abundantly malondialdehyde ( mda ) ( 23 ) . therefore , content of sperm lipid peroxidation was determined by measuring mda as described by rao et al .
sperm samples were lysed using rapid freeze and thaw method , respectively at 80 c and 35 c , for least three times .
the samples were centrifuged at 4000 g for 10 minutes , and the supernatant was used for the assays .
the mda level of each sample was measured by its color reaction with thiobarbituric acid reagent at 100 c for one hour , which its maximum absorbance is at 534 nm
( 23 ) .
the concentration of mda was calculated using the extinction coefficient of 1.5610
mol.l.cm
and expressed as nmol of mda equivalents per 10 sperm .
no assay is difficult because it decomposes rapidly into nitrite ( no2
) and nitrate ( no3
) in biological solutions .
therefore , no2
and no3
assay is often used as a measure of no radical production .
sperm specimens lysed through rapid freeze - thawing at 80 c and 35 c , for three times respectively .
the sperm no2
levels were measured using the griess reagent , as previously described ( 24 , 25 ) .
the griess reagent consists of sulfanilamide ( 58 mm in 3 m hcl ) and n-1-naphtylethylenediamine ( 722 m ) .
it is necessary to use protein - free samples for no2
and no3
assays ; therefore , somogyi method is used to eliminate protein interference ( 26 ) .
briefly , 8 ml distilled water , 0.5 ml zinc sulphate ( 10% ) , and 0.5 ml naoh ( 0.5 n ) , were added to 1 ml of semen sample .
the mixture was later centrifuged for 10 minutes at 4000 g , and finally the supernatant was collected .
no2
concentration of sperm samples was determined by spectrophotometry at 540 nm with the addition of griess reagent to deproteinized samples converting no2
into a deep purple azo compound . in this study , 0.7850 m concentration of sodium nitrite was used for plotting the standard curve , and the results were reported as nmol/10 sperm .
the first step is the reduction of no3
to no2
using copper coated cadmium granules , as previously reported ( 25 ) .
t - test was employed for comparisons between sperm nitrite , nitrate , and malondialdehyde levels in smoker and nonsmoker men .
the coefficients of correlation were calculated analyzed by linear ( pearson ) analysis . according to
the series studied included a total of 147 normozoospermic men who were , divided into two groups of smokers ( n = 64 ) and nonsmokers ( n=83 ) .
smokers had consumed 7 to 40 cigarettes per day ( 167.5 cigarettes / day ) for a duration of 1 to 20 years ( 64 years ) . comparing sperm motility , lipid peroxidation levels ( using mda assay ) , and
no levels ( in forms of nitrite and nitrate ) between the two groups showed a significant decrease in motility ( p=0.004 ) and a significant increase in concentrations of mda , nitrite , and nitrate ( p < 0.001 ) in sperm of smokers ( table 1 ) .
correlations between sperm motility and levels of mda , nitrite , and nitrate in sperm of smokers have been shown in figure 1 .
this figure , shows a significantly negative correlation between sperm motility and concentrations of mda ( r=0.32 ; p=0.01 ) , nitrite ( r=0.34 ; p=0.006 ) , and nitrate ( r=0.38 ; p=0.002 ) in sperm of male smoker .
a significant positive relation was found between mda levels in sperm of the smoker group with the number of cigarettes smoked per day ( r = 0.48 ; p < 0.001 ) and/ or duration of cigarette smoking per year ( r=0.66 ; p < 0.001 ) ( figure 2 ) .
however , the relationship was not statistically significant between nitrite and nitrate content of sperm cells with the number of consumed cigarettes per day ( r=0.07 ; p=0.5 and r=0.02 ; p=0.8 , respectively ) and/or cigarette smoking duration of per year ( r=0.04 ; p=0.7 and r=0.01 ; p=0.9 , respectively ) ( data not shown ) .
finally , a positive but not significant relation was observed between concentration of mda in sperm with both nitrite ( r=0.20 ; p=0.1 ) and nitrate levels ( r=0.19 ; p=0.1 ) in sperm of smoker men ( figure 3 ) .
the relationship between sperm motility and concentration of sperm mda , a ) nitrite , b ) and nitrate ( c ) in male smokers the relationship between sperm mda concentration and the number of cigarette , a ) and duration of smoking , b ) in male smokers the relationship between sperm mda and sperm nitrite , a ) and nitrate concentrations , b ) in male smokers semen analysis and comparison of motility and concentration of mda , nitrite , and nitrate in the sperm of normozoospermic smokers and non - smokers mda : malondialdehyde concentration ; no2
: nitrite concentration ; no3
: nitrate concentration .
cigarette smoke contains several oxidant chemicals ( 15 , 44 ) , which could have an important role in lipid peroxidation processes within sperm . in the present study , we observed a significantly decreased sperm motility ( by 9% ) and a significantly increased mda , nitrite , and nitrate concentrations ( by 48% , 47% , and 43% , respectively ) , in the sperm of normozoospermic smokers in comparison with non - smoker men .
several reports support the negative effect of cigarette smoke on sperm motility ; for instance , calogero et al .
( 27 ) demonstrated cigarette smoke extract suppressing sperm motility in a concentration- and time- dependent manner .
( 28 , 29 ) also reported that in vitro and/or in vivo tobacco smoke treatment decrease the percentage of motile sperm and motility parameters in adult rhesus monkeys .
however , the literature concerning the effects of cigarette smoking on sperm mda , nitrite , and nitrate concentration in fertile men on smoking is limited .
non the less , there were some studies on levels of these substances in semen and/or other parts of the reproductive system of smoker and/or non - smoker infertile men ( 3033 ) .
numerous studies have shown oxidative stress to play a key role in the pathophysiology of sperm in human ( 4 , 34 , 35 ) .
hsieh et al . ( 36 ) obtained a negative correlation between mda concentration and motility of sperm in oligoasthenospermic men .
they suggested that increased mda levels could inhibit sperm motility by pathologically affecting sperm membrane .
in contrast , some other researchers have reported that mda levels in the seminal plasma were not correlated with motility or concentration of sperm ( 37 , 38 ) .
several reports have indicated that these deleterious reactive compounds are usually suppressed by the coordinated functioning of seminal enzymatic and nonenzymatic antioxidants .
for instance , geva et al . ( 3 ) demonstrated that use of antioxidants led to a reduction in mda , which was correlated with improvement in the prevalence of fertilization .
other reports indicate that enzymatic antioxidants , such as glutathione peroxidase ( 40 ) , and sod ( 41 ) , as well as nonenzymatic antioxidants , such as vitamin e ( 42 ) , and vitamin c ( 43 ) , could increase sperm motility by reducing mda activity . in the present study , evaluation of the mda content of sperm had a significant ( p=0.01 ) negative correlation with sperm motility in normozoospermic smokers .
in addition , we observed a positive and significant relationship between both the number of cigarette smoked per day ( p < 0.001 ) and duration of cigarette smoking per year ( p < 0.001 ) with concentration of mda in sperm .
these results suggest that cigarette smoke could increase sperm mda levels in a concentration- and time - dependent manner .
nitric oxide ( no ) is a large molecule which plays an important role in sperm physiology ( 45 ) .
lewis et al . ( 46 ) showed that sperm were sources of no and constitutive nitric oxide synthase ( nos ) which is present in two isoforms similar to those present in both endothelial ( ecnos ) and brain ( bnos ) cells .
no itself is a highly reactive , short - lived , and lipophilic molecule with a half - life of just a few seconds which makes it difficult to measure ( 47 ) .
therefore , its metabolites , nitrite and nitrate , were studied in the present study .
our study indicated a significant negative correlation between sperm motility and levels of both nitrite ( p=0.006 ) and nitrate ( p=0.002 ) in sperm of normozoospermic smokers .
however , no significant correlation was found between sperm nitrite and/ or nitrate concentration with the number and/or duration of cigarette smoking .
similarly , except one report ( 48 ) that demonstrated sperm motility could not be affected by no levels , other investigations have shown decreased sperm motility in the presence of high concentrations of no - releasing compounds , such as sodium nitroprussie ( 4951 ) . thus , result of this study being in agreement with other reports , we suggest that cigarette smoking can increase no concentration in sperm in a concentration- and time- independent manner .
weinberg et al . ( 51 ) demonstrated that no could reduce atp levels in cells via a decrease in atp content and/or production as approximately 90% of the energy in sperm is produced as atp ( 51 ) ; therefore , this mechanism may probably be responsible for a significant negative correlation observed between sperm nitrite and nitrate levels with sperm motility , in this study .
finally , this investigation indicated a positive but insignificant correlation between mda content of sperm with nitrite and nitrate levels in male smokers .
this suggests that the elevated lipid per oxidation in male smokers sperm has not solely occured by no production .
therefore , cigarette smoking probably could increase mad / lipid per oxidation in sperm via production of no and other oxidative agents . in the present study , we excluded samples presenting leukocytospermia , thereby , allowing us to demonstrate these molecular alterations had been caused by tobacco and not mediated by leucocytes .
in support of our hypothesis , there are a lot of reports that demonstrate production of different oxidative agents , such as superoxide anion ( o2
) , hydrogen peroxide ( h2o2 ) , proxyl radical ( roo ) and hydroxyl radical ( oh ) in the male reproductive system ( 5254 ) .
in conclusion , the present study indicate an association between cigarette smoking and an increase in mda / lipid peroxidation levels duo to an increase in the concentration of no and probably other oxidative factors in the sperm of normo zoospermic cigarette smokers .
in addition , it was also demonstrated that high levels of mda and no have a significant negative correlation with sperm motility .
therefore , we suggest cigarette components may potentially affect motility of sperm via increase in mda / lipid peroxidation , and no concentrations in sperm
. thus , high levels of sperm mda and no may be one of the possible factors of fertility in male smokers
. however , further investigations are needed to elucidate the exact effect of cigarette smoking on sperm activity in male smokers . | backgroundnitric oxide ( no ) is synthesized from l - arginine by a family of enzymes known as nitric oxide synthases .
low concentrations of no is essential in biology and physiology of spermatozoa , but high amounts of no is toxic and has negative effects on sperm functions .
moreover , sperm membrane contains high concentrations of polyunsaturated fatty acids that are highly susceptible to oxidative damage that interferes with fertilization ability .
therefore , we investigated the correlation between levels of sperm malondialdehyde ( mda ) and no with sperm motility in male smokers.methodssemen samples were collected from normozoospermic smoker ( n=64 ) and nonsmoker ( n=83 ) men .
the content of sperm lipid peroxidation was determined by measuring malondialdehyde ( mda ) .
the sperm no were also measured using griess reagent .
data was analyzed by spss , ( version 15.0 ) , using independent t - test and pearson analysis.resultsthe mean mda and no concentrations in the sperm of normozoospermic male smokers were significantly higher than the control group or normozoospermic nonsmokers , ( p < 0.001 ) .
a significant negative relationship was noted between sperm motility and sperm mda levels ( r=0.32 , p=0.01 ) ; and sperm motility and sperm no concentration ( for nitrite , r=0.34 , p=0.006 and for nitrate , r=0.38 , p=0.002).conclusionit was concluded that the increase in mda and no production in sperm can influence sperm motility in normozoospermic smokers .
therefore , it seems that cigarette smoking may affect the fertility of male smokers via increasing the amount of sperm mda / lipid peroxidation and no concentrations . | Introduction
Methods
Sperm lipid peroxidation determination
Sperm NO analysis
Statistical analysis
Results
Discussion
Conclusion |
health care administrators have experienced a significant increase in the cost of treatment due to the costly new biologic therapies tumor necrosis factor alpha ( tnf ) inhibitors and other biologic agents . due to this challenge , in 2006 , the ministry of health and care services and norwegian directorate of health took an initiative to develop guidelines for the use of these drugs . in this
setting , cost - effective treatment and similar access to therapy within the norwegian regions were the main focus of the national health authorities .
furthermore , patients access was based on specific selection criteria published as national guidelines.14 the guidelines ( table 1 ) were made by national groups with representatives from the norwegian medicines agency ; norwegian knowledge centre for the health services ; norwegian associations for rheumatologists , dermatologists , and gastroenterologists ; norwegian regional health authority trusts ; and norwegian directorate of health . according to these guidelines , tnf antagonists are generally considered as second- and/or third - line therapy .
for example , patients suffering from ra are considered candidates for this therapy when the disease is active and they do not respond to disease - modifying antirheumatic drugs ( dmards ) ( ie , methotrexate ) , antiflogistics , or intra - articular injection of steroids .
regular follow - ups are requested on a clinical as well as an economic basis , with the first check - up at 3 months . in cases of no clinical effect
furthermore , the inhibitors are considered second- or third - line therapy in patients suffering from crohn s disease ( cd ) , ulcerous colitis ( uc ) , and/or psoriatic disease . according to the order from the department of health ,
the four norwegian regional health authority trusts took over the financing of the tnf inhibitors and other biologic therapy in june 2006 .
consequently , the various pharmacies in norway delivering these drugs to the patients ( outpatient setting ) and/or to the hospitals have their expenses refunded by the trusts . in northern norway ,
the regional health authority trust constitutes four hospital trusts and one pharmacy trust . due to the fact that hospital trusts now have to cover the expenses
, they have a common concern about cost - effectiveness and whether national guidelines are followed and good clinical practice is achieved .
, biologic therapies have caused a significant rise in the cost of therapy for rheumatic disorders and ibd . to compensate for this situation ,
national guidelines , summaries of present knowledge , and recommendations for therapy have been introduced.15 these recommendations list the first and second choice of treatment . in this survey , we aimed to clarify the medical cost of these drugs in all norwegian health regions and further elucidate the status in the northern region .
we aimed to clarify whether patients in the various regions had similar access to biologic treatment and discuss strategies for follow - up of this high - cost therapy .
in the time period between january 1 , 2008 and december 31 , 2008 , the total cost of tnf inhibitors and other biologic agents in norway was registered prospectively by the drug procurement cooperation ( www.lisnorway.no ) ( dpc ) .
this database has a nationwide coverage , includes all biologic drugs refunded by the specialist health care team , and is run in cooperation by the four norwegian regional health authorities .
the dpc s main goal is to provide costly drugs to the hospital trusts at a low price by regularly announcing tenders for the supply .
the expenses were calculated in norwegian krone ( nkr ) and converted into euros ( ) at a rate of 1 = 9.84 nkr based on data from the national bank of norway on december 30 , 2008 ( www.norges-bank.no ) .
the figures were calculated in absolute amounts spent and adjusted to the number of inhabitants in each region .
the total costs should have been correlated to the number of patients in each region , but there were no national incidence data available .
we also accessed data from the northern norway regional health authority concerning resources spent in the region on tnf inhibitors and other biologic agents .
furthermore , the accounts at the university hospital of north norway ( unn ) trust were analyzed for 10 months ( january october ) in 2007 and 2008 , respectively .
microsoft office excel 2007 ( microsoft corporation , redmond , wa , usa ) was used for the database and calculations .
we had no access to any individual patient data , and approval from the regional committees for medical and health research ethics ( rek ) was not necessary .
microsoft office excel 2007 ( microsoft corporation , redmond , wa , usa ) was used for the database and calculations .
we had no access to any individual patient data , and approval from the regional committees for medical and health research ethics ( rek ) was not necessary .
the total expenditure on the tnf inhibitors and other biologic agents in the four norwegian health regions is shown in figure 1 and table 2 .
a somewhat more frequent use of etanercept in the western region was observed initially , but the discrepancy was minimized during the study period .
the national consumption pattern disclosed corresponds well with the recommendations given by the drug procurement cooperation ( www.lisnorway.no ) ( table 1 ) .
the amount was significantly higher ( 1.56 times higher ) in the northern region compared with the other regions . employing the consumption rates revealed in the southeast , western , and central regions in northern norway
looking at the figures from the accounts of the four hospital trusts within northern norway ( figure 2 ) , the costs increased by 26%54% ( mean 34.5% ) from 2007 to 2008 , indicating a harmonization of the costs within the region .
the two southern located trusts experienced a major increase of costs ( helgeland hospital trust 54% , nordland hospital trust 42% ) compared with the others ( unn trust 26% , finnmark hospital trust 32% ) .
the cost per inhabitant in 2008 was still highest at the unn trust , but the gap was about to be closed .
the figures ( cost/1000 inhabitants ) were helgeland hospital trust 4002 , nordland hospital trust 4429 , unn trust 5161 , and finnmark hospital trust 3575 .
these figures document minor differences in resources spent on tnf inhibitors and other biologic agents within the northern region .
furthermore , people living in the neighbourhood of the university hospital seem to use these drugs more frequently than people living in other areas . however , this statement has to be handled with significant caution and should be considered within the framework that the unn trust serves as a regional university hospital for northern norway and frequently takes part in research projects on new drugs .
thus , patients referred from other areas of northern norway and treated as inpatients will influence the balance of costs between hospitals . in a similar way , the nordland hospital trust serves as a central hospital in the county of nordland and supports the helgeland hospital trust in its care of these patients . focusing on the departments at the unn trust , we disclosed the departments of rheumatology and gastroenterology as the major consumers of these drugs .
the department of gastroenterology spent 758,346 and 1,022,181 on these drugs during the first 10 months in 2007 and 2008 , respectively ( an annual increase of 35% ) .
the corresponding figures at the department of rheumatology were 1,602,281 and 1,861,079 , indicating a 16% annual increase .
these two departments accounted for three - quarters of all resources spent on tnf inhibitors and other biologic agents at the unn trust .
in this study , we have documented a similar pattern of use of various tnf inhibitors and other biologic agents in all norwegian health regions .
the amount spent on these drugs in northern norway was more than 1.5 times higher than in the other norwegian health regions .
the similar pattern of use indicated that national guidelines were followed.15 this was probably due to the fact that clinicians have taken an active part in the development of guidelines .
the obedience to guidelines will be important when the new tender for the supply of biologic agents is announced .
a common national strategy for the use of these drugs is of utmost importance ( to achieve maximum value for money ) when negotiating with pharmaceutical companies .
the cost - effectiveness of the biologic agents has been the focus of several investigators.614 according to brennan et al6 and chen et al7 figures in rheumatology ( after ra patients have failed at least two traditional dmards ) have been calculated at around 24,000 per quality - adjusted life - year ( qaly ) . the incremental cost - effectiveness ratio ( icer ) for etanercept
has been reported to be lower ( 24,000/qaly ) than the corresponding figures of adalimumab ( 30,000/qaly ) and infliximab ( 38,000/qaly ) , respectively.7 however , these figures should be viewed with caution , as drug prices vary between countries and over time . concerning as ,
cost - effectiveness analyses have led to diverging results.10,11 mcleod et al10 concluded that none of the three anti - tnf agents ( adalimumab , etanercept , infliximab ) was likely to be considered cost - effective .
bravo vergel and hawkins11 concluded that only etanercept ( 26,361/qaly ) remained cost - effective .
the cost - effectiveness issue in the treatment of ibd has been the focus of at least four different groups.1215 they all investigated tnf inhibitors in the treatment of cd .
arseneau et al12 calculated the cost / qaly between $ 355,450 and $ 377,000 and concluded that the icer of infliximab for treating cd perianal fistulae over a 1-year period may not justify the higher cost .
the icer figures for chronic active cd were calculated as 6700 , 10,400 , and 84,400/qaly , depending on whether it was a single - dose treatment , retreatment , or maintenance treatment , respectively .
in fistulating cd , the cost / qaly was calculated to be 102,000 to 123,000 and 82,000 to 96,000 for the most favorable retreatment assumptions .
similar figures have been shown by others.1315 all these reports documented that maintenance treatment using infliximab was not cost - effective in the case of cd . in this
setting , the annual increase of total costs ( 35% ) of tnf inhibitors at the department of gas - troenterology at the unn trust should be further analyzed .
most likely , this is caused by a new indication of uc , which together with cd is the main indication of anti - tnf in gastrointestinal diseases . in northern norway ,
we did not reveal any increasing cost pattern from south to north within the northern region .
jnsson et al16 investigated international differences in the use of tnf inhibitors and disclosed that the us had the most expensive use of these drugs , with norway not far behind .
it has been suggested that a focus on the societal rather than a health care cost prospective may be one of the explanations.16 the notable sequelas of uncontrolled rheumatic inflammation include joint damage and functional disability , which , in turn , cause severe economic consequences not only to the patients and their families but also to society .
therefore , pharmacoeconomic analysis should take into account all relevant costs.17 a possible explanation for the significant spending of these drugs in northern norway may be a higher incidence of ibd ( personal communication from professor jon florholmen , unn ) and rheumatologic disease.18,19 a somewhat higher incidence of as has been reported in northern norway.20 moreover , as is the disease with better survival on tnf inhibitors in comparison with ra and psa.21 looking at ibd , a difference between north and south in europe has been reported , although the gap is being reduced as the incidence of ibd is increasing in southern europe while it is stable in the north.22 however , known differences in prevalence and incidence can not fully explain the different pattern of use within norway .
however , the clinical impression is that ibd is more frequent and more severe in the northern region than in the southern part of norway ( personal communication from professor jon florholmen , unn ) .
unpublished data from the norwegian disease - modifying antirheumatic drug register on all disease - modifying treatment given to patients with ra , psa , and as have shown that the center representing north norway ( unn ) is not more liberal in starting treatment with tnf inhibitors than centers located in the southern and central regions .
this is shown by the disease activity at onset of treatment , measured by disease activity score 28 , and by number of prior dmards at onset of tnf inhibitor treatment .
a third explanation could be that the institutions in the north are at the forefront of modern therapy in this setting and the time gap will be closed as the other regions change their practice . during the study period , we did not reveal any sign of a gap being closed , but the study period was short .
furthermore , the distinctive characteristic of northern norway may explain some of the differences in cost .
this region constitutes half of the area of norway but includes only 10% of the population . with a scattered population ,
keeping this fact in mind , clinicians may be less concerned about keeping patients off tnf inhibitors because these drugs usually offer the best symptom control and consequently cost of travel may be avoided .
an alternative treatment , repeated intra - articular injections by corticosteroids for patients with arthritis , would imply considerably more traveling in north norway .
it could be speculated that the introduction of tnf inhibitors may decrease consumption of other health care resources and therefore should be encouraged .
this was not observed by juillard - condat et al.23 however , other investigators have shown savings in this setting . in a dutch study , welsing et al24 concluded that the greater effectiveness of the tnf inhibitors reduced medical and nonmedical costs compared with usual treatment by about 16% and 33% , respectively .
the norwegian orthopaedic association started the norwegian arthroplasty register in 1987.25 the register was extended to include arthroplasties in all joints in 1994 .
the aim of the registry was to detect inferior implants , cements , and operation techniques as early as possible . in the future ,
the need for arthroplasty and possible savings , following the introduction of biologic agents , should be focused on .
an increase in total cost of disease due to tnf inhibitors was established in a danish study.26 this study concluded that the implementation of tnf inhibitors in the treatment of ra would impose additional cost per year on the danish health care service in the range of 67 million to 188 million for a progressive scenario .
health care budgets in norway have been put under high pressure over the last few years , and resources have been allocated to achieve maximum value for money .
the pressure has been even stronger during recent months due to the worldwide financial crisis . in this
setting , raised expenses on drugs ( such as tnf inhibitors ) have to be covered by savings in other settings .
there is currently a need for health care administrators to get an overview of the use of tnf inhibitors and other biologic agents in the treatment of ibd , skin diseases , and rheumatic disease .
this situation calls for a national prescription database and/or a registry that makes it possible to compare the estimated annual cost and number of patients treated , time on therapy , actual cost per patient , and number of patients with a prescription for a defined time period .
the registry should also include data on disease activity and damage , quality of life , and working capacity and disability . especially in the care of ibd , maintenance therapy must be avoided from a cost - effectiveness point of view . | objective : costly biologic therapies have improved function and quality of life for patients suffering from rheumatic and inflammatory bowel diseases . in this survey , we aimed to document and analyze the costs.methods:in 2008 , the total costs of tumor necrosis factor alpha inhibitors and other biologic agents in norway were registered prospectively .
in addition to costs , the pattern of use in the four norwegian health regions was analyzed .
the expenses were calculated in norwegian krone and converted into euros.results:the pattern of use was similar in all four regions , indicating that national guidelines are followed . whereas the cost was similar in the southeast , western , and central regions , the expenses per thousand inhabitants were 1.56 times higher in the northern region .
this indicates that patients in the northern region experienced a lower threshold for access to these drugs .
the gap in costs between trusts within northern norway was about to be closed .
the departments of rheumatology and gastroenterology had the highest consumption rates.conclusion:the total cost of biologic agents was significant .
northern norway had among the highest consumption rates worldwide .
this can partly be explained .
further exploration calls for a national registry for the use of these drugs . | Introduction
Materials and methods
Statistical analysis and authorization
Results
Discussion
Conclusion |
back pain
disorders , followed by more than 3 months of physical inactivity , lead to a reduction in
spinal alignment and deformation of musculoskeletal structures , due to the negative effects
on bone mineral density , decreases in muscle strength and muscle atrophy . indeed ,
the contractions of the transverse and multifidus muscles are responsible for maintaining
the stability of the lumbar spine3 .
previous studies have demonstrated that the weakened lumbar muscles in patients with low
back pain , especially the multifidus , do not heal naturally .
.
indicated that the strength of the most important deep lumbar stabilizer muscles does not
improve with resistance exercise in patients with low back pain .
furthermore , in cases of
reduced strength because of pain , hides et al .
reported that selective strengthening
exercise for the multifidus is problematic because it is difficult to detect capacity
degradation due to the mechanical movement of deep muscles4,5,6 .
the deep stabilizer muscles in patients with low back pain are weak and unbalanced ,
consequently causing a reduction in proprioceptive sense ( posture or kinesthetic ) , which can
subsequently lead to stability problems in the spine and the recurrence of back pain7 .
it has been suggested that in order to
stabilize the trunk , co - contractions of the deep superficial muscles of the lumbar region
are necessary in order to strengthen the deep stabilizer muscles that are directly attached
to the spine8 .
the sling exercise
technique is suitable for these purposes and is a concept of active neuromuscular control .
using the appropriate tools and a swaying line , this method of exercise revitalizes the
muscle by stimulating the nerve root with static - dynamic muscle contraction movement , and at
the same time
this technique is effective for soft tissue relaxation in
dysfunction and pain due to musculoskeletal problems ; it increases the joint range of motion
and traction and stabilizes and strengthens muscle tissue10 . through the movement involved in push - ups plus sling exercise , it
is possible to apply a stabilized movement to induce the contraction of trunk muscles .
while
this approach has been reported to functionally improve back pain in some patients ,
objective evaluation and application of this treatment is scarce11 .
therefore , in this study , we investigated whether training methods that contribute to
stabilization of the trunk , such as push - ups plus sling exercise that focuses on
strengthening the stabilizer muscles of the shoulder joint , minimize the load on the spine
in patients with low back pain .
thirty patients with 3 months of low back pain agreed to participate in this study .
exclusion criteria included structural problems , such as bone and nerve fracture , disc
herniation , and previous lower limb and spine surgery .
the subjects understood the principal
objective of this study and provided their written informed consent before participating in
the study this protocol was approved by the institutional review board of dongshin
university and was conducted in accordance with the ethical standards of the declaration of
helsinki .
the patients were divided into 3 exercise groups : general physical therapy ( pt ; n = 10 ) ,
general physical therapy and lumbar stability using sling exercises ( sling ex ; n = 10 ) , and
general physical therapy and push - ups plus sling exercises ( sling ex+pu ; n = 10 ) .
group i : general physical therapy ( gpt ) ,
group ii : gpt + lumbar stabilization exercise using sling ,
group iii : gpt + push - up plus exercise using sling . mean sd .
group i : general physical therapy ( gpt ) ,
group ii : gpt + lumbar stabilization exercise using sling ,
group iii : gpt + push - up plus exercise using sling general physical therapy similar to that commonly used in patients with chronic low back
pain , including administration of a hot pack ( 80 c , 10 minutes ) , intermittent / continuous
traction ( 2,0002,500 hz , 15 minutes ) , and ultrasound ( 0.81 mhz , 5 minutes ) , was used . as
shown in table 2table 2 .
sling exercise programslevelcontentlumbar stabilization exercise1prone lumbar settingbridging exercise2supine pelvic liftsupine pelvic lift and abduction3side lying hip abductionside lying hip adductionpush up plus exercise1quadrupedal position2prone position3standing positionsets / repetitions5 times/3 sec/3 setsrest30 sec / set , lumbar stability exercises in the sling ex and sling ex+pu groups were
performed for 30 minutes at level 1 during week 1 , levels 12 for weeks 23 , levels 13 for
weeks 45 , and levels 13 for week 6 .
the subjects performed the exercise programs for 30
minutes 3 times a week for 6 weeks . the oswestry disability index ( odi ) , surface
electromyographic ( semg ) activity of the lumbar muscles , and
cross - sectional area of the
multifidus muscle on computed tomography ( ct ) were evaluated before and at 2 , 4 , and 6 weeks
of therapy . a one - way anova with a tukey s multiple range post - hoc test ,
comparisons of groups at each time - point were conducted using a paired t - test .
all statistical analyses were performed using
the statistical package for the social sciences ( spss ) version 12.0 software .
a significant decrease in odi was seen in all 3 therapy groups . however , after 2 weeks ,
greater decreases in odi were noted in the sling ex and sling ex+pu groups , and these
changes persisted for the entire 6-week period ( table
3table 3 .
oswestry disability index over time in each therapy group ( unit : % rms)before2 weeks4 weeks6 weeksgroup 44.83.2 41.95.838.95.00**37.35.00***group 45.13.638.24.1**31.54.2***25.14.1***group 46.43.136.53.9***32.23.7***27.33.0 * * * ) . while no changes in semg activity were seen in the pt group ( tables 4 , 5 , 6 , 7 , 8table 4 .
erector spinae muscle activity over time in each therapy group ( unit :
% rms)before2 weeks4 weeks6 weeksgroup 97.98.0 96.89.696.28.795.110.5group 99.37.3110.59.5**120.410.0***129.612.1***group 98.612.2104.512.9***111.613.0***118.313.4***table 5 .
rectus abdominis muscle activity over time in each therapy group ( unit :
% rms)before2 weeks4 weeks6 weeksgroup 90.56.5 89.26.790.36.590.77.8group 89.05.894.36.3***100.56.8***111.97.3***group 91.75.5103.29.1***112.99.8***123.08.9***table 6 .
external oblique muscle activity over time in each therapy group ( unit :
% rms)before2 weeks4 weeks6 weeksgroup 95.95.9 95.95.097.34.696.84.0group 95.27.1103.07.1**107.36.6***113.57.8***group 94.97.8105.26.2**113.07.4***120.08.1***table 7 .
internal oblique muscle activity over time in each therapy group ( unit :
% rms)before2 weeks4 weeks6 weeksgroup 93.75.4 93.55.694.15.394.56.7group 92.55.797.16.4***103.26.4***111.26.7***group 93.49.099.78.5***105.79.5***114.712.0***table 8 .
the change in multifidus muscle cross - sectional area in each therapy group
( unit : mm)leftrightgroup 0.20.50.20.5group 11.53.811.23.2group 7.52.07.02.1mean sd .
group i : general physical therapy ( gpt ) ,
group ii : gpt + lumbar stabilization exercise using sling ,
group iii : gpt + push - up plus exercise using sling .
p <
0.05 , p < 0.01 ; p < 0.001(one way anona ) . p
< 0.05 , p < 0.01 ; p < 0.001 : pt vs. sling ex and
sling ex+pu ( tukey post hoc test ) .
p < 0.05 , p < 0.01 ;
p < 0.001 : sling ex vs. sling ex+pu ( tukey post hoc test ) . * p <
0.05,**p < 0.01 ; * * * p < 0.001 ( paired t - test ) ) , after 6 weeks of therapy , the erector spinae semg activity was increased in
the other 2 groups , with a greater change in the sling ex group compared with the sling
ex+pu group ( table 4 ) . however , in comparison
with the other 2 groups , the sling ex+pu group demonstrated greater increases in the semg
activities of the rectus abdominis and internal and external oblique muscles of the abdomen .
the cross - sectional area of the multifidus muscle on ct was unchanged in the pt group and
significantly increased in both the sling ex and the sling ex+pu groups . in selective
examination of the sling exercise therapy groups ,
the increase in the cross - sectional area
of the multifidus muscle was significantly greater in the sling ex group than in the sling
ex+pu group .
group i : general physical therapy ( gpt ) ,
group ii : gpt + lumbar stabilization exercise using sling ,
group iii : gpt + push - up plus exercise using sling .
p <
0.05 , p < 0.01 ; p < 0.001(one way anona ) .
p
< 0.05 , p < 0.01 ; p < 0.001 : pt vs. sling ex and
sling ex+pu ( tukey post hoc test ) .
p < 0.05 , p < 0.01 ;
p < 0.001 : sling ex vs. sling ex+pu ( tukey post hoc test ) . *
p <
0.05,**p < 0.01 ; * * * p < 0.001 ( paired t - test )
the superficial multifidus muscle is a deep spinal muscle that covers multiple levels of
the spine and , along with the erector spinae , plays a role in rotating and extending the
spine .
the deep multifidus muscle has a high reaction rate and contributes to stabilization
of the spine12 .
previous studies have
indicated that lumbar segmental stabilization using multifidus strength training decreases
pain and reduces the relapse rate of back pain13,14,15 . in this study , we investigated whether push - ups plus sling
exercise , which focuses on the movement of the shoulder joint stabilizer muscles , could
minimize the load on the spine and thereby enhance the superficial and deep trunk muscles
and stabilize the trunk
. examination of the odi results over time showed a significant decrease in both sling
exercise groups after 2 weeks of therapy .
however , by 6 weeks , the decrease in odi was
greater in the sling ex+pu group than in the sling ex group .
these findings are important
given that a previous study demonstrated that patients with a lower odi score are more
likely to respond favorably to stabilization exercise16 .
to evaluate the stabilization of the spine , we examined the semg activities of the rectus
abdominis , erector spinae , and internal and external oblique muscles of the abdomen .
interestingly , the increase in erector
spinae semg activity was greater in the sling ex group than in the sling ex+pu group
. in
contrast , the sling ex+pu group demonstrated greater increases in the semg activities of the
rectus abdominis and internal and external oblique muscles of the abdomen .
in line with
these findings , a previous study , which examined the muscle activities of the rectus
abdominis and internal and external oblique muscles of the abdomen during movement of the
arms and legs , demonstrated that these muscles provide stability to the pelvis and trunk and
also maintain the stability of the spine ( particularly the oblique muscles of the
abdomen)17 .
therefore , sling ex+pu
therapy , involves exercise on an unstable surface and arm motion with the trunk fixed ,
causes high activation of the rectus abdominis and internal and external oblique muscles of
the abdomen .
the multifidus muscle cross - sectional area showed no changes in the pt group , whereas
significant increases were seen in the sling ex and the sling ex+pu therapy groups , with the
greater increase occurring the sling ex group .
michael and andre have also reported that
stabilization exercise of the lumbar region using an unstable support surface ( swiss ball )
maintained the stability of local muscles by activating muscles that are not normally
used18 .
the results of the present study indicate that sling ex+pu , with a focus on strengthening
the stabilizer muscles of shoulder joint , improves functional movement by activating the
rectus abdominis and the internal and external abdominal oblique muscles .
this approach
could be used during early rehabilitation for patients with chronic low back pain when
weight - bearing exercise can not be used because of pain and limited mobility .
we believe that
sling ex+pu is similar to lumbar stabilization exercise and can lead to increases in lumbar
strength . the limitations of this study were examination of 1 region alone and the
short - term follow - up period . the influences of spinal stabilization muscles other than the
multifidus were also not considered . | [ purpose ] the purpose of this study was to examine the effect of lumbar stability exercises on
chronic low back pain by using sling exercise and push - ups .
[ subjects ] thirty adult
subjects with chronic back pain participated , with 10 adults being assigned to each of 3
exercise groups : general physical therapy ( pt ) , lumbar stability using sling exercises
( sling ex ) , and sling exercise plus push - ups ( sling ex+pu ) .
each group trained for 30
minutes 3 times a week for 6 weeks . the oswestry disability index ( odi ) , surface
electromyographic ( semg ) activity of the lumbar muscles , and
cross - sectional area of the
multifidus muscle on computed tomography ( ct ) were evaluated before and at 2 , 4 , and 6
weeks of therapy .
[ results ] a significant decrease in odi was seen in all therapy groups ,
and this change was greater in the sling ex and sling ex+pu groups than in the pt group .
no changes in semg activity were noted in the pt group , whereas significant increases in
the semg activities of all lumbar muscles were found in the other 2 groups .
the increases
in the semg activities of the rectus abdominis and internal and external oblique muscles
of the abdomen were greater in the sling ex+pu group than in the other 2 groups .
[ conclusion ] these findings demonstrate that sling ex+pu , similar to normal lumbar
stabilization exercise , is effective in activating and improving the function of the
lumbar muscles .
these results suggest that sling ex+pu has a positive impact on
stabilization of the lumbar region . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
sensory gating , which is often described as an ability to filter out irrelevant information that is repeated in close temporal proximity , is essential for the selection , processing , and storage of more salient information [ 13 ] . on the one hand
, it protects the brain from an overload of sensory information . on the other hand
, it allows the brain to make adjustments in response to changes in the environment .
the prefrontal cortex ( pfc ) is the major projection area of dopaminergic neurons , which are located in ventral tegmental area .
dopaminergic neurons are uniformly coupled with the pyramidal neuron synapses ; the couplings may play a preferential role in determining the activities of receptors .
many cognitive functions are dominated by the pfc , such as attention , working memory , and executive function .
disorders in these functions lead to a variety of psychiatric diseases , such as schizophrenia and attention - deficit hyperactivity disorder .
studies have found that the pfc and auditory cortex are related to p50 inhibition by the auditory evoked potential ( aep ) and magnetic field in healthy people ; focal lesions in the pfc induce a weaker suppression in patients than in healthy people . by observing scalp recordings from patients with epilepsy , korzyukov and
colleagues found that the pfc and the medial temporal lobe may be substantial contributors to the process of sensory gating .
dopamine hyperactivity is one of the most important causes that leads to information processing deficit and schizophrenia .
many studies have shown that in the dopaminergic system in the nucleus accumbens , in which the prepulse inhibition ( ppi ) has been adjusted , injection of dopamine and quinpirole into the nucleus accumbens causes serious damage to the ppi , and administration of haloperidol can reverse this damage .
the effects of the dopaminergic system on sensory gating show that defects in suppression may be related to the use of the dopamine receptor agonists . for example ,
bromocriptine and apomorphine induce the disruption of p50 gating suppression in the healthy human . in rats and humans ,
sensory gating is impaired by administration of apomorphine , reducing the first sound ( s1 ) amplitude in the cortex and hippocampus .
disruption of gating , with its clinical symptoms of cognitive dysfunction such as sensory and attention deficit , has been considered to be a biological marker for schizophrenia .
such symptoms are due to the diminished inhibitory capacity of the brain , which leads to an overload of irrelevant information and its habituation deficits .
p50 suppression is not only used for the diagnosis of schizophrenia but also for relative risk assessment , which is also used in the diagnosis of schizotypal personality and antipsychotic - free subjects at risk , as well as in first - episode and chronic patients .
sensory gating can occur at different stages of information processing , such as in the aep ( the mid - latency 10 to 250 ms ) , and between the subconscious ( p50 ) and conscious ( n100 , p200 ) stages .
we examined the n100 ( 100 ms latency ) waveform from the auditory event - related potential ( erps ) induced by the paired - click paradigm . in the paired - click paradigm , franks et al .
reported directly the second sound ( s2 ) on testing ( t ) , the first sound on conditioning ( c ) , and the suppression ratio ( s2/s1 or t / c ) in a study of manic and healthy participants .
this study showed that sensory gating can be defined as the amplitude ratio ( s2/s1 ) .
low ratios represent strong suppression , and a large quantity of irrelevant information is filtered .
therefore , the purpose of the present study was to test the effect of sensory gating under anesthesia in the pfc of monkeys following injection of different drugs at several doses .
a total of 10 cynomolgus monkeys ( experimental group : n=6 ; control group : n=4 ; female , mean age of 12.22.44 years , mean weight of 4.520.77 kg ) from the breeding colonies at the hainan jingang biological technology co. , ltd . were used in the experiments .
the monkeys were individually housed under standard conditions ( 12-h light / dark cycle with light on from 07:00 to 19:00 , humidity 60% , 212c ) .
the experiments were conducted in accordance with the guidelines for the national care and use of animals approved by the chinese national animal research authority .
the cynomolgus monkeys were anesthetized with tiletamine sodium 1.25 mg / kg injected intramuscularly ( i.m . ) ( virbac s.a . , france ) .
the surgery was carried out under deep anesthesia , which was monitored by the toe - pinch reflex test .
the monkeys were placed on a comfortable soft platform , and propofol was intravenously ( i.v . ) injected ( 1 mg / ml 0.9% nacl ) , using a micro - pump at a speed of 40 ml / h .
the skull was penetrated with copper wire ( diameter 0.48 mm ) in the bilateral frontal , temporal , parietal , occipital , left earlobe , and forehead areas , using the earlobe as the reference electrode , with the forehead as the ground .
all socket connectors ( flexible flat cable connector , 15 pins , shenzhen king - hunter technology co. , ltd . )
the eeg signals from all electrodes were amplified and digitized by a biophysical amplifier ( synakps 2 , neuroscan instrument co. , ltd . ,
usa ) , which included a 32-bit a / d , band filter : 0.540 hz , with an analog notch filter at 50 hz notch , sampling rate : 1000 hz .
the research - grade eeg system ( neuroscan version 4.5 ) used an eeg electrode fitted with eight copper wire electrodes located at f3 , f4 , ft7 , ft8 , cp3 , cp4 , o1 , and o2 .
when experiments were completed , the recording electrodes were removed and the monkeys were gently returned to their cages .
the sound stimuli of the paired - click paradigm was produced by e - prime 2.0 ( psychology software tools , inc . ) .
two different sound stimuli ( 2000 hz , 50 ms duration ) were delivered with a 500 ms inter - stimulus interval at 75 db and infrequently with a 2 to 3 s random inter - trial interval to prevent habituation .
the sound paired - click stimuli were presented with a loudspeaker located approximately 10 cm from the monkey s head .
the first step was manual rejection of the disturbance waveform ; the data were band - filtered by a digital filter from 1 hz , rejecting the lowest - frequency waves .
the next step was extraction of the erps located between 100 ms and 500 ms , reduced artifacts outside of 50 mv to 50 mv . in the next step
, aep data were collected to include 100 ms prior to the stimulus and lasting for 500 ms after each stimulus was completed ; the data were band - filtered by a digital filter from 40 hz , rejecting the highest - frequency waves . at
last , the waveforms of the s1 and s2 were extracted , which were computed by averaging 500 aep trials .
we defined the positive waveform that occurred at latency in 10 to 60 ms following the auditory stimuli as the p50 component , and the negative waveform that occurred within 60 to 150 ms as the n100 .
the waveform amplitudes evoked by the s1 and s2 were determined as the absolute difference between the peaks of the p50 and n100 components .
a value of 0 for s2/s1 indicates essentially a strong suppression , whereas a value of 1 indicates no sensory gating .
bromocriptine ( tocris , uk ) was dissolved in sterile saline and dosed at 0.315 mg / kg , 0.625 mg / kg , and 1.25 mg / kg .
haloperidol ( sigma , germany ) was dissolved in sterile saline and dosed at 0.001 mg / kg , 0.01 mg / kg , and 0.05 mg / kg .
phencyclidine ( pcp ; chemsky [ shanghai ] international co. , ltd . ) was dosed at 0.3 mg / kg .
after 30 min . typically , the monkeys were injected with drugs , then allowed to recover for 14 days before the next drug treatment .
all data were subsequently processed with spss software version 19.0 ( spss statistics , ibm , usa ) . to avoid the effect of intersubject variation , the effect of drug administration ( 30 to 60 min ) on the s2/s1 ratio was calculated as the percent difference score from baseline ( 0 to 30 min ) .
the effects of haloperidol ( 0.001 , 0.01 , 0.05 mg / kg ) , bromocriptine ( 0.313 , 0.625 , 1.25 mg / kg ) , and pcp ( 0.3 mg / kg ) on auditory sensory gating were contrasted with the effect of saline by one - way anova with repeated measure where appropriate .
the effects of the cortical areas , including the right temporal lobe , the right pfc ( rpfc ) , the left occipital lobe , and the left parietal lobe , on the amplitude , s1 , and s2 were investigated using one - way anova with repeated measure where appropriate .
the effects of different doses of one drug were compared by one - way anova with repeated measure where appropriate and the least significant difference ( lsd ) post hoc test .
two - way anova with repeated measure where appropriate and the lsd post hoc test were used to compare haloperidol with bromocriptine .
a total of 10 cynomolgus monkeys ( experimental group : n=6 ; control group : n=4 ; female , mean age of 12.22.44 years , mean weight of 4.520.77 kg ) from the breeding colonies at the hainan jingang biological technology co. , ltd . were used in the experiments .
the monkeys were individually housed under standard conditions ( 12-h light / dark cycle with light on from 07:00 to 19:00 , humidity 60% , 212c ) .
the experiments were conducted in accordance with the guidelines for the national care and use of animals approved by the chinese national animal research authority .
the cynomolgus monkeys were anesthetized with tiletamine sodium 1.25 mg / kg injected intramuscularly ( i.m . ) ( virbac s.a . , france ) .
the surgery was carried out under deep anesthesia , which was monitored by the toe - pinch reflex test .
the monkeys were placed on a comfortable soft platform , and propofol was intravenously ( i.v . ) injected ( 1 mg / ml 0.9% nacl ) , using a micro - pump at a speed of 40 ml / h .
the skull was penetrated with copper wire ( diameter 0.48 mm ) in the bilateral frontal , temporal , parietal , occipital , left earlobe , and forehead areas , using the earlobe as the reference electrode , with the forehead as the ground .
all socket connectors ( flexible flat cable connector , 15 pins , shenzhen king - hunter technology co. , ltd . ) connected to the copper wire electrodes were inserted into an electrode pedestal .
the eeg signals from all electrodes were amplified and digitized by a biophysical amplifier ( synakps 2 , neuroscan instrument co. , ltd . ,
usa ) , which included a 32-bit a / d , band filter : 0.540 hz , with an analog notch filter at 50 hz notch , sampling rate : 1000 hz .
the data were subsequently saved and displayed on a computer . the research - grade eeg system ( neuroscan version 4.5 )
used an eeg electrode fitted with eight copper wire electrodes located at f3 , f4 , ft7 , ft8 , cp3 , cp4 , o1 , and o2 .
when experiments were completed , the recording electrodes were removed and the monkeys were gently returned to their cages .
the sound stimuli of the paired - click paradigm was produced by e - prime 2.0 ( psychology software tools , inc . ) .
two different sound stimuli ( 2000 hz , 50 ms duration ) were delivered with a 500 ms inter - stimulus interval at 75 db and infrequently with a 2 to 3 s random inter - trial interval to prevent habituation .
the sound paired - click stimuli were presented with a loudspeaker located approximately 10 cm from the monkey s head .
the first step was manual rejection of the disturbance waveform ; the data were band - filtered by a digital filter from 1 hz , rejecting the lowest - frequency waves .
the next step was extraction of the erps located between 100 ms and 500 ms , reduced artifacts outside of 50 mv to 50 mv . in the next step ,
aep data were collected to include 100 ms prior to the stimulus and lasting for 500 ms after each stimulus was completed ; the data were band - filtered by a digital filter from 40 hz , rejecting the highest - frequency waves . at
last , the waveforms of the s1 and s2 were extracted , which were computed by averaging 500 aep trials .
we defined the positive waveform that occurred at latency in 10 to 60 ms following the auditory stimuli as the p50 component , and the negative waveform that occurred within 60 to 150 ms as the n100 .
the waveform amplitudes evoked by the s1 and s2 were determined as the absolute difference between the peaks of the p50 and n100 components .
a value of 0 for s2/s1 indicates essentially a strong suppression , whereas a value of 1 indicates no sensory gating .
bromocriptine ( tocris , uk ) was dissolved in sterile saline and dosed at 0.315 mg / kg , 0.625 mg / kg , and 1.25 mg / kg .
haloperidol ( sigma , germany ) was dissolved in sterile saline and dosed at 0.001 mg / kg , 0.01 mg / kg , and 0.05 mg / kg .
phencyclidine ( pcp ; chemsky [ shanghai ] international co. , ltd . ) was dosed at 0.3 mg / kg .
after 30 min . typically , the monkeys were injected with drugs , then allowed to recover for 14 days before the next drug treatment .
all data were subsequently processed with spss software version 19.0 ( spss statistics , ibm , usa ) . to avoid the effect of intersubject variation , the effect of drug administration ( 30 to 60 min ) on the s2/s1 ratio was calculated as the percent difference score from baseline ( 0 to 30 min ) .
the effects of haloperidol ( 0.001 , 0.01 , 0.05 mg / kg ) , bromocriptine ( 0.313 , 0.625 , 1.25 mg / kg ) , and pcp ( 0.3 mg / kg ) on auditory sensory gating were contrasted with the effect of saline by one - way anova with repeated measure where appropriate .
the effects of the cortical areas , including the right temporal lobe , the right pfc ( rpfc ) , the left occipital lobe , and the left parietal lobe , on the amplitude , s1 , and s2 were investigated using one - way anova with repeated measure where appropriate .
the effects of different doses of one drug were compared by one - way anova with repeated measure where appropriate and the least significant difference ( lsd ) post hoc test .
two - way anova with repeated measure where appropriate and the lsd post hoc test were used to compare haloperidol with bromocriptine .
bromocriptine ( 0.313 mg / kg ) had no obvious effect on sensory gating in the pfc or other areas compared with the control ; therefore , low - dose bromocriptine did not significantly disrupt the gating in the pfc .
there was a significant effect of bromocriptine ( 0.625 mg / kg ) in the right temporal lobe relative to the control group ( p=0.03 ) .
these results showed that the mid - dose bromocriptine disrupted sensory gating in the right temporal lobe , but not in the pfc .
there was no significant difference in sensory gating in the presence of bromocriptine ( 1.25 mg / kg ) compared with the control ( p>0.05 ) ; thus , high - dose bromocriptine did not damage the pfc . with the increase in dose ,
increases in the ratios in cp3 ( left parietal lobe ) , f3 ( left pfc ) , f4 ( rpfc ) , and ft7 ( left temporal lobe ) were seen .
the ratio was the highest following administration of mid - dose bromocriptine in the rest of the areas , but the differences were not significant ( f ( 1 , 15)=1.444 ; p>0.05 ) ( figure 1 ) . haloperidol ( 0.001 mg / kg ) was compared with the control in all areas . in the rpfc , sensory gating was significantly higher following haloperidol administration compared with the control ( p<0.05 ) ; in other areas , there was no significant difference . at a low dose ,
sensory gating in the left occipital lobe was less suppressed following haloperidol administration ( 0.01 mg / kg ) compared with the control ( p<0.05 ) .
mid - dose haloperidol impaired sensory gating in the left occipital lobe , but did not cause significant differences in other parts of the brain .
haloperidol ( 0.05 mg / kg ) had no significant effect on sensory gating compared with the control in all areas .
there was a significant difference between the high- and low - dose haloperidol treatments in the left parietal lobe ( p<0.05 ) , left temporal lobe ( p<0.05 ) , and rpfc ( p=0.052 ) , whereby gating was significantly lower following the high - dose treatment compared with the low dose ( figure 2 ) .
in contrast to haloperidol , bromocriptine had a significant effect on the sensory gating ratio in the low - dose group in the left parietal lobe ( p<0.05 ) and the rpfc ( p=0.055 ) .
in the high - dose treatment , bromocriptine , in contrast to haloperidol , had significant effects on the rpfc ( p<0.05 ) and the left temporal lobe ( p<0.05 ) .
the dose effects of bromocriptine and haloperidol in the pfc and other areas differed between drug treatments . increasing the dose of bromocriptine
was followed by an increase in gating , but sensory gating with the haloperidol treatments declined ( figure 3 ) .
the effect of pcp ( 0.3 mg / kg ) administration on sensory gating was compared with that of the control .
however , the effect of pcp ( 0.3 mg / kg ) in the left parietal lobe was significantly higher than that of the control ( p<0.05 ) .
sensory gating in areas other than left temporal lobe also was higher than that in the control , but there was no significant difference .
these results showed that pcp ( 0.3 mg / kg ) did not cause significant suppression of sensory gating in the pfc ; however , in the left parietal lobe , sensory gating was impaired ( figure 4 ) .
the amplitude of s1 was lower in the bromocriptine group than in the control group ; however , the amplitude of s2 was not significantly different .
low - dose haloperidol disrupted sensory gating in the rpfc ; this was mainly reflected in an increase in the amplitude of s2 , while the increase in the amplitude of s1 was smaller .
further examination of the amplitude of s2 with mid - dose haloperidol indicated that when the differences in the amplitude of s2 and s1 in the haloperidol group relative to the control in the left occipital lobe were compared , s1 was increased to a lesser extent .
pcp impaired sensory gating in the left parietal lobe , which was mainly reflected in an increase in s2 ; the change in s1 was not significant ( figure 5 ) .
bromocriptine ( 0.313 mg / kg ) had no obvious effect on sensory gating in the pfc or other areas compared with the control ; therefore , low - dose bromocriptine did not significantly disrupt the gating in the pfc .
there was a significant effect of bromocriptine ( 0.625 mg / kg ) in the right temporal lobe relative to the control group ( p=0.03 ) .
these results showed that the mid - dose bromocriptine disrupted sensory gating in the right temporal lobe , but not in the pfc .
there was no significant difference in sensory gating in the presence of bromocriptine ( 1.25 mg / kg ) compared with the control ( p>0.05 ) ; thus , high - dose bromocriptine did not damage the pfc . with the increase in dose ,
increases in the ratios in cp3 ( left parietal lobe ) , f3 ( left pfc ) , f4 ( rpfc ) , and ft7 ( left temporal lobe ) were seen .
the ratio was the highest following administration of mid - dose bromocriptine in the rest of the areas , but the differences were not significant ( f ( 1 , 15)=1.444 ; p>0.05 ) ( figure 1 ) .
haloperidol ( 0.001 mg / kg ) was compared with the control in all areas . in the rpfc ,
sensory gating was significantly higher following haloperidol administration compared with the control ( p<0.05 ) ; in other areas , there was no significant difference . at a low dose ,
sensory gating in the left occipital lobe was less suppressed following haloperidol administration ( 0.01 mg / kg ) compared with the control ( p<0.05 ) .
mid - dose haloperidol impaired sensory gating in the left occipital lobe , but did not cause significant differences in other parts of the brain .
haloperidol ( 0.05 mg / kg ) had no significant effect on sensory gating compared with the control in all areas .
there was a significant difference between the high- and low - dose haloperidol treatments in the left parietal lobe ( p<0.05 ) , left temporal lobe ( p<0.05 ) , and rpfc ( p=0.052 ) , whereby gating was significantly lower following the high - dose treatment compared with the low dose ( figure 2 ) .
in contrast to haloperidol , bromocriptine had a significant effect on the sensory gating ratio in the low - dose group in the left parietal lobe ( p<0.05 ) and the rpfc ( p=0.055 ) .
in the high - dose treatment , bromocriptine , in contrast to haloperidol , had significant effects on the rpfc ( p<0.05 ) and the left temporal lobe ( p<0.05 ) .
the dose effects of bromocriptine and haloperidol in the pfc and other areas differed between drug treatments . increasing the dose of bromocriptine
was followed by an increase in gating , but sensory gating with the haloperidol treatments declined ( figure 3 ) .
the effect of pcp ( 0.3 mg / kg ) administration on sensory gating was compared with that of the control .
however , the effect of pcp ( 0.3 mg / kg ) in the left parietal lobe was significantly higher than that of the control ( p<0.05 ) .
sensory gating in areas other than left temporal lobe also was higher than that in the control , but there was no significant difference .
these results showed that pcp ( 0.3 mg / kg ) did not cause significant suppression of sensory gating in the pfc ; however , in the left parietal lobe , sensory gating was impaired ( figure 4 ) .
the amplitude of s1 was lower in the bromocriptine group than in the control group ; however , the amplitude of s2 was not significantly different .
low - dose haloperidol disrupted sensory gating in the rpfc ; this was mainly reflected in an increase in the amplitude of s2 , while the increase in the amplitude of s1 was smaller .
further examination of the amplitude of s2 with mid - dose haloperidol indicated that when the differences in the amplitude of s2 and s1 in the haloperidol group relative to the control in the left occipital lobe were compared , s1 was increased to a lesser extent .
pcp impaired sensory gating in the left parietal lobe , which was mainly reflected in an increase in s2 ; the change in s1 was not significant ( figure 5 ) .
in this study , we confirmed that sensory gating in the right temporal lobe was disrupted by mid - dose bromocriptine .
low - dose haloperidol damaged sensory gating in the rpfc , and mid - dose haloperidol disrupted gating in the left occipital lobe .
the effect of high - dose haloperidol was less suppressive than that of other doses , as shown in the left parietal lobe , right prefrontal , lobe and left temporal lobe .
low - dose bromocriptine more strongly suppressed sensory gating , but high - dose haloperidol was more suppressive .
the pfc plays a leading role in the generation of p50 sensory gating . through eeg studies , brain functional imaging of near - infrared spectroscopy , and functional magnetic resonance imaging ,
many studies have demonstrated that p50 gating mainly originates in the dorsolateral pfc ( dlpfc ) , and the effects of the dlpfc in sensory gating of schizophrenia patients are more significant .
showed that haloperidol can impair normally high gating , consistent with our observation that haloperidol ( 0.001 mg / kg ) disrupted gating in the rpfc .
this study demonstrated that disruption of gating in the pfc by haloperidol at a high dose ( 0.05 mg / kg ) was not significant .
many studies have reported that dysfunction of the dopaminergic system in the brain impaired sensory gating .
their experiments showed that the dopamine receptor agonists and antagonists impair gating in the healthy subject .
for example , the injection of amphetamine into rats impairs n30 gating and ppi .
the increase of s1 amplitude results from the injection of haloperidol , which improves brainstem gating in rats .
dopaminergic receptors were stimulated by drugs , which caused a deficit in sensory gating and ppi .
this was because dopamine was overactive , and oversuppression occurred in the ventral tegmental area , ventral striatal area , nucleus accumbens , and olfactory tubercle .
our studies found that mid - dose bromocriptine impaired gating in the right temporal lobe ; the effects of other doses were not significant .
this may have been due to the inverted - u dose - response of dopaminergic receptors .
the inverted - u dose - response in the pfc was confirmed in the cognitive function of animal models and humans .
many studies have reported that eeg changes are nonuniform in different areas ; for example , the state - dependent sleep changes in auditory sensory gating in the rat are mainly reflected in the frontal and parietal regions .
this may be due , firstly , to the inconsistency of signal sources on recording of the eeg ; for example , the signal of the frontal cortex may be derived from frontal cortex , the signal of the parietal areas may originate in the hippocampus , and possible sources of the temporal signal are in the superior temporal gyrus .
the signal of the occipital areas was possibly derived from the auditory nerve or the middle and lower part of the brainstem .
previous studies found that damage to sensory gating ( s2/s1 ) was mainly reflected in two aspects .
on the one hand , the amplitude of s1 was reduced and that of s2 was unchanged . on the other hand ,
the amplitude of s1 was reduced in the bromocriptine group in the right temporal lobe .
however , the amplitude of s2 was increased in the haloperidol and pcp groups in the rpfc and left parietal lobes . in animal models ,
the dopamine system in the brain is overactive to disrupt gating by reducing the amplitude of s1 .
for example , systematic injection of quinpirole into the nucleus accumbens is accompanied by a decrease in the s1 amplitude .
the increase in the s2 amplitude was the gating component impaired by disorders of the cholinergic system .
interestingly , a subtype of dopamine d2 receptors were found in the cell body , dendrites , and axons of cholinergic interneurons in the nucleus accumbens of rats .
therefore , the use of d2 receptor antagonists induces the release of acetylcholine in the striatum .
this also shows that injection of haloperidol and pcp may increase the function of the cholinergic system , which can induce an increase in sensory gating via a decrease in response to the s2 .
our study confirmed that low - dose haloperidol impaired sensory gating in the rpfc , and it was disrupted by the mid - dose in the left occipital lobe .
bromocriptine impaired sensory gating by the inverted - u response in a region - dependent pattern in the right temporal region , but not in the pfc .
therefore , our results show that the dopaminergic system plays an important role in information processing and sensory gating in the pfc , while this regulation is dose - dependent and region - dependent , which might imply that they modulate the different stages that receive further processing due to novel information . | backgroundsensory gating , often described as the ability to filter out irrelevant information that is repeated in close temporal proximity , is essential for the selection , processing , and storage of more salient information .
this study aimed to test the effect of sensory gating under anesthesia in the prefrontal cortex ( pfc ) of monkeys following injection of bromocriptine , haloperidol , and phencyclidine ( pcp).material / methodswe used an auditory evoked potential that can be elicited by sound to examine sensory gating during treatment with haloperidol , bromocriptine , and pcp in the pfc in the cynomolgus monkey .
scalp electrodes were located in the bilateral pfc and bilateral temporal , bilateral parietal , and occipital lobes .
administration of bromocriptine ( 0.313 mg / kg , 0.625 mg / kg , and 1.25 mg / kg ) , haloperidol ( 0.001 mg / kg , 0.01 mg / kg , and 0.05 mg / kg ) , and the n - methyl - d - aspartic acid receptor antagonist pcp ( 0.3 mg / kg ) influenced sensory gating.resultswe demonstrated the following : ( 1 ) administration of mid - dose bromocriptine disrupted sensory gating ( n100 ) in the right temporal lobe , while neither low - dose nor high - dose bromocriptine impaired gating .
( 2 ) low - dose haloperidol impaired gating in the right prefrontal cortex .
mid - dose haloperidol disrupted sensory gating in left occipital lobe .
high - dose haloperidol had no obvious effect on sensory gating .
( 3 ) gating was impaired by pcp in the left parietal lobe.conclusionsour studies showed that information processing was regulated by the dopaminergic system , which might play an important role in the pfc .
the dopaminergic system influenced sensory gating in a dose- and region - dependent pattern , which might modulate the different stages that receive further processing due to novel information . | Background
Material and Methods
Subjects
Apparatus for electroencephalogram recording
Stimulus paradigm
Data analysis
Drugs and doses
Statistical analysis
Results
Effects of bromocriptine administration on the PFC
Effects of administration of haloperidol in the PFC
The effects of administration of bromocriptine in contrast to haloperidol in the PFC
The effects of administration of PCP in the PFC
Drug-dependent changes in the N100 amplitude of S1 And S2 in different cortical areas
Discussion
Conclusions |
the determination of cytotoxicity is part of the initial evaluation process stipulated by iso standards .
however , it is not only the absence of a toxic effect that describes the term biocompatibility , but also the presence of a positive influence in terms of biofunctionality ( e.g. , promotion of wound healing ) .
cell culture systems may be of value in testing the biocompatibility of drugs , biomaterials or treatment techniques , as they allow the direct measurement of cytotoxicity and effect on cellular growth or the determination of tissue / material interactions . at present , a wide variety of self - initiated and commercially available test methods are used .
these include different types of cell lines , such as cultured fibroblasts from human skin , buccal mucosa , periodontal membrane , embryonic lung , epithelial and hela cells , cultures of human keratinocytes and hacat cells , different murine cell lines ( c3h - l , balb / c 3t3 , l929 , liver and spleen cell lines , others ) , t - lymphocytes from lymph nodes and macrophages obtained by lavage .
all of these cells are suitable for biocompatibility tests . nevertheless , the general opinion is that toxicity tests in vitro are more convincing when performed with cells that are homologous with the human tissue concerned . in accordance ,
appropriate cell lines for use in cytotoxicity and tolerance tests concerning the skin would be human dermal fibroblasts and human epidermal keratinocytes .
1 ) ) . their main function is the maintenance of the structural integrity through continuous secretion of macromolecules of the extra - cellular matrix ( collagen , glycosamine glycanes and glycoproteins ) .
, fibroblasts can be isolated from skin biopsies or phimoses through enzymatic digestion of the dermis or by explant method .
they are available as the primary cell line nhdf ( normal human dermal fibroblasts ) from promocell ( heidelberg , germany ) .
keratinocytes are the most common cell type of the epidermis ( figure 1 ( fig .
they develop from epidermal stem cells located in the stratum basale ( lowest layer of the epidermis ) and differentiate into corneocytes during their movement to the stratum corneum ( upper layer of the epidermis ) .
there , they form a barrier which protects the body from foreign bodies and germs as well as dehydration .
keratinocytes take an active part in the immune response , inflammatory processes , and wound healing as they are able to synthesize a broad range of cytokines , growth factors , and complement factors . like fibroblasts
, they can be isolated from skin biopsies and phimoses through enzymatic treatment of the epidermis .
promocell ( heidelberg , germany ) also offers a primary keratinocyte cell line ( nhek , normal human epidermal keratinocytes ) .
after a few passages , the cells lose their proliferation ability , probably due to chromosomal alterations .
those developing changes can influence experiment outcomes , which means there is a relatively small time frame for repeating tests .
hacat cells are spontaneously immortalized keratinocytes . they originate from the isolate of the distal periphery of a melanoma located on the upper half of the back of a 62-year - old male patient and have been described in detail by boukamp et al . .
while they demonstrate a transformed phenotype in vitro , they are not tumorogenic , exhibit a steady expression of specific keratins and other identification markers , and show normal differentiation .
fusenig , dkfz , heidelberg , germany ) are cultured in dulbecco s modified eagle s medium ( dmem , promocell , heidelberg , germany ) , supplemented with 10% fetal calf serum ( fcs , promocell , heidelberg , germany ) and 1% antibiotic - antimycotic mix ( psf , promocell , heidelberg , germany ) at 37c in a humidified atmosphere containing 5% co2 .
primary fibroblasts ( nhdf , promocell , heidelberg , germany ) and keratinocytes ( nhek , promocell , heidelberg , germany ) can be maintained in appropriate basal media supplemented with growth factors but without antibiotics under the same conditions .
for experiments , the cells are seeded at the required density into 96-well plates , 12-well plates , object slides etc . after allowing the cells to attach to their new surface
therefore , the cell culture medium is replaced by either fresh culture medium ( negative control / medium control ) or the test substance dissolved in culture medium or the material extract prepared according to the din standard en iso 10993 - 12 .
the subsequent assessment of the in vitro cytotoxicity of the substance is often a qualitative analysis based on the morphological examination of cell damage and growth after direct or indirect contact with the material . in general , methods involving the measurement of cellular growth ( cell counting or confluency evaluation ) are more sensitive and discriminating than those in which the test materials are placed directly in the cell cultures to determine the zone of growth inhibition .
however , dead cells lose their membrane integrity and are stained blue . this method is inexpensive and easy to perform , but unsuitable for high - throughput screening ; it is only recommendable for suspension cells , as adherent cells need to be trypsinized prior to counting .
determination of cell proliferation and cell viability has become the key technology to assess the growth behavior in a cell culture .
they are based on the determination of nucleic acids , metabolic activity , and protein content , or membrane integrity . a small selection ( table 1 ( tab .
1 ) ) , such as pico green dna cell proliferation assay , atplite luminescence atp detection assay , bc assay : protein quantitation kit , alamarblue proliferation assay and live / dead staining with syto-13 and ethd-2 , is discussed here .
picogreen ( molecular probes inc , or , usa ) is a reagent for quantifying dsdna , which provides a stable measurement parameter .
the relative fluorescence units measured correlate with the number of cells present ( figure 2 ) .
cell number and picogreen fluorescence exhibit a linear relationship for low and medium cell densities , but which is lost for high cell densities . on the other hand
, the assay shows a high sensitivity for low cell numbers , as it is able to detect dsdna concentrations as low as 25 pg / ml . equally sensitive is atplite ( perkin elmer life sciences , ma , usa ) , an atp monitoring system based on the detection of light generation by firefly luciferase . in an atp - dependent reaction ,
the amount of emitted light is directly proportional to the atp concentration ( figure 2 ( fig .
atp is present in all metabolically active cells and declines rapidly when cells undergo necrosis and apoptosis .
however , it allows only the measurement of viable cells and not the quantification of the total cell number .
another possibility is the determination of the protein content , which naturally corresponds directly to the cell number ( figure 2 ( fig .
the bc assay ( uptima interchim , france ) is derived from the biuret reaction ( cu + e cu ) using bicinchoninic acid .
it is based on the measurement of the formation of a water soluble , purple - colored complex of bicinchoninic acid and cu .
the test is sensitive , but not as sensitive as the picogreen or atp assay , and low cell numbers do not provide the necessary protein concentration .
in contrast to the first three methods , the alamarblue proliferation assay ( biosource , ca , usa ) is a non - destructive method .
the test uses the indicator dye resazurin , which is dark blue in color and possesses little intrinsic fluorescence until it is reduced by the metabolic activity of viable cells to resorufin , which is pink and highly fluorescent .
non - viable cells rapidly lose their metabolic capacity , do not reduce the dye , and thus do not generate a fluorescent signal .
the assay allows simultaneous incubation and measurement but requires precisely defined time and temperature conditions , as the resorufin will continually develop .
3 ) shows a cell standard curve incubated with alamarblue measured after 3 h ( figure 3 a ( fig .
all of the cell quantification methods discussed have advantages and disadvantages ( summarized in table 1 ( tab .
1 ) ) , and the most suitable test method for a given study must be chosen . besides , it is often necessary and always recommendable to verify the test results with at least two different assays . in that way , it is possible to exclude outside influences or false interactions of the components .
the assays can be used to determine the cell proliferation over a particular time period ( figure 4 ( fig .
so far , we have been able to demonstrate proliferative as well as cytotoxic effects of various test substances on keratinocytes and fibroblasts .
cell proliferation tests are able to express toxic effects via loss of proliferation ability or reduction of living cells .
these negative effects can be quantified by methods that determine cytotoxicity through the measurement of necrosis or apoptosis ( table 1 ( tab .
1 ) ) . staining with syto-13 and ethidiumhomodimer-2 enables the direct count of viable and dead cells .
syto-13 and ethidiumhomodimer-2 ( molecular probes inc , or , usa ) are green and red fluorescent dyes that bind to nucleic acids . while syto-13 can permeate membranes and
is therefore able to stain living cells , ethidiumhomodimer-2 can not , and can only enter dead cells , but possesses a higher binding affinity to dna than syto-13 .
treatment of the cells with a toxic substance leads to a reduction in the ratio of green to red cells ( figure 7 ( fig .
lactate dehydrogenase ( ldh ) in the supernatant is a sign for necrotic degradation of the cells depending on the release of ldh from the cytosol after cell membrane damage .
the cytotoxicity detection kit ( roche , in , usa ) is a colorimetric assay which measures the activity of released ldh .
cytotoxic substances which affect membrane integrity cause a rapid release of ldh compared to the medium control ( figure 8 ( fig .
this process is accompanied by a rapid decline of cell viability ( figure 8 ( fig .
this method is quick and easily adaptable for different types of cells and test conditions , but the assay is sensitive to interference from compounds with inherent ldh activity , such as serum - supplemented culture media .
frequently , the treatment with a substance does not kill the cells directly , but induces cellular damage that subsequently leads to pathological events .
the members of the caspase family play a key effector role in mammalian cell apoptosis .
the caspase - glo 3/7 , 8 and 9 assays ( promega gmbh , mannheim , germany ) provide specific luminogenic substrates that form a luminescence signal proportional to the amount of caspase activity present ( figure 9 ( fig .
the release of inflammatory cytokines accompanies the reaction of the cells to the test substance ( figure 10 ( fig . 10 ) ) .
cells do not function independently from each other , they communicate through the secretion of interleukins such as il-6 and il-8 .
interleukin 6 is a cytokine that provokes a broad range of cellular and physiological responses , e.g. inflammation , haematopoiesis and neuronal differentiation .
interleukin 8 is a member of the superfamily of chemokines and acts as a chemo - attractant for neutrophils ; it plays a crucial role in many inflammatory events .
the cytokine concentration in the supernatant can be easily measured by elisas specific for il-6 and il-8 ( milenia biotec , bad nauheim , germany ) .
materials and substances used , for example , in the treatment of wounds do not only influence cell proliferation and viability , but also affect their migration capacity .
fibroblasts and keratinocytes enable the maintenance of the barrier function of normal skin . upon wounding ,
this shield is disrupted and fibroblasts and keratinocytes migrate into the wound area to regenerate the skin .
therefore , the cells must dissociate from one another and break the cell - matrix contacts to detach from the basal lamina .
migration into the wounded area entails remodelling of the extra - cellular matrix by its proteolytic degradation as well as by de novo synthesis and deposition of newly formed matrix components .
both degradation and reformation of the matrix are functions performed by dermal fibroblasts and epidermal keratinocytes .
the mechanical scratch wounding of confluent monolayers ( figure 11 ( fig . 11 ) ) serves as a model to study cell migration at the wound margins .
the method allows the direct measurement of cell migration and layer regeneration . using the scratch apparatus described by bth et al .
so far , all featured test methods utilize cell monolayers which are an exclusively in vitro condition .
evidence suggests that cells behave differently within these two dimensions than in their familiar 3-dimensional in vivo environment .
for instance , fibroblasts reach confluency in monolayer cultures with a high proliferation rate and display protein biosynthesis levels that do not resemble the in vivo situation . on the other hand , fibroblasts embedded in 3-d collagen gels exhibit lower biosynthetic activity , regulated growth , and stellate shape ; they also have protruding dendritic extensions and a state of differentiation close to the in vivo situation .
to mimic the conditions which cells experience in vivo , the use of 3-d cell cultures is becoming increasingly popular .
ordered collagen matrices ( orc ) use the long range assembly properties of type i collagen monomers , and the cells are seeded afterwards into the collagen gel formed .
( figure 12 ( fig . 12 ) ) is formed as a tissue - like structure from a collagen gel contracted by fibroblasts . with 3-d cultures it is possible to study collagen degradation as well as polymerization and deposition of newly synthesized collagen .
they allow the study of tissue remodelling in vitro and provide an assay system for wound contraction .
it is even possible to prepare skin - equivalent tissue by co - culturing with keratinocytes . of course , these are time - consuming and elaborate preparations that require operator experience and the appropriate evaluation methods .
it has been reported that the use of physical wound treatment techniques , such as wira ( water filtered infrared a ) and hvpc ( high - voltage pulsed current ) , have a positive effect in terms of accelerated wound healing or additional antibacterial activity .
for the measurement of the effect of these physical methods in vitro or for the application of a new plasma device on cells , several considerations must be taken into account to design an appropriate study .
for one , it is necessary to scale the in vivo application parameters to the in vitro use on cell monolayers ( figure 13 ( fig . 13 ) ) . for assessment of the bactericidal activity of hvpc
, cotton pieces were soaked with bacterial suspension , placed on a steel plate , and covered with the electrode ( 42 ma , 128 hz , 30 min ) .
cells need stable culture conditions that guarantee the establishment of an appropriate cell environment during the experiment .
therefore , in vitro studies that have been performed to assess the bactericidal activity of hvpc can not be applied one - to - one on human cell cultures . in conclusion , for testing physical treatment methods , the choice will probably tend to the use of a 3-d culture system which more closely resembles the in vivo situation , although cellular effects are far easier to detect and quantify in cell monolayers than in 3-d cultures .
hopefully , this summary of methods for measuring cell and tissue compatibility will provide ideas for the design of conclusive in vitro studies . | biocompatibility is one of the main requirements for the safe use of medical devices .
determination of cytotoxicity is part of the initial evaluation stipulated by iso standards for the biological evaluation of medical devices .
the use of cell cultures to test the biocompatibility of drugs , biomaterials or treatment techniques used in various disciplines is gaining in importance .
a wide variety of self - initiated and commercially available cell lines has been evaluated and used : cultured fibroblasts from human skin , buccal mucosa , periodontal membrane , embryonic lung , epithelial and hela cells ; cultures of human keratinocytes and hacat cells ; different murine cell lines ( c3h - l , balb / c 3t3 , l929 and others ) as well as murine cells cultured from liver and spleen ; t - lymphocytes from lymph nodes and macrophages obtained by lavage.all of the above cells are suitable for use in biocompatibility tests . nevertheless , the general opinion is that toxicity tests in vitro will be more convincing when performed with cells that are homologous with the human tissue concerned . in accordance ,
appropriate cell lines for use in cytotoxicity and tolerance tests concerning the skin would be human dermal fibroblasts and human epidermal keratinocytes , as they take an active part in the immune response , inflammatory processes , and wound healing.the evaluation of the in vitro cytotoxicity of a biomaterial is often a qualitative analysis based on the morphological examination of cell damage and growth after direct or indirect contact with the material .
different commercial assays based on the determination of nucleic acids , metabolic activity , protein content or membrane integrity are available to measure cell proliferation and cell viability . a small selection
pico green dna cell proliferation assay , atplite luminescence atp detection assay , bc assay : protein quantitation kit , alamarblue proliferation assay and live / dead staining with syto-13 and ethd-2 are discussed concerning sensitivity , reliability and applicability . | Introduction
Measurement of cell and tissue compatibility
Determination of regeneration processes
Assessment of the potential of testing physical wound treatment techniques |
liver ischemia and reperfusion ( i / r)-induced injury is a major complication associated with hemorrhagic or endotoxin shock and thermal injury as well as liver transplantation and resectional surgery . the vast majority of studies performed in experimental animals have used in situ models of liver i / r suggesting that data obtained from these investigations are most relevant to pathophysiological situations or surgical manipulations involving warm
( 37c ) i / r such as hemorrhagic or endotoxin shock or resectional surgery .
indeed , the pathophysiological mechanisms responsible for warm i / r - induced liver injury may be significantly different than those that occur with the cold ischemia ( 4c ) associated with liver storage prior to transplantation . nevertheless , a great deal of mechanistic information has been derived from in situ i / r studies .
these studies demonstrate that reperfusion of ischemic tissue initiates a cascade of molecular and cellular events that culminate in the superoxide anion radical ( o2 ) dependent , nuclear transcription factor-b ( nf-b)-mediated expression of both injurious and protective mediators in favor of the former ( fig . 1 ) .
post - ischemic liver injury is biphasic in nature consisting of an acute or early phase and a subacute or late phase .
the early phase of injury occurs in the absence of leukocyte infiltration and is thought to be initiated by a rapid alteration in the redox state of the tissue in favor of a more oxidative environment .
the late phase of injury is dependent upon the production of several different cytokines and chemokines that promote the infiltration of large numbers polymorphonuclear neutrophils ( pmns ) and lymphocytes into the liver interstitium via the up - regulation of endothelial cell adhesion molecules and formation of chemotactic gradients .
interstitial pmns become fully activated and release copius amounts of reactive oxygen species ( ros ) together with extracellular matrix degrading enzymes such as collagenase and matrix metalloproteases .
the net result of this inflammatory infiltrate is an amplification of the acute injurious response resulting in extensive inflammatory tissue injury . over the past 10 years
, an emerging body of experimental data suggest that endothelial cell nitric oxide synthase ( enos)-derived no may limit ros- and pmn - mediated tissue injury thereby regulating the subsequent inflammatory response in vivo .
indeed , it is becoming clear that reperfusion of ischemic tissue induces a rapid reduction in the bio - availability of no which is thought to represent an important initiating event in the pathophysiology of post - ischemic injury in a variety of different tissues including the liver , heart , kidney and gut .
this chapter presents evidence supporting the hypothesis that : a ) i / r enhances the generation of the o2 and b ) the i / r - induced decrease in the bioavailability of no is mediated directly or indireclty by the o2-dependent decomposition of endogenous no .
in addition to reducing the steady state levels of this protective nitrogen oxide , post - ischemic overproduction of o2 as well as other ros would also alter the redox potential of the liver creating a more oxidative environment within the major cell types of liver including the hepatocytes , kupffer cells ( kcs ) and sinusoidal endothelial cells ( secs ; fig . 1 ) . increasing
the redox potential within these cells would activate nf-b thereby promoting the expression of pro - inflammatory cytokines ( e.g. , tnf- , il-12 , il-1 ) that may injure the tissue directly in the acute phase as well as induce chemokine and endothelial cell adhesion molecule expression in the later subacute phase .
activation of this transcription factor would also induce the expression of certain protective and anti - apopototic genes that could limit tissue injury following i / r .
the evidence implicating endogenous enos - dependent and - independent generation of no as important regulatory pathways that limit the early and late phases of i / r - induced liver injury are also presented in this review .
the early phase of hepatocellular i / r occurs within 16 h following reperfusion and is associated with kupffer cell ( kc ) and possibly lymphocyte activation .
data obtained from several different laboratories demonstrate that this acute , pmn - independent injury may be initiated by alterations in the redox state of the post - ischemic liver such that the redox potential becomes more positive thereby creating a more oxidative environment .
exactly how this happens has been the subject of active debate for more than a decade .
we do know that one of the earliest events associated with i / r is a remarkable dysfunction of the sec characterized by profound decreases in the steady state production of enos - derived no .
this decrease in steady state levels of no occurs very quickly ( within 5 min of reperfusion ) and appears to be due to decreased synthesis of no , enhanced inactivation of no by certain ros or both .
co - incident with the decrease in no production is the enhanced production of ros such as o2 and hydrogen peroxide ( h2o2 ) during the first few minutes of reperfusion .
the cellular source(s ) of these ros are not known with certainty but several candidate enzymes / pathways have been suggested including hepatocyte xanthine oxidase and/or mitochondrial electron transport as well as kc- and sec - associated nadph oxidase .
interest in xanthine oxidase has waned over the years in favor of mitochondrial sources of ros and/or kc - associated nadph oxidase .
it is well known that certain oxygen - or hemoprotein - derived free radicals such as o2 and ferryl ( fe ) hemoproteins may interact with and decompose no .
in addition , h2o2 can indirectly decrease steady state levels of no via the peroxidase - catalyzed consumption of no in different tissues .
therefore , i / r - induced over - production of ros not only reduces ( or eliminates ) the important vasoactive mediator and cytoprotective no free radical , but it also induces the rapid oxidation of cellular gsh creating a more positive redox potential within the liver . indeed , a number of different studies , using pharmacologic interventions or genetic approaches have demonstrated that the protective effects of certain nonenzymatic or enzymatic antioxidants administered prior to inducing liver ischemia correlates with restoration of gsh levels within the tissue .
there is a large body of work suggesting that the major ros responsible for post - ischemic hepatocellular injury is o2 .
a variety of different studies have demonstrated that adenoviral transfection of the liver with manganese superoxide dismutase ( mnsod ) as well as exogenous administration of long - lived sods attenuate i / r - induced injury suggesting that o2 per se rather than secondary oxidants is directly or indirectly responsible for i / r - induced tissue damage ( fig . 2 ) .
a number of cellular sources of o2 have been proposed to account for post - ischemic tissue injury in the absence of an inflammatory infiltrate .
the most likely candidate is kc - associated nadph oxidase although sec nadph oxidase may play an as yet - to - be discovered role .
this multimeric o2-producing enzymatic complex has been well - characterized in phagocytic leukocytes and kupffer cells .
several studies have determined that kc - derived nadph oxidase is critically involved in promoting post ischemic liver injury .
exactly how cell - derived o2 promotes liver injury is not known but emerging data suggest that multiple mechanisms are likely involved .
several studies had originally suggested that o2 dependent lipid peroxidation represented an important pathway for i / r - induced damage ; however other investigations cautioned that oxidative degradation of membrane lipids may , in reality , be a consequence rather than a cause of i / r - induced liver injury .
more recent studies suggest that o2 may directly or indirectly mediate cell and tissue injury by rapidly interacting with and damaging mitochondrial membrane proteins leading to the loss of inner membrane potential and atp generating capacity .
another mechanism that has been proposed to account for o2 dependent injury to the postischemic liver is through the generation of even more potent oxidants via its interaction with no .
this interaction is known to produce the potent cytotoxic oxidizing and nitrating specie peroxynitrite ( onoo ) and its conjugate acid peroxynitrous acid ( onooh ) .
although an attractive hypothesis , the data do not support a role for this pathway .
indeed , we have found that i / r - induced liver injury in enos - deficient ( enos ) mice or wild type mice pretreated with the nos inhibitor n(g)-nitro - l - arginine methyl ester ( l - name ) exacerbates liver injury suggesting that no is protective in nature ( fig .
in addition to its direct biochemical effects , o2 may mediate hepatocellular damage indirectly via the up - regulation ( or down - regulation ) of certain redox - sensitive genes known to be important in cell proliferation , apoptosis and the inflammatory response .
in addition to the above mentioned mechanisms , there is good evidence to suggest that i / r - induced o2 production may mediate hepatocellular injury and inflammation by creating a more oxidative environment within the different cells of the liver thereby activating redox - sensitive transcription factors such as nf-b .
studies from several different laboratories have shown that i / r - induces the rapid up regulation of several different cytokines ( tnf- , il-1 , il-2 , il-6 , il-8 , il-12 ) , endothelial cell adhesion molecules ( icam-1 , vcam-1 , e - selectin and mucosal addressin cell adhesion molecule-1 ) , cxc chemokines ( macrophage inflammatory protein-2 , keritinocyte - derived chemokine , and epithelial neutrophil - activating peptide ) as well as nitric oxide synthase-2 ( inos ) and cyclooxygenase-2 ( cox2 ) .
the expression of the majority of these genes is regulated by the transcription factor nf-b and thus there is great interest in understanding how the molecular events associated with i / r - mediated activation of nf-b and the subsequent induction and roles of the different cytokines , chemokines , adhesion molecules and enzymes ( fig .
indeed , it is now well - appreciated that the expression of tnf- , il-1 and il-12 are important for mediating post - ischemic liver injury .
a large body of experimental data suggest o2 and/or h2o2 may act as downstream signaling and/or costimulatory molecules required for the activation of nf-b .
although an attractive hypothesis that continues to receive substantial attention in the literature , there is in fact little data directly demonstrating that ros mediate nf-b activation in vivo . it may be that ros function to activate nf-b indirectly by promoting the expression of certain cytokines and/or mediators ( e.g. , tnf- and il-1 ) which can in turn activate this transcription factor .
indeed , it has been demonstrated that a major mediator of post - ischemic liver injury is tnf-. the mechanisms by which tnf- promotes hepatocellular injury are not entirely clear but are most likely multifactorial .
previous studies have suggested that much of the post - ischemic liver injury may be a result of tnf--induced endothelial cell and hepatocyte apoptosis .
it is known that tnf- will induce hepatocyte injury via the ros - dependent activation of numerous cell signaling pathways resulting in apoptosis .
however , jaeschke et al . found , using well - defined histopathological criteria , that virtually all liver injury occurred by
nevertheless , it should be remembered that cells that begin to die by apoptosis may also undergo necrosis if intracellular atp is low or absent as would be the case during ischemia .
it is clear that i / r - induced activation of nf-b within kcs and secs induces an injurious / pro - inflammatory response via the upregulation of numerous cytokines , enzymes and chemokines ( fig . 1 ) .
however , the function of nf-b in hepatocytes appears to be more complicated ( fig . 1 ) .
emerging evidence suggests that nf-b activation may be to protect a variety of cells from the injurious effects of certain pro - inflammatory cytokines such as tnf-. it is well known that pre - treatment of cultured cells with non - lethal concentrations of tnf- protects these cells from a subsequent challenge by lethal amounts of this same cytokine and that this protective response is dependent upon nf-b activation . in addition , ikk has been shown to be required for embryonic development of skin and limbs whereas ikk appears essential for normal liver development with massive liver apoptosis , injury and embryonic lethality occurring in ikk-deficient mice .
similar effects have been observed in mice that genetically lack the p65 ( rela ) component of nf-b . taken together
, these studies suggest that nf-b activation is essential for normal liver development and that inhibition of nf-b nuclear translocation promotes liver injury via massive apoptosis .
studies by brenner and coworkers have confirmed that over - expression of liver ib- in mice inhibited nf-b activation resulting in massive hepatocyte injury and apoptosis following partial hepatectomy in these animals .
it is known that nf-b activation induces several anti - apoptotic genes including mnsod , a zinc finger protein termed a20 , cellular inhibitors of apoptosis-2 ( c - iap-2 ) , and bcl-2 family members such as a1 .
most of these genes ( i.e. mnsod , a20 and a1 ) are also known to inhibit nf-b activation and thus may control the expression of certain pro - inflammatory genes ( fig . 1 ) .
despite the recent body of experimental data demonstrating that nf-b may limit hepatocyte injury , there is also an equally convincing body of data demonstrating that i / r - induced , tnf--mediated liver injury is mediated by nf-b activation .
for example , it has been shown that much of the post - ischemic liver injury is abrogated in tnf--deficient mice .
these are important studies as they demonstrate that i / r - induced liver injury is attenuated in the complete absence of nf-b activation ie in the absence of both the injurious / inflammatory and the protective responses .
it may be that the balance between injurious vs protective genes upregulated during i / r as well as the cellular location of nf-b activation dictates whether nf-b activation will promote injury or protection .
a more direct approach to the role of hepatocyte nf-b in the pathophysiology of post - ischemic tissue injury has been provided by investigators who showed that i / r - induced injury was attenuated in mice with hepatocyte - specific ablation of ikk. they found that the protective effect was associated with decreased expression of hepatocyte - derived tnf-. in apparent contradiction to these results , these same investigators reported that i / r - induced liver injury in mice with a hepatocyte - specific deletion of ikk ( nemo ) was dramatically increased compared to wild type mice suggesting a protective effect of hepatocyte nf-b . in an attempt to reconcile these seemingly contradictory results
, the authors suggested that hepatocyte - specific deletion of ikk may possess some residual nf-b activity whereas ikk ( nemo)-deficient hepatocytes have no detectable nf-b activity and thus no protective response to limit liver damage . taken together , these data suggest that further work is needed to define the role of hepatocyte nf-b .
there is a large body of work suggesting that the major ros responsible for post - ischemic hepatocellular injury is o2 .
a variety of different studies have demonstrated that adenoviral transfection of the liver with manganese superoxide dismutase ( mnsod ) as well as exogenous administration of long - lived sods attenuate i / r - induced injury suggesting that o2 per se rather than secondary oxidants is directly or indirectly responsible for i / r - induced tissue damage ( fig . 2 ) .
a number of cellular sources of o2 have been proposed to account for post - ischemic tissue injury in the absence of an inflammatory infiltrate .
the most likely candidate is kc - associated nadph oxidase although sec nadph oxidase may play an as yet - to - be discovered role .
this multimeric o2-producing enzymatic complex has been well - characterized in phagocytic leukocytes and kupffer cells .
several studies have determined that kc - derived nadph oxidase is critically involved in promoting post ischemic liver injury .
exactly how cell - derived o2 promotes liver injury is not known but emerging data suggest that multiple mechanisms are likely involved .
several studies had originally suggested that o2 dependent lipid peroxidation represented an important pathway for i / r - induced damage ; however other investigations cautioned that oxidative degradation of membrane lipids may , in reality , be a consequence rather than a cause of i / r - induced liver injury .
more recent studies suggest that o2 may directly or indirectly mediate cell and tissue injury by rapidly interacting with and damaging mitochondrial membrane proteins leading to the loss of inner membrane potential and atp generating capacity .
another mechanism that has been proposed to account for o2 dependent injury to the postischemic liver is through the generation of even more potent oxidants via its interaction with no .
this interaction is known to produce the potent cytotoxic oxidizing and nitrating specie peroxynitrite ( onoo ) and its conjugate acid peroxynitrous acid ( onooh ) .
although an attractive hypothesis , the data do not support a role for this pathway .
indeed , we have found that i / r - induced liver injury in enos - deficient ( enos ) mice or wild type mice pretreated with the nos inhibitor n(g)-nitro - l - arginine methyl ester ( l - name ) exacerbates liver injury suggesting that no is protective in nature ( fig .
in addition to its direct biochemical effects , o2 may mediate hepatocellular damage indirectly via the up - regulation ( or down - regulation ) of certain redox - sensitive genes known to be important in cell proliferation , apoptosis and the inflammatory response .
in addition to the above mentioned mechanisms , there is good evidence to suggest that i / r - induced o2 production may mediate hepatocellular injury and inflammation by creating a more oxidative environment within the different cells of the liver thereby activating redox - sensitive transcription factors such as nf-b .
studies from several different laboratories have shown that i / r - induces the rapid up regulation of several different cytokines ( tnf- , il-1 , il-2 , il-6 , il-8 , il-12 ) , endothelial cell adhesion molecules ( icam-1 , vcam-1 , e - selectin and mucosal addressin cell adhesion molecule-1 ) , cxc chemokines ( macrophage inflammatory protein-2 , keritinocyte - derived chemokine , and epithelial neutrophil - activating peptide ) as well as nitric oxide synthase-2 ( inos ) and cyclooxygenase-2 ( cox2 ) .
the expression of the majority of these genes is regulated by the transcription factor nf-b and thus there is great interest in understanding how the molecular events associated with i / r - mediated activation of nf-b and the subsequent induction and roles of the different cytokines , chemokines , adhesion molecules and enzymes ( fig .
indeed , it is now well - appreciated that the expression of tnf- , il-1 and il-12 are important for mediating post - ischemic liver injury .
a large body of experimental data suggest o2 and/or h2o2 may act as downstream signaling and/or costimulatory molecules required for the activation of nf-b . although an attractive hypothesis that continues to receive substantial attention in the literature , there is in fact little data directly demonstrating that ros mediate nf-b activation in vivo
. it may be that ros function to activate nf-b indirectly by promoting the expression of certain cytokines and/or mediators ( e.g. , tnf- and il-1 ) which can in turn activate this transcription factor .
indeed , it has been demonstrated that a major mediator of post - ischemic liver injury is tnf-. the mechanisms by which tnf- promotes hepatocellular injury are not entirely clear but are most likely multifactorial .
previous studies have suggested that much of the post - ischemic liver injury may be a result of tnf--induced endothelial cell and hepatocyte apoptosis .
it is known that tnf- will induce hepatocyte injury via the ros - dependent activation of numerous cell signaling pathways resulting in apoptosis .
however , jaeschke et al . found , using well - defined histopathological criteria , that virtually all liver injury occurred by
nevertheless , it should be remembered that cells that begin to die by apoptosis may also undergo necrosis if intracellular atp is low or absent as would be the case during ischemia .
it is clear that i / r - induced activation of nf-b within kcs and secs induces an injurious / pro - inflammatory response via the upregulation of numerous cytokines , enzymes and chemokines ( fig . 1 ) .
however , the function of nf-b in hepatocytes appears to be more complicated ( fig . 1 ) .
emerging evidence suggests that nf-b activation may be to protect a variety of cells from the injurious effects of certain pro - inflammatory cytokines such as tnf-. it is well known that pre - treatment of cultured cells with non - lethal concentrations of tnf- protects these cells from a subsequent challenge by lethal amounts of this same cytokine and that this protective response is dependent upon nf-b activation . in addition , ikk has been shown to be required for embryonic development of skin and limbs whereas ikk appears essential for normal liver development with massive liver apoptosis , injury and embryonic lethality occurring in ikk-deficient mice .
similar effects have been observed in mice that genetically lack the p65 ( rela ) component of nf-b . taken together
, these studies suggest that nf-b activation is essential for normal liver development and that inhibition of nf-b nuclear translocation promotes liver injury via massive apoptosis .
studies by brenner and coworkers have confirmed that over - expression of liver ib- in mice inhibited nf-b activation resulting in massive hepatocyte injury and apoptosis following partial hepatectomy in these animals .
it is known that nf-b activation induces several anti - apoptotic genes including mnsod , a zinc finger protein termed a20 , cellular inhibitors of apoptosis-2 ( c - iap-2 ) , and bcl-2 family members such as a1 .
most of these genes ( i.e. mnsod , a20 and a1 ) are also known to inhibit nf-b activation and thus may control the expression of certain pro - inflammatory genes ( fig . 1 ) .
despite the recent body of experimental data demonstrating that nf-b may limit hepatocyte injury , there is also an equally convincing body of data demonstrating that i / r - induced , tnf--mediated liver injury is mediated by nf-b activation .
for example , it has been shown that much of the post - ischemic liver injury is abrogated in tnf--deficient mice .
these are important studies as they demonstrate that i / r - induced liver injury is attenuated in the complete absence of nf-b activation ie in the absence of both the injurious / inflammatory and the protective responses .
it may be that the balance between injurious vs protective genes upregulated during i / r as well as the cellular location of nf-b activation dictates whether nf-b activation will promote injury or protection .
a more direct approach to the role of hepatocyte nf-b in the pathophysiology of post - ischemic tissue injury has been provided by investigators who showed that i / r - induced injury was attenuated in mice with hepatocyte - specific ablation of ikk. they found that the protective effect was associated with decreased expression of hepatocyte - derived tnf-. in apparent contradiction to these results , these same investigators reported that i / r - induced liver injury in mice with a hepatocyte - specific deletion of ikk ( nemo ) was dramatically increased compared to wild type mice suggesting a protective effect of hepatocyte nf-b . in an attempt to reconcile these seemingly contradictory results
, the authors suggested that hepatocyte - specific deletion of ikk may possess some residual nf-b activity whereas ikk ( nemo)-deficient hepatocytes have no detectable nf-b activity and thus no protective response to limit liver damage . taken together , these data suggest that further work is needed to define the role of hepatocyte nf-b .
the early phase of i / r injury is a pmn - independent process in which kcs are activated , ros are generated by these resident phagocytes and by dysfunctional mitochondria in kcs , secs and hepatocytes and proinflammatory cytokines ( tnf- , il-12 , il-1 ) are synthesized .
this initial response induces the upregulation of additional inflammatory mediators including activated complement factors ( e.g. , c5a ) , platelet activating factor and different cxc chemokines , such as interleukin-8 ( il-8 ) , macrophage inflammatory protein-2 ( mip-2 ) , keratinocyte - derived chemokine , and cytokine - induced neutrophil chemoattractant ( cinc-1 ) .
these inflammatory mediators promote the enhanced expression of cd11b / cd18 which prime pmns and promote their extravasation from the sinusoids and post - sinusoidal venules into the tissue thereby initiating the late or subacute phase of post - ischemic tissue injury .
this inflammatory response is characterized by the activation of pmns resulting in the production of large amounts of ros and release of a variety of different proteases ( gelatinase , collagenase ) .
the net result of this acute inflammatory response is oxidant - mediated mitochondrial dysfunction as well as degradation of the interstitial matrix and damage to the parenchymal cells .
in addition , tlr4-mediated production of cytokines and inflammatory mediators may represent an additional important pathway for the amplification of the inflammatory response .
although it is well - recognized that pmns play an important role in the pathophysiology of iiver i / r injury , other leukocytes may also be involved in this innate immune response .
for example , it has been reported that cd4 + t - cells play an important role in recruiting pmns into the post - ischemic liver and that cd4 + t - cell - deficiency remarkably attenuated both pmn infiltration and tissue injury following i / r .
however , more recent studies have found that cd4 + t - cell - deficient mice responded to liver i / r with an exacerbation in liver injury despite the fact that these mice displayed fewer pmns infiltrating the post - ischemic liver .
the mechanisms responsible for these seemingly contradictory results are not clear at the present time but may relate to the suppressive or regulatory nature of the infiltrating cd4 + t - cells in the later study .
it is becoming increasingly appreciated that i / r results in rapid endothelial dysfunction in a number of different tissues that is characterized by a marked decrease in steady - state production of endothelial cell - derived no .
the decrease in no bioavailability occurs within the first few minutes following reperfusion and appears to be due to decreased synthesis of no , enhanced inactivation of no by o2 ( or other ros ) or both . because endogenous no is known to interact with and
rapidly decompose certain ros such as o2 , ho , and ferryl hemoproteins , decreased steady state production of no would in all likelihood increase the redox potential of hepatocytes , kcs and/or secs in favor of a more oxidative environment .
indeed , we have demonstrated that many of the pathophysiological characteristics induced by i / r ( i.e. oxidative stress , leukocyte adhesion , enhanced vascular permeability ) may be recapitulated in normal tissue by administration of certain nos inhibitors suggesting that enos - derived no may act as an endogenous anti - inflammatory mediator in the microcirculation . the use of certain nos inhibitors to evaluate the role of no in vivo has proven problematic .
it is known for example that l - name and l - nmma are nonspecific inhibitors of both enos and inos as well as potent vasoconstrictors in vivo .
thus , the non - selective nature of these nos inhibitors coupled to the fact that their exacerbatory effects may be due to their vasoconstrictor effects in a model of hypoperfusion makes their usefulness less than ideal to probe the role for no in liver i / r .
the potentially protective role of endogenous no in liver i / r injury is supported by studies demonstrating enhanced hepatocellular injury in post - ischemic animals rendered deficient in enos ( fig . 3 ) .
in addition , investigators have demonstrated that no donors or over - expression of liver enos protects mice from liver i / r - induced injury .
although enos has been shown by several groups of investigators to play a critical protective role in liver
studies from our laboratory suggest that i / r - induced liver injury is enhanced in inos - deficient mice however interpretation is complicated by the fact that we could not demonstrate upregulation of inos message in post - ischemic livers of wild type mice suggesting that the source of inos is not in the liver ( e.g. , in the intestine ) or that a small population of cells such as kcs or secs upregulate inos which we are unable to detect in whole liver preparations . the mechanisms by which endogenous no protects the liver against the injurious effects of i / r are now beginning to be defined ( fig . 5 )
no is known to interact with and decompose o2 or other reactive radicals or oxidants thereby limiting the formation of o2-derived oxidants and preventing the downstream oxidant - induced signaling pathways .
if this antioxidant / free radical scavenging activity of no is a major protective mechanism , then one would predict that administration of a long lived sod such as the fusion protein sod2/3 to enos - deficient mice would attenuate i / r - induced injury to the liver .
in fact , we found that although sod2/3 markedly attenuates post - ischemic injury in wild type mice , it does not attenuate liver injury in enos - deficient animals .
these data suggested that the protective effect of sod2/3 in wild type mice is due to its ability to increase the bioavailability of no by preventing the o2 mediated inactivation of no .
we then return to the question : how does no limit i / r - induced tissue damage ?
for example , decaterina and coworkers provide evidence that no enhances the denovo synthesis and/or stabilization of the natural inhibitor ib-. in addition , no - mediated s - nitrosation of a specific cysteine on the p50 and/or p65 subunits of nf-b results in inhibition of binding of this heterodimer to it consensus sequences upstream of the different pro - inflammatory genes .
another possible mechanism may be that no - dependent activation of soluble guanylyl cyclase ( sgc ) with the subsequent production of the vasorelaxant cgmp may protect against reperfusion injury by enhancing blood flow thereby limiting the degree of ischemia to the liver .
it has also been proposed that no - mediated activation of protein kinase g via the sgc / cgmp pathway opens mitochondrial katp channels thereby reducing calcium accumulation within the mitochondria and preventing the loss of cytochrome c from the mitochondrial intermembranal space .
alternatively or in addition to , no may reversibly inhibit mitochondrial respiration via interaction with complex i and/or cytochrome c oxidase .
this would inhibit apoptosis , maintain small but significant amounts of oxygen during ischemia and allow for a more controlled resumption of respiration following reperfusion .
similar observations have been made with no - dependent s - nitrosation of caspase-3 resulting in inactivation of this enzyme and inhibition of apoposis .
this would minimize free radical - mediated damage to the mitochondrial membrane and preserve cellular function .
no may attenuate the later stages of post - ischemic tissue damage by inhibiting platelet / leukocyte - endothelial cell interactions .
there is ample evidence demonstrating that enos - derived no as well as exogenous no - releasing compounds attenuate i / r - induced liver injury .
more recent studies demonstrate that animals and humans are capable of producing substantial quantities of no via enos - independent pathways that utilize nitrogen oxides that are produced from the decomposition of no or are ingested in the diet . like any important signaling molecule , the generation and lifetime of no is very short - lived in vivo .
a physiologically - relevant set of pathways for the inactivation of no are mediated by metal - catalyzed oxidation reactions .
the copper - containing protein ceruloplasmin has been shown to rapidly oxidize no to no2 under physiological conditions .
in addition to ceruloplasmin , ferrous dioxygenated hemoglobin ( hb - feo2 ; oxyhemoglobin ) or myoblobin rapidly decomposes no to yield nitrate .
historically , it has been assumed that blood and tissue nitrate and nitrite simply reflect inert endproducts of no metabolism and food contaminants .
however , it is now recognized that these oxidized metabolites of nitrogen can be metabolized in vivo to regenerate no ( fig .
because the rate of no generation from nitrite is linearly dependent on reductions in tissue po2 and ph , nitrite may be metabolized to no in ischemic tissue ( via deoxyhb or mb ) and exert no - dependent protective effects site - specifically .
indeed , the tissue - specific reduction of nitrite to generate cytoprotective no has important clinical significance in that treatment of different ischemic disorders with nitrite would reduce the untoward cardiovascular effects of systemic delivery of inhaled no or intravenous administration of s - nitrosothiols .
a growing number of studies demonstrate that nitrite is cytoprotective in different models of tissue i / r including the liver , heart , kidney and brain ( reviewed in ref .
the clinical usefulness of nitrite is currently being investigated as a potential therapeutic agent to treat ischemic disorders .
there is now overwhelming evidence supporting the concept that i / r - induced tissue damage is associated with the o2-dependent decrease in steady - state levels of no .
furthermore , accumulating evidence suggests that enos - dependent as well as enos - independent generation of no acts to limit post - ischemic tissue injury .
the mechanisms by which this nitrogen oxide protects the post - ischemic liver remain to be defined and are the subject of active investigation .
therapeutic uses of no or nitrite are currently underway in several different laboratories to treat a variety of ischemic disorders . | liver ischemia and reperfusion - induced injury is a major clinical complication associated with hemorrhagic or endotoxin shock and thermal injury as well as liver transplantation and resectional surgery .
data obtained from several different studies suggest that an important initiating event in the pathophysiology of ischemia and reperfusion - induced tissue injury is enhanced production of superoxide concomitant with a decrease in the bioavailability of endothelial cell - derived nitric oxide .
this review will summarize the evidence supporting the hypothesis that the redox imbalance induced by alterations in superoxide and nitric oxide generation creates a more oxidative environment within the different cells of the liver that enhances the nuclear transcription factor-b - dependent expression of a variety of different cytokines and mediators that may promote as well as limit ischemia and reperfusion - induced hepatocellular injury .
in addition , the evidence implicating endothelial cell nitric oxide synthase - dependent and -independent generation of nitric oxide as important regulatory pathways that act to limit ischemia and reperfusion - induced liver injury and inflammation is also presented . | Introduction
Ischemia and Reperfusion Increases Reactive Oxygen Production, Decreases Nitric Oxide Bioavailability and Alters the Redox State of the Liver:
Superoxide is the major ROS involved in post-ischemic liver injury
Role of Leukocytes in I/R Injury
Suppression of I/R-Induced Liver Damage by Endogenous or Exogenous Nitric Oxide
Concluding Remarks |
the presence of microorganisms in the root canal system plays a fundamental role in the pathogenesis of apical periodontitis .
the elimination of all microorganisms from the root canal system is accomplished by mechanical instrumentation supported by various irrigating solutions and placement of intracanal medications .
calcium hydroxide ( ca(oh)2 ) is a commonly used intracanal medicament because of its proven antimicrobial activity , for its capacity to neutralize bacterial endotoxin and stimulate apical and periapical repair .
ca(oh)2 paste should be removed before the obturation of root canal because the remnants of ca(oh)2 on the canal walls influence dentine bond strength and negatively affect the quality of root filling .
sodium hypochlorite ( naocl ) and ethylenediaminetetraacetic acid ( edta ) irrigation solutions are commonly used for the removal of residual ca(oh)2 . passive ultrasonic irrigation ( pui ) , rotary nickel titanium instruments , and apical patency file have been used to activate the effectiveness of irrigants .
although various irrigants and methods have been proposed for the removal of ca(oh)2 dressing , there is still no general consensus about which technique is best . the endovac system ( discus dental , culver city , ca , usa ) was newly introduced and designed to safely deliver irrigants to the apical terminus of root canals .
the recommended protocol for the use of apical negative pressure irrigation includes two main phases : macro- and microirrigation .
this system is composed by a macro- and microcannula that make the irrigating solution circulate due to difference of pressure caused by the vacuum inside the root canal system . measuring 0.32 mm in diameter , the microcannula can be placed to the working length ( wl ) , provided the canal is prepared to a minimum size specified by the international organization for standardization 35 .
the other new branch is represented by the self - adjusting file ( saf ) system which is a hollow file designed as a compressible , thin - walled , pointed cylinder 1.5 mm in diameter ; and composed of 120 m thick nickel - titanium lattice .
it is operated with a continuous flow of naocl that is delivered into the root canal through the hollow file and claimed to be activated by sonic agitation of the irrigant .
the purpose of this study was to compare the efficacy of saf with endovac and canalbrush in removing ca(oh)2 from root canal walls .
sixty freshly extracted human mandibular canine teeth with intact mature apices were used for evaluation .
all teeth were single rooted with a single canal , which was confirmed with multiple angulated radiographs . following extraction ,
teeth were scaled with ultrasonic instruments , washed with distilled water , and immersed in 10% formalin solution until use .
the crowns were removed at the cementoenamel junction with a water - cooled diamond disc ( kg sorensen , barueri , sp , brazil ) at low speed to obtain a standardized root length of 15 mm . a size 15 k - file ( dentsply maillefer , ballaigues , switzerland )
was passively introduced into each canal until its tip was just visible at the apical foramen .
all canals were prepared by the same operator using protaper nickel - titanium rotary instruments ( dentsply maillefer , baillagues , switzerland ) to size # 40 as the master apical file . during the preparation ,
the root canal was irrigated with 2 ml of 2.5% naocl solution after each instrument .
the irrigant was delivered via a 27-gauge needle , which was placed passively into the canal , up to 3 mm from the apical foramen without binding .
when instrumentation was completed , final irrigation was applied using 5 ml 17% edta and 5 ml 2.5% naocl and then dried with paper points .
ca(oh)2 ( calcicur , voco , cuxhaven , germany ) was placed into each canal via a lentulo spiral to the wl until the medicament was visible at the apical foramen .
the access cavities were sealed with cotton pellet and temporary filling ( cavit , espe , seefeld , germany ) .
all samples were stored at 37 1c and 100% relative humidity for 7 days .
sixty specimens were randomly assigned into four experimental groups based on irrigant agitation protocols ( n = 15 ) .
initially , a size # 40 hedstrm file was inserted into the root canals to the wl and up and down strokes were performed to disrupt and loosen the medication .
the groups were as follows : in group 1 , conventional syringe irrigation ( no activation ) : 27-gauge conventional irrigation needle was inserted as deep apically as possible without binding , and irrigation was performed with 10 ml of 2.5% naocl solution ; in group 2 , root canals were irrigated with 5 ml of 2.5% naocl , and a medium - sized canalbrush ( coltne / whaledent , langenau , germany ) was placed in a slow - speed handpiece ( 600 rpm ) and advanced to the wl .
canalbrush was applied into the canals for 30 s with circumferential motion , and a final irrigation of 5 ml of 2.5% naocl was used .
the brush was used at full wl with a gentle up and down motion . in group 3 , the apical negative pressure irrigation ( endovac , discus dental , culver city , ca )
the macrocannula tip was used to deliver irrigant up and down the canal for 1 min .
each cycle of microcannula irrigation consisted of the tip being placed at full wl for 6 s and then withdrawn 2 mm from full wl for 6 s. this was repeated five times during a period of 30 s. a total of 10 ml of 2.5% naocl was used . in group 4 ,
saf ( redent - nova , raanana , israel ) with 2.0 mm diameter and 25 mm length was operated in the canal using an rdt3 nx handpiece head ( re - dent nova , raanana , israel ) with endodontic motor ( x - smart , dentsply maillefer , ballaigues , switzerland ) , that produced 5,000 vibrations per min with an amplitude of 0.4 mm .
the saf was used for 4 min with 2.5% naocl at a flow rate of 2.5 ml / min .
after the irrigation procedures , the canals were dried with paper points , and longitudinal grooves were prepared on the buccal and lingual surfaces of each root with a diamond disk at a slow speed without penetrating the canal .
the teeth were split along their long axis in a buccolingual direction using a hammer and chisel . for scanning electron microscopic ( quanta 400f field emission sem , fei , oregon , usa ) analysis ,
the samples were dehydrated and coated with gold - palladium particles , and a magnification of 1,000 was used to evaluate the cleanliness of the canal walls at the apical andcoronal thirds of root canal .
the images of canal surfaces were selected from apical and coronal thirds ( 3 and 11 mm shorter from the apex , respectively ) for sem evaluation . a scoring system , which was previously described by kuga et al . ,
the scores used were as follows : score 0 : absence of residues , score 1 : small amount of residues ( up to 20% of the surface covered ) , score 2 : moderate amount of residues ( 20 - 60% of the surface covered ) , score 3 : large amount of residues ( more than 60% of the surface covered ) .
all ca(oh)2 removing procedures were completed by one operator and sem evaluations were performed by a second examiner who was blind in respect of test groups .
all statistical tests were performed using the software , statistical packages for social sciences ( spss ) , version 19.0 for windows ( spss inc . , chicago , il ) .
sample sem images of apical and coronal thirds of each group were shown in figure 1 .
the mean scores and standard deviations ( sds ) for the coronal and apical thirds of groups were shown in tables 1 and 2 , respectively . comparing the irrigant agitation protocols with respect to ca(oh)2 removal , all irrigant agitation protocols successfully removed ca(oh)2 at the coronal third .
no statistically significant difference ( p > 0.05 ) was found among the irrigant agitation protocols at the coronal third .
group 1 ( no activation ) demonstrated the highest score values , but it was not statistically different from other groups . whereas , group 3 ( apical negative pressure irrigation ) and group 4 ( sonic agitation ) demonstrated the lowest scores in coronal thirds . at the apical third ,
no difference was found between groups 1 ( no activation ) and 2 ( rotary brush agitation ) .
groups 3 ( apical negative pressure irrigation ) and 4 ( sonic agitation ) were significantly efficient for the removal of ca(oh)2 at the apical third in comparison with groups 1 and 2 ( p < 0.05 ) . in group 4 ( sonic agitation )
scanning electron microscopy images of the coronal and apical thirds in each group the mean scores and standard deviations of the coronal thirds .
a different superscript in the column of mean values indicates statistically significant different values the mean scores and standard deviation ( sd ) of the apical thirds .
the most commonly used intracanal medicament is ca(oh)2 because it is effective against the majority of endodontic pathogens .
however , prior to definite obturation of the root canal , ca(oh)2 medicament should be removed completely in order to provide maximum interface between the root canal wall and filling material .
previous studies reported that residual ca(oh)2 on the root canal wall could influence the penetration of sealer into dentinal tubules , reduce the bond strength of a resin - based sealer , and interfere with the sealing ability of a silicon - based sealer .
various methods have been used to investigate the amount of residues on the canal walls , such as the uses of digital photographs , stereo microscopes , scanning electron microscopes , microtomography computed tomography ( micro - ct ) , and spiral ct . in our study , scanning electron microscopy was used at 1,000 magnification , and the remnants of ca(oh)2 on root canal walls were evaluated using a scoring method similar to that used in previous studies .
naocl irrigation without activation was reported to be an inadequate method for the removal of ca(oh)2 from the root canals because of limited ability to dissolve inorganic materials .
margelos et al . , showed that using 15% edta solution or naocl alone as irrigants does not remove ca(oh)2 efficiently from the root canal ; however , the combination of two irrigants with hand instrumentation improves the removal efficiency .
therefore , in the current study , we purposed to evaluate the efficacy of different irrigant agitation protocols using with naocl on removal of ca(oh)2 from root canals .
result obtained in the present study showed that least efficient was syringe irrigation for ca(oh)2 removal .
additionally , in group 1 , naocl was delivered to the root canal by 27-gauge irrigation needle .
the syringe needle irrigation may be effective in cleaning the coronal third of root canal but not in the apical third since the irrigant can progress only 1 mm further from the tip of the needle .
therefore , adequate delivery of the irrigant to the wl with needle irrigation may not be obtained .
this could be another reason for less removal of ca(oh)2 from the apical part of the root canal in group 1 .
these studies reported that canalbrush was not effective in the removal of ca(oh)2 due to its packing effect .
the apical third of the root canals in group 2 ( canalbrush ) showed higher average scores with significant differences compared to apical negative pressure irrigation and sonic agitation groups .
canalbrush did not remove ca(oh)2 from the apical third , but conversely packed the ca(oh)2 to the apical part of the canals . in our study , canalbrush tip was fractured in the apical third of one specimen like in a previous study . since the canalbrush is radiolucent and impossible to discern in radiographs , in the presence of fractured tip , there is a chance of pushing it through the apical foramen with files during the root canal instrumentation . in all groups ,
the coronal third demonstrated significantly better results in the removal of ca(oh)2 than apical third .
lee et al . , reported that this result may be related with the higher velocity and volume of irrigant flow created at the coronal part of the root canal during irrigation procedure . in all of the experimental groups , a statistical difference in cleanliness was observed between the coronal versus apical third , except in group 4 . in the sonic agitation saf group , no statistical difference was found between the apical and coronal thirds .
the results of the present study showed better efficacy of endovac and saf groups compared with other groups in the apical of the root canal .
the saf is a hollow file which allows for the continuous irrigation of the root canal throughout the procedure , with additional activation of the irrigant by its vibrating motion that creates turbulence in the root canal . in the present study ,
thus , it allows for the delivery of the irrigant to wl and provides constant irrigant turnover at apical third . according to the results of the present study , the use of saf , in continuous flow with vibrating motion and effective replacement of naocl in the apical part of the canal ,
the endovac system was introduced newly ; and to our knowledge , no study has evaluated ca(oh)2 removal efficacy .
endovac is a negative pressure irrigation system that has been designed to deliver irrigants safely into the apical portion of the canal in order to optimize the removal of the smear layer and minimize the extrusion of solution through the apical foramen .
the placement of the microcannula to wl as recommended by the manufacturer enables the irrigant to penetrate almost to the wl , to be suctioned in sufficient volume , and flow to remove smear layers and displace debris . in our study , the effectiveness of endovac on removal of ca(oh)2 from apical third is related with its better mechanical flushing action and vacuum aspiration effect .
additionally , the orifices of the microcannula may provide a portal of exit for ca(oh)2 , resulting in effective removal from apical third of root canal . in the present study , even with the use of different irrigant agitation protocols complete removal of ca(oh)2 was not possible in the apical third of the root canal
. this could possibly be due to the nature of dentinal tubules which are irregular and less in number .
whittaker and kneale also suggested relatively fewer dentinal tubules per unit area being present in the apical third of the root canal wall .
within limitations of this study , we showed that all irrigant agitation techniques were equally effective in removing ca(oh)2 from the coronal third of the root canal .
however , in the apical third of the root canal , present study demonstrated that none of the methods completely removed ca(oh)2 from the canal walls .
saf and endovac showed significantly better performance in removing ca(oh)2 from apical third of the root canals compared with canalbrush . | objectives : this study comparatively evaluated the efficacy of self - adjusting file ( saf ) , endovac , and canalbrush irrigant agitation protocols in removing calcium hydroxide ( ca(oh)2 ) from the root canals.materials and methods : sixty extracted human mandibular canine teeth were instrumented with protaper rotary instruments to size # 40 and dressed with ca(oh)2 . the roots were randomly assigned to four groups according to irrigant agitation protocol used ( n = 15 ) . in group 1 : conventional syringe irrigation ( no activation , control ) ; group 2 : rotary brush agitation ( canalbrush ) ; group 3 : apical negative pressure irrigation ( endovac system ) ; and group 4 : sonic agitation ( saf ) were used .
scanning electron microscopic ( sem ) evaluation was done for assessment of ca(oh)2 removal in the coronal and apical thirds .
statistical analysis was performed by wilcoxon and kruskal - wallis tests.results:there were statistically significant differences among the groups ( p = 0.218 ) .
a statistically significant difference was seen between the test groups in ca(oh)2 removal from the apical third of the canal ( p < 0.05 ) . in the coronal third
, there was no difference between the groups ( p > 0.05 ) .
the most efficient ca(oh)2 removal in apical third was recorded in group 3 ( endovac ) and group 4 ( saf ) ( p < 0.05 ) . in group 4 ( sonic agitation ) , there was no significantly difference between ca(oh)2 removal in coronal and apical thirds.conclusions:saf and endovac showed significantly better performance than canalbrush and conventional syringe irrigation in removing ca(oh)2 from apical third of the root canals . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
a central aim of untargeted metabolomics and metabonomics studies is the identification of marker metabolites which play a crucial role in the experimental context [ 1 , 2 ] .
mass spectrometry combined with either gas chromatography ( gc / ms ) or liquid chromatography ( lc / ms ) has become a key technology for metabolome analysis under different experimental conditions [ 3 , 4 ] . a typical data set after peak detection and sample alignment [ 57 ] consists of several thousand marker candidates which are characterized by a retention time ( rt ) , a mass - to - charge value ( m / z ) , and a multivariate intensity profile of abundance levels per condition , respectively .
the experimental conditions are represented by replicate samples and may correspond to environmental disease or genetic perturbations [ 911 ] . in order to obtain a high - quality data set of experiment - related marker candidates , the raw data set is usually ranked and filtered using supervised machine learning techniques such as random forest classification [ 12 , 13 ] or statistical analysis based on anova or kruskal - wallis tests [ 1416 ] .
the filtered marker candidates are then annotated according to known metabolites from public biological and biomedical compound databases [ 1721 ] .
a central task of annotation is the calculation of actual molecular masses corresponding to each marker candidate by correcting the m / z ratios according to the ionization mode , potential adduct formation , and included natural isotopes .
this problem can be addressed by applying the ionization rules [ xm+y ] , where x denotes the number of combined target molecules , y the mass of attached molecules ( adduct formation ) , and z the degree of ionization ( e.g. , single or double ) .
additionally , the number of included isotopes has to be estimated in order to query databases which contain monoisotopic compound masses . based on a potential ionization rule with parameters x , y , z and the number of included isotopes , the corresponding compound mass can be calculated . for the corrected masses which can not be assigned to particular compounds ,
the number of considered formulas can be significantly reduced by incorporating information from preprocessing as well as rules for heuristic filtering of molecular formulas , respectively .
a major step in this process is the estimation of the number of included carbon atoms based on the intensity profiles of previously detected isotopologues .
there are a great number of software packages available , which provide tools for statistical analysis of multivariate experimental data [ 25 , 26 ] . a number of tools for peak detection and sample alignment of mass spectrometry data , such as metalign or openms , also support the deconvolution of isotopologues and statistical analysis [ 27 , 28 ] . for the xcms platform , a package for the annotation of lc / esi - ms mass signals based on adduct rules has been implemented .
the calculation of possible ionization products and the rule - based heuristic filtering of molecular formulas is provided by several software packages [ 22 , 24 ] . however , to the best of our knowledge , there is no software available which incorporates all of these methods in a single user - friendly tool as offered by marvis - filter .
in the following sections , the algorithm for adduct / isotope correction and the implementation of marvis - filter are described in detail .
the algorithm is based on the input of the retention times , m / z ratios , and raw intensity profiles of all marker candidates in a data set and calculates as output the potential monoisotopic mass , ionization rule , and number of included c - isotopes for every candidate .
the approach is based on a greedy strategy which minimizes the number of potential molecular masses and simultaneously maximizes the similarity of intensity profiles between candidates with a similar retention time and actual mass .
this concept follows the paradigm that in mass spectrometry analysis a metabolite is usually represented by several marker candidates with a similar retention time and intensity profile , but different m / z ratios according to the various possibilities of ionization and number of included isotopes . as parameters
, the algorithm expects a list of ionization / adduct rules sorted according to their relevance , the assumed maximal number of c - isotopes per marker candidate , a mass tolerance , an rt tolerance , and a minimal cosine similarity of intensity profiles .
the isotopologues correction is restricted to the detection of c. for storage of pairwise cosine similarities between candidate profiles , the algorithm utilizes a five - dimensional matrix m. each entry m(m , a1,i1,a2,i2 ) corresponds to the maximal cosine similarity between the intensity profile of candidate m , assuming ionization rule a1 and i1c - isotopes , and another candidate , which has a similar retention time ( within tolerance ) and corrected mass ( within tolerance ) assuming ionization rule a2 and i2c - isotopes . for each candidate m , the algorithm then chooses the ionization rule and number of c - isotopes which is supported by the highest sum of cosine similarities . in the following
calculate all possible masses by applying all ionization rules and number of c - isotopes to all candidate m / z ratios .
consider all pairs of potential masses under the following constraints and fill m with pairwise cosine similarities of corresponding candidate profiles .
consider only pairs of different marker candidates.consider only pairs within the mass and rt tolerance.consider only pairs with at least the requested cosine similarity .
for each entry in m hold only the maximum cosine similarity .
consider only pairs of different marker candidates .
calculate the reduced three - dimensional matrix m with summed entries :
( 1)m(m , a1,i1)red=a2,i2m(m , a1,i1,a2,i2 ) .
choose for each candidate m : the adduct rule and isotope number with the maximal sum of similarities cmax = maxa1,i1(m(m , a1,i1 ) ) .
if cmax = 0 , use the first ionization rule and zero c - isotopes as default . calculate the masses according to chosen rules and isotope numbers . in order to avoid apparently false associations between marker candidates , negative cosine similarities are disregarded . if for a given candidate different selections of the ionization rule and the number of isotopes maximize the sum of cosine similarities , the ionization rules with the highest relevance and the minimal number of c - isotopes are selected . following the annotation of the ionization rules and c - isotopes ,
the number of carbon atoms per candidate is estimated by comparing the raw intensities of marker candidates with zero predicted c - isotopes ( i m ) and the respective marker candidates including one c - isotope ( im+1 ) according to the following formula :
( 2)nc=98.9 im+11.1 i m ,
corresponding to the natural abundances of carbon isotopes .
given a pair of candidates , annotated as isotopologues ( m and m + 1 ) and with the same ionization rule , a robust estimation of the number of carbon atoms is obtained by calculating the median nc over all samples included in both intensity profiles .
marvis - filter is implemented in the matlab and c programming language and has been compiled together with the marvis - cluster tool for microsoft windows xp / vista/7 .
execution of the software requires installation of the matlab compiler runtime , which is provided with the software .
the installation packages , the documentation , and example data sets can be downloaded from the project home page http://marvis.gobics.de/. for data import and export marvis - filter uses the csv ( comma separated values ) file format , which can easily be processed by statistical analysis software and spreadsheet applications .
marvis - filter also supports the direct import of aligned mass spectrometry samples from markerlynx application manager of masslynx ( waters corporation , milford ) . for interactive analysis ,
ranking and filtering of multivariate intensity profiles marvis - filter provides the well - known one - way anova and kruskal - wallis tests combined with methods for p value adjustment for multiple - hypothesis testing [ 30 , 31 ] .
based on customizable lists of ionization rules , the adduct / isotope correction can be performed on raw or filtered data sets .
the ionization rules are imported as text files and can easily be adapted or extended .
( 1 ) displays the adjusted p values ( y - axis ) of all candidate intensity profiles in the current data set sorted in ascending order .
the data set can interactively be filtered according to a user - defined significance level by selecting a marker , sliding the red separator line or jumping to a predefined level .
the profile plot ( 2 ) shows the raw intensity profile of the currently selected marker candidate .
intensity values of replicated samples belonging to the same experimental conditions are marked in the same color .
( 3 ) displays information about all marker candidates of the data set arranged according to the p values and characterized by the m / z ratio , rt and additional user - defined scores , which can be imported along with the data set .
after adduct and isotope correction , the additional annotations are displayed in this listbox as well .
data set clipboard listbox ( 4 ) shows data sets which are currently held in the marvis clipboard . the current ( filtered or unfiltered ) data set can simply be added or removed to / from this list .
the data set clipboard supports an adduct and isotope correction of selected data sets in a batch mode .
data sets which were corrected based on different sets of ionization rules ( e.g. , positive and negative ionization ) may be combined into one single data set . for selected candidate profiles , bar plots , standard error plots , and boxplots can easily be inspected and exported in various image formats . for detailed analysis , the user can zoom into all plots . additionally , marvis - filter provides a convenient interface for quick candidate search based on the i d , rt , m / z , or mass value .
marvis - filter also provides a molecular formula calculator , which is based on the seven golden rules and utilizes the estimated number of carbon atoms per marker candidate obtained after adduct and isotope correction .
marvis - filter and marvis - cluster are combined in the marvis - suite which features the direct data exchange between preprocessing in marvis - filter and convenient visualization of multivariate intensity profiles and high - level cluster analysis in marvis - cluster .
the functionality of marvis - filter is demonstrated using two data sets of a metabolomic case study for plant wounding experiments .
the data sets are available on the project homepage http://marvis.gobics.de/ together with a detailed description of the extraction and uplc - tof method . additionally , the data sets are available for import in marvis - filter after installation of the marvis - suite ( wound_neg_raw.csv and wound_pos_raw.csv in the examples directory ) .
the case study reflects a wounding time course of arabidopsis thaliana wild - type ( wt ) plants as well as of mutant plants ( dde 2 - 2 ) , which are deficient in the biosynthesis of the plant wound hormone jasmonic acid and its derivatives .
the first four conditions reflect the metabolic situation within a wounding time course of wild - type ( wt ) plants , starting with the unwounded control plants ( abbreviation wt_0 ) followed by the plants harvested 0.5 ( wt_30 ) , 2 ( wt_2 ) , and 5 hours past wounding ( wt_5 ) . the conditions 5 to 8 represent the analogous time course for the jasmonate deficient mutant plant dde 2 - 2 ( aos_0 , aos_30 , aos_2 , aos_5 ) .
the two data sets are imported sequentially in marvis - filter using the import raw csv data entry in the file menu with the following options : delimiter : , ; start row : 5 , start column : 3 ; i d label :
i d ; generate ids : activated ; x column : 2 ; x label : rt ; y column : 3 ; y label : m / z ; condition identifiers :
wt_0 , wt_30 , wt_2 , wt_5 , aos_0 , aos_30 , aos_2 , aos_5 .
after data import , the marker candidates are sorted and ranked according to the p values of a kruskal - wallis test and the bonferroni - holm adjustment for multiple hypothesis testing by selecting the corresponding checkboxes in the filter dialog and the adjustment for multiple testing dialog . adduct and isotope correction are performed on the full data sets separately using predefined sets of adduct rules for the negative ( table 2 ) and positive ionization mode ( table 3 ) , an rt tolerance of 0.04 minutes , a mass tolerance of 0.005 da , a minimal cosine similarity of 0.75 , and a maximum number of two c - isotopes per candidate .
the adduct rules had been determined in previous targeted uplc - tof - ms experiments .
after correction , the data sets are filtered according to a significance level for adjusted p values of 0.01 ( goto level entry in selection
table 1 shows the initial number of imported marker candidates and the number of high - quality marker candidates after filtering . finally , the two data sets in the marvis clipboard are concatenated using the combine button .
the combined data set can be sorted according to a user - defined method once again and is then presented in a new marvis - filter window . after selecting the whole data set
, the combined subset of 3504 high - quality marker candidates can be exported as a csv file , and clustered as well as visualized using marvis - cluster ( goto marvis - cluster entry in the marvis - suite menu ) .
figure 2 shows the results from clustering of the filtered and combined data in marvis - cluster .
the corrected , filtered , and combined data sets were used to identify metabolites which show a significant change of abundance in the wound time course in wt and/or jasmonate deficient mutant plants .
first , the corrected masses of marker candidates were matched to molecular masses of all compounds recorded in the kegg and aracyc database or literature based on a tolerance of 0.005 da .
the identity of marker candidates was confirmed based on the isotopic pattern and coelution with identical standards or ms / ms fragmentation .
thus , a number of oxylipins could be identified as wound - induced metabolite markers ( see table 4 ) .
oxylipins are metabolites deriving from lipid peroxidation and are involved in regulating developmental processes as well as environmental responses , like the inflammatory or wound response , in nearly every organism . among these bioactive lipids ,
mammals use predominantly c20 fatty acids ( eicosanoids ) , while in plants c18 fatty acids are most abundantly used for the biosynthesis of oxylipins or so - called octadecanoids . the identified oxylipins ( see table 4 ) are part of the -linolenic acid metabolism or members of the compound class of mono- and digalactosyldiacylglycerols .
they are described in the context of plant wounding [ 33 , 34 , 36 ] .
thirteen of the fifteen identified oxylipins could only be detected in either the negative or the positive ionization mode .
the findings are supported by very low adjusted p values from the kruskal - wallis test of the intensity profiles ( see previous section and table 4 ) .
marvis - filter combines essential preprocessing tools for mass spectrometry data analysis within a single user - friendly tool .
large data sets from the negative and positive ionization mode can easily be imported , corrected , filtered , and combined .
lists of ionization rules for adduct correction can be customized , extended , and commented in a convenient way using a standard text editor . within the marvis - suite filtered and combined data sets can directly be clustered , visualized , and analyzed in detail using the marvis - cluster tool . in a case
study 75 high - quality marker candidates could be clearly assigned to fifteen compounds of the oxylipin class based on the adduct and isotope correction in marvis - filter .
the combination of data sets deriving from the negative and positive ionization mode is an important step for further data analysis . in the case study , most of the identified metabolites
the significance of the selected wound markers is supported by a high number of annotated and assigned ions / marker candidates and by very low adjusted p values from the kruskal - wallis test .
the statistical filtering of marker candidates reduced the complexity of the data sets from about 48000 to 3500 significant candidates ( about 7 percent ) . | statistical ranking , filtering , adduct detection , isotope correction , and molecular formula calculation are essential tasks in processing mass spectrometry data in metabolomics studies . in order to obtain high - quality data sets , a framework which incorporates all these methods is required .
we present the marvis - filter software , which provides well - established and specialized methods for processing mass spectrometry data . for the task of ranking and filtering multivariate intensity profiles ,
marvis - filter provides the anova and kruskal - wallis tests with adjustment for multiple hypothesis testing .
adduct and isotope correction are based on a novel algorithm which takes the similarity of intensity profiles into account and allows user - defined ionization rules .
the molecular formula calculation utilizes the results of the adduct and isotope correction . for a comprehensive analysis
, marvis - filter provides an interactive interface to combine data sets deriving from positive and negative ionization mode . the software is exemplarily applied in a metabolic case study , where octadecanoids could be identified as markers for wounding in plants . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions |
the current standard of care for neovascular age - related macular degeneration ( namd ) is treatment with agents ( such as ranibizumab ) that inhibit vascular endothelial growth factor ( vegf ) , a potent angiogenic mediator associated with age - dependent choroidal neovascularization.1 ranibizumab was approved for use in namd largely due to the findings from two pivotal clinical studies.2,3 in these studies , monthly ranibizumab ( 0.3 or 0.5 mg ) was shown to be superior to sham or photodynamic therapy for the 12-month primary efficacy outcome , and visual and anatomical benefits were sustained over a 24-month follow - up.24 based on these studies , monthly dosing with ranibizumab was recommended.5 however , this approach can be unsustainable in many clinical practices .
less frequent dosing is now the norm , and the label for all the currently available anti - vegf agents recommends an initial loading phase followed by as - needed ( prn ) dosing with frequent monitoring.5,6 however , it is acknowledged that prn or quarterly dosing may be less effective compared with monthly dosing , and optimal regimens need to be established.711 some studies show that the visual benefit declines during the maintenance phase despite an initial marked improvement in acuity at the completion of the loading phase.7,8,10,11 in contrast , the sustain study found that ranibizumab prn was comparable with monthly dosing.12 the aura study was undertaken to monitor ranibizumab use and treatment outcomes in a real - life setting .
aura was a large , retrospective study conducted in eight countries ( canada , france , germany , ireland , italy , the netherlands , the uk , and venezuela ) that evaluated the real - life clinical use of ranibizumab treatment for patients with namd who were diagnosed and treated by their own physicians.13 in the global population ( all eight included countries ) , there was a mean increase in visual acuity of + 2.4 letters during year 1 , which decreased to + 0.6 letters during year 2 .
patients received approximately seven injections over a 2-year period , but there was variability between countries .
the findings also indicated that monitoring strategies and retreatment frequencies varied between countries , which may explain the poorer visual outcomes in the overall aura population .
the aim of the present report is to describe the outcomes of the uk cohort of aura and to compare with the global aura cohort ( canada , france , germany , ireland , italy , the netherlands , and venezuela ) .
the study design has been reported elsewhere.13 in brief , patients who were diagnosed with namd and given ranibizumab injections during january 1 , 2009 to august 31 , 2009 were eligible ; the diagnosis and subsequent decision to treat was made by the patient s own physician . for inclusion , patients were required to have received one or more ranibizumab injections with documented follow - up to the end of their treatment and/or monitoring , or until august 31 , 2011 , whichever came first ( figure s1 ) .
patients who switched to bevacizumab or pegaptanib sodium , but received ranibizumab at some point , remained in the study . only one eye was included per patient , which was defined as the eye treated at the start of ranibizumab therapy . in patients with bilateral disease
patients taking part in an investigational study of any other drug or device were excluded .
approval from the country - specific independent ethics committees or institutional review boards was received based on the requirements of local law and/or regulations , and written consent was obtained from each patient prior to inclusion .
we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research .
this paper reports the findings from the uk cohort only , but makes explicit comparisons with the global results .
all data , including demographics , were collected retrospectively from patients medical records ( paper or electronic ) , which included results from examinations performed during routine clinical practice .
the primary assessment was the mean change in visual acuity over the 2-year period ( with key assessments at year 1 [ day 360 ] and year 2 [ day 720 ] ) after the start of ranibizumab treatment .
as the study was observational , visual acuity was measured using a variety of vision charts , but was converted to a standardized visual acuity scoring system ( letter count ) .
the conversion chart used is provided in the online supplementary material for the primary manuscript.13 secondary assessments included resource utilization during the 2-year period ( ie , the number of clinic visits , use of optical coherence tomography [ oct ] and visual acuity tests , and the number of injections ) .
the mean change in visual acuity over the 2-year period was calculated for patients who received one or more doses of ranibizumab treatment and had one or more post - baseline assessments of visual acuity for the treated eye .
this was defined as the effectiveness analysis set , and it was estimated that a guiding sample size of 399 subjects per country was required to estimate the change from baseline in the standardized visual acuity score within a 95% confidence interval ( ci ) of 1.5 letters .
mean change in visual acuity was assessed using a last observation carried forward ( locf ) analysis to account for missing data .
secondary assessments were analyzed using frequency tables or summary statistics and were also performed using the effectiveness analysis set .
the study design has been reported elsewhere.13 in brief , patients who were diagnosed with namd and given ranibizumab injections during january 1 , 2009 to august 31 , 2009 were eligible ; the diagnosis and subsequent decision to treat was made by the patient s own physician . for inclusion , patients were required to have received one or more ranibizumab injections with documented follow - up to the end of their treatment and/or monitoring , or until august 31 , 2011 , whichever came first ( figure s1 ) .
patients who switched to bevacizumab or pegaptanib sodium , but received ranibizumab at some point , remained in the study . only one eye was included per patient , which was defined as the eye treated at the start of ranibizumab therapy . in patients with bilateral disease
patients taking part in an investigational study of any other drug or device were excluded .
approval from the country - specific independent ethics committees or institutional review boards was received based on the requirements of local law and/or regulations , and written consent was obtained from each patient prior to inclusion .
we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research .
this paper reports the findings from the uk cohort only , but makes explicit comparisons with the global results .
all data , including demographics , were collected retrospectively from patients medical records ( paper or electronic ) , which included results from examinations performed during routine clinical practice .
the primary assessment was the mean change in visual acuity over the 2-year period ( with key assessments at year 1 [ day 360 ] and year 2 [ day 720 ] ) after the start of ranibizumab treatment . as the study was observational , visual acuity
was measured using a variety of vision charts , but was converted to a standardized visual acuity scoring system ( letter count ) .
the conversion chart used is provided in the online supplementary material for the primary manuscript.13 secondary assessments included resource utilization during the 2-year period ( ie , the number of clinic visits , use of optical coherence tomography [ oct ] and visual acuity tests , and the number of injections ) .
the mean change in visual acuity over the 2-year period was calculated for patients who received one or more doses of ranibizumab treatment and had one or more post - baseline assessments of visual acuity for the treated eye .
this was defined as the effectiveness analysis set , and it was estimated that a guiding sample size of 399 subjects per country was required to estimate the change from baseline in the standardized visual acuity score within a 95% confidence interval ( ci ) of 1.5 letters .
mean change in visual acuity was assessed using a last observation carried forward ( locf ) analysis to account for missing data .
secondary assessments were analyzed using frequency tables or summary statistics and were also performed using the effectiveness analysis set .
a total of 774 patients were screened , and 461 of these were enrolled from 13 centers in the uk .
a total of 313 patients were excluded for the following reasons : participation in an investigational study ( n=40 ) , no diagnosis of namd ( n=3 ) , no informed consent form signed ( n=257 ) , and start of ranibizumab therapy outside inclusion dates ( n=13 ) . of the enrolled patients , 410 received one or more doses of ranibizumab , had one or more post - baseline measurements of visual acuity for the treated eye , and were included in the effectiveness analysis set ( thus meeting the prespecified sample size of 399 subjects ) .
the baseline characteristics of patients included in the effectiveness analysis sets in the uk and global cohorts are shown in table 1 .
the cohorts were well - matched with regard to age , sex , oct volume ( center point thickness ) , and mean visual acuity ( letter count ) at baseline . although the aim of the study was to follow patients receiving ranibizumab treatment , five patients received at least one injection of bevacizumab and one patient received one injection of pegaptanib sodium
the majority of patients ( 99.3% overall : 99.8% at year 1 and 99.5% at year 2 ) did not receive any treatment change .
three patients switched from ranibizumab to bevacizumab due to nonresponse ( n=2 ) and reason not known ( n=1 ) .
visual acuity ( letter count ) improved from baseline following initiation of ranibizumab treatment , peaking at day 270 with a mean ( standard deviation [ sd ] ) gain in visual acuity of + 6.5 ( 14.4 ) letters in the overall uk cohort .
the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 , but there was a small decline in visual acuity over time , and the mean ( sd ) change was + 4.1 ( 16.9 ) at year 2 in the uk cohort ( figure 1a ) .
the majority of patients in the uk cohort ( 355/410 ; 86.6% ) received a loading phase ( ie , first three ranibizumab injections within 90 days ) .
the mean change in visual acuity was greater in these patients compared with patients who did not receive a loading phase ; however , a small decline in visual acuity was observed during the follow - up in both groups ( figure 1a ) . the mean change in visual acuity from baseline was + 6.8 ( 15.5 ) letters in year 1 and + 4.8 ( 17.1 ) letters in year 2 in patients receiving a loading phase , and + 0.5 ( 13.7 ) and 0.2 ( 14.3 ) in patients who did not receive a loading phase .
the mean improvement in visual acuity was also greater in the uk compared with the global population ( figure 1b ) ; there was a decline in both the uk and global populations , but this occurred earlier in the global population ( from approximately day 120 onward ) and was more marked . compared with the other countries enrolled in aura , the uk demonstrated the greatest visual acuity improvements over time , followed by the netherlands ( figure 1c ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was higher for the uk than for the global population ( figure 2 ) . in the uk ,
mean ( sd ) improvement in visual acuity was also higher in patients with a baseline score of < 35 letters ( + 15.1 [ 16.9 ] at year 1 and + 11.6 [ 16.9 ] at year 2 ) ( figure 2 ) .
overall , 43.4% of patients in the uk cohort received ranibizumab treatment within 2 weeks of receiving their namd diagnosis ( figure 3 ) , and the mean ( sd ) treatment duration was 634.7 ( 292.4 ) days . in the uk cohort ,
the mean ( sd ) number of visits to the ophthalmologist was 18.4 ( 5.0 ) over the full 2-year observation period ( figure 4a ) .
there was a higher mean ( sd ) number of visits in the first year ( 10.4 [ 2.2 ] ) than in the second year ( 8.0 [ 3.3 ] ) .
similar findings were observed for the mean ( sd ) number of visual acuity tests ( 10.0 [ 2.4 ] in year 1 and 7.8 [ 3.3 ] in year 2 ) and oct tests ( 9.2 [ 2.4 ] in year 1 and 7.4 [ 3.0 ] in year 2 ) ( figure 4a ) .
patients from the uk received a mean ( sd ) of 9.0 ( 4.7 ) injections during the full 2-year period ( 5.8 [ 2.4 ] in year 1 and 3.2 [ 2.8 ] in year 2 ) ( figure 4a ) .
there were more visits , tests , and injections in the uk compared with the global population during year 1 and year 2 ( figure 4b and c ) .
the mean number of injections in the uk compared with the global population ( excluding the uk ) was 5.8 versus 4.8 in year 1 and 3.2 versus 2.0 in year 2 .
in the global cohort , 80.2% ( n=1,786/2,227 ) completed a 3-month loading phase , and 19.9% ( 355/1,786 ) of these patients were from the uk .
the mean ( sd ) time from the end of the loading phase to the next injection ( maintenance phase ) was 102.8 ( 121.4 ) days in the uk and 134.0 ( 141.2 ) days in the global cohort ( excluding the uk ) .
the mean ( sd ) duration between visits in the maintenance phase was 42.1 ( 11.7 ) days in the uk compared with 59.3 ( 51.0 ) in the global cohort ( excluding the uk ) .
the mean ( sd ) duration between injections in the maintenance phase was 100.0 ( 80.3 ) in the uk cohort versus 104.1 ( 98.7 ) days in the global cohort ( excluding the uk ) .
a total of 365 patients ( 89.0% ) completed the observation period ( august 31 , 2011 ) .
the remaining patients ( n=45 ; 11.0% ) discontinued the study prior to this for a number of reasons , including permanent discontinuation of anti - vegf ( n=34 ; 8.3% ) ( due to treatment failure [ n=22 ] , patient decision [ n=2 ] , stable disease [ n=6 ] , and other reason [ n=4 ] ) , change of treating physician ( n=5 ; 1.2% ) , withdrawal by patient ( n=1 ; 0.2% ) , lost to follow - up ( n=1 ; 0.2% ) , other reason ( n=3 ; 0.7% ) , and missing ( n=1 ; 0.2% ) .
a total of 774 patients were screened , and 461 of these were enrolled from 13 centers in the uk .
a total of 313 patients were excluded for the following reasons : participation in an investigational study ( n=40 ) , no diagnosis of namd ( n=3 ) , no informed consent form signed ( n=257 ) , and start of ranibizumab therapy outside inclusion dates ( n=13 ) . of the enrolled patients , 410 received one or more doses of ranibizumab , had one or more post - baseline measurements of visual acuity for the treated eye , and were included in the effectiveness analysis set ( thus meeting the prespecified sample size of 399 subjects ) .
the baseline characteristics of patients included in the effectiveness analysis sets in the uk and global cohorts are shown in table 1 .
the cohorts were well - matched with regard to age , sex , oct volume ( center point thickness ) , and mean visual acuity ( letter count ) at baseline . although the aim of the study was to follow patients receiving ranibizumab treatment , five patients received at least one injection of bevacizumab and one patient received one injection of pegaptanib sodium
the majority of patients ( 99.3% overall : 99.8% at year 1 and 99.5% at year 2 ) did not receive any treatment change .
three patients switched from ranibizumab to bevacizumab due to nonresponse ( n=2 ) and reason not known ( n=1 ) .
visual acuity ( letter count ) improved from baseline following initiation of ranibizumab treatment , peaking at day 270 with a mean ( standard deviation [ sd ] ) gain in visual acuity of + 6.5 ( 14.4 ) letters in the overall uk cohort .
the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 , but there was a small decline in visual acuity over time , and the mean ( sd ) change was + 4.1 ( 16.9 ) at year 2 in the uk cohort ( figure 1a ) .
the majority of patients in the uk cohort ( 355/410 ; 86.6% ) received a loading phase ( ie , first three ranibizumab injections within 90 days ) .
the mean change in visual acuity was greater in these patients compared with patients who did not receive a loading phase ; however , a small decline in visual acuity was observed during the follow - up in both groups ( figure 1a ) .
the mean change in visual acuity from baseline was + 6.8 ( 15.5 ) letters in year 1 and + 4.8 ( 17.1 ) letters in year 2 in patients receiving a loading phase , and + 0.5 ( 13.7 ) and 0.2 ( 14.3 ) in patients who did not receive a loading phase .
the mean improvement in visual acuity was also greater in the uk compared with the global population ( figure 1b ) ; there was a decline in both the uk and global populations , but this occurred earlier in the global population ( from approximately day 120 onward ) and was more marked . compared with the other countries enrolled in aura , the uk demonstrated the greatest visual acuity improvements over time , followed by the netherlands ( figure 1c ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was higher for the uk than for the global population ( figure 2 ) . in the uk ,
mean ( sd ) improvement in visual acuity was also higher in patients with a baseline score of < 35 letters ( + 15.1 [ 16.9 ] at year 1 and + 11.6 [ 16.9 ] at year 2 ) ( figure 2 ) .
overall , 43.4% of patients in the uk cohort received ranibizumab treatment within 2 weeks of receiving their namd diagnosis ( figure 3 ) , and the mean ( sd ) treatment duration was 634.7 ( 292.4 ) days . in the uk cohort ,
the mean ( sd ) number of visits to the ophthalmologist was 18.4 ( 5.0 ) over the full 2-year observation period ( figure 4a ) .
there was a higher mean ( sd ) number of visits in the first year ( 10.4 [ 2.2 ] ) than in the second year ( 8.0 [ 3.3 ] ) .
similar findings were observed for the mean ( sd ) number of visual acuity tests ( 10.0 [ 2.4 ] in year 1 and 7.8 [ 3.3 ] in year 2 ) and oct tests ( 9.2 [ 2.4 ] in year 1 and 7.4 [ 3.0 ] in year 2 ) ( figure 4a ) . patients from the uk received a mean ( sd ) of 9.0 ( 4.7 ) injections during the full 2-year period ( 5.8 [ 2.4 ] in year 1 and 3.2 [ 2.8 ] in year 2 ) ( figure 4a ) .
there were more visits , tests , and injections in the uk compared with the global population during year 1 and year 2 ( figure 4b and c ) .
the mean number of injections in the uk compared with the global population ( excluding the uk ) was 5.8 versus 4.8 in year 1 and 3.2 versus 2.0 in year 2 . in the global cohort
, 80.2% ( n=1,786/2,227 ) completed a 3-month loading phase , and 19.9% ( 355/1,786 ) of these patients were from the uk .
the mean ( sd ) time from the end of the loading phase to the next injection ( maintenance phase ) was 102.8 ( 121.4 ) days in the uk and 134.0 ( 141.2 ) days in the global cohort ( excluding the uk ) .
the mean ( sd ) duration between visits in the maintenance phase was 42.1 ( 11.7 ) days in the uk compared with 59.3 ( 51.0 ) in the global cohort ( excluding the uk ) .
the mean ( sd ) duration between injections in the maintenance phase was 100.0 ( 80.3 ) in the uk cohort versus 104.1 ( 98.7 ) days in the global cohort ( excluding the uk ) .
a total of 365 patients ( 89.0% ) completed the observation period ( august 31 , 2011 ) .
the remaining patients ( n=45 ; 11.0% ) discontinued the study prior to this for a number of reasons , including permanent discontinuation of anti - vegf ( n=34 ; 8.3% ) ( due to treatment failure [ n=22 ] , patient decision [ n=2 ] , stable disease [ n=6 ] , and other reason [ n=4 ] ) , change of treating physician ( n=5 ; 1.2% ) , withdrawal by patient ( n=1 ; 0.2% ) , lost to follow - up ( n=1 ; 0.2% ) , other reason ( n=3 ; 0.7% ) , and missing ( n=1 ; 0.2% ) .
patients from aura who were diagnosed with namd and treated with ranibizumab by their own ophthalmologists according to routine practice were retrospectively monitored to determine long - term effectiveness of ranibizumab from a uk perspective . compared with the global population ,
the uk - specific outcomes were better and may have been achieved by higher monitoring and retreatment rates .
also , most patients in the uk ( 86.6% ) received a loading phase ( ie , three injections within 90 days ) , which is more than was observed in the global population ( 80.2% ) .
the improvement in visual acuity was higher in patients who received a loading phase in both populations , but there was still decline over time , which was more marked in the global population .
the mean number of ophthalmology visits in years 1 and 2 was 10.4 and 8.0 in the uk compared with 8.6 and 4.9 in the global population , respectively .
the mean number of injections was also higher in the uk ( 5.8 in year 1 and 3.2 in year 2 ) compared with the global population ( 5.0 in year 1 and 2.2 in year 2 ) .
these findings reflect how ranibizumab injection frequency and long - term monitoring affect visual outcomes in real - life , and are an indication of how the uk situation compares with the outcomes from other countries . although less frequent ranibizumab dosing is not as effective as monthly dosing , four to five injections in the 9 months following a 3-month loading phase is still expected to maintain visual acuity based on clinical study outcomes .
the catt study showed that the mean change in visual acuity was + 8.8 letters with monthly ranibizumab ( mean number of injections was 22.4 ) and + 6.7 letters with prn ranibizumab ( mean number of injections was 12.6 ) over a 2-year period.14 in the anchor study , ranibizumab 0.5 mg monthly was associated with a mean change in visual acuity of + 11.3 letters in year 1 and + 10.7 letters in year 2.2,15 the corresponding values in the marina study were + 7.2 letters in year 1 and + 6.6 in year 2.3 in comparison , the current observational study showed that the mean change in visual acuity was + 6.8 letters in year 1 and + 4.8 letters in year 2 ( with a loading phase ) , with an average of nine injections over a 2-year period .
the emr study , a national namd database study of ranibizumab in the uk that included information from 11,135 patients ( 12,951 eyes ) and more than 300,000 clinic visits showed that mean visual acuity only improved by + 2 letters at the end of year 1 and by + 1 letter at the end of year 2 ( the mean baseline visual acuity was 55 letters).16 however , the median number of visits and injections were similar to those observed in the current study ( 9.2 and five in year 1 and 8.2 and four in year 2 , respectively ) .
the differences in the improvement in visual acuity between the present study and the national amd database study may be attributed to differences in the clinical characteristics of the sites providing data for the two studies and/or the exclusion criteria applied in aura , as 40.4% of the cases were excluded from the original screened sample in the present study , contributing to selection bias . a comparison of the uk results with the other individual countries in the aura study ( figure 1c ) showed notable differences in visual acuity over time , including better outcomes with the uk cohort . in the uk ,
treatment of namd with appropriate imaging is undertaken to specified standards that are agreed with the local national health service provider , while in other countries , there may be limits in terms of the numbers of treatments that may be administered , and monitoring may also occur less frequently .
the national institute for health and care excellence criteria17 may have also helped select the most appropriate patients for treatment , based on guidance ta155 , to receive intravitreal anti - vegf injections : patients must have best - corrected visual acuity between 6/12 and 6/96 , with no permanent structural damage to the central fovea .
royal college of ophthalmologists clinical guidelines also specify the need for frequent monitoring and appropriate treatment.6 uk clinical commissioning groups conduct regular audits , and it is likely that this results in improved adherence with protocols .
consequently , monthly monitoring and relatively frequent treatment are likely to be more prevalent in the uk , resulting in better functional outcomes compared with other countries .
it was moderately sized , employed a stringent approach to extraction of data ( ensuring that all clinical information was captured at every visit ) , and involved multiple centers , which were geographically dispersed throughout the uk .
it may also better reflect real - world outcomes than randomized studies , and could help inform policy and provide benchmarks for uk audits .
limitations include its retrospective nature , observational design , use of different methods for assessing visual acuity outcomes ( although they were standardized ) , and its conduct between 2009 and 2011 ; it is likely that with increasing experience of anti - vegf therapy and the addition of other therapeutic agents , treatment of namd will have changed in recent years .
the study also used an locf approach , and thus excluded data from patients with poorer or declining visual outcomes ( ie , those who discontinued early due to treatment failure or for reasons not stated ) .
however , observational data ( also reported in the global paper ) showed a similar pattern in visual acuity outcomes compared with the locf analysis . in the uk cohort ,
visual acuity changed from 55.0 letters ( n=408 ) at baseline to 62.1 letters ( + 7.1 ) at year 1 ( n=263 ) and to 60.8 letters ( + 5.8 letters ) at year 2 ( n=209 ) using observational data ( data not shown ) .
if patients had bilateral disease , then the eye with the worst visual acuity was included into the analysis . in the uk cohort ,
there are also limitations associated with evaluating the uk subgroup independently of the global population .
one of the major drawbacks of the uk aura cohort is that the 13 centers that were included may not be representative of uk practice as a whole .
it is likely that only better - organized centers with excellent record keeping and adequate research staff were recruited to the study .
the relatively good results seen with ranibizumab in the uk cohort does not exclude the possibility that results with ranibizumab over the whole of the uk may be closer to those seen in larger real - world studies such as the emr study , which found that real world outcomes did not match the results in randomized studies.16 the ongoing luminous study ( nct01318941 ) , which includes patients from a larger number of uk centers , may also provide additional information from a real - world perspective . in summary , visual outcomes in real - life clinical experience in the uk cohort of aura were only slightly lower than those initially observed in clinical studies such as catt and marina,3,14 but there was still some decline over time .
this study also showed that anti - vegf agents are effective in patients with poor baseline visual acuity , which is similar to what was reported in another uk study.18 the findings in the uk also highlight the role of the loading phase with ranibizumab treatment .
this is consistent with results observed in interventional studies with ranibizumab and bevacizumab.1921 however , the reasons for this are not fully understood , and may be related to a preconditioning effect of the loading phase , resulting in modification of subsequent disease course .
overall , our results demonstrate that good outcomes are achievable in real - life settings , particularly if high quality control by payers ( such as the situation in the uk ) means that physicians adhere to protocols for monthly monitoring ( including injections being around or above seven in the first year ) ; however , this is associated with high resource utilization and increased burden for patients and health care providers , and may depend on reimbursement . in countries where monitoring and treatment were less frequent , and audits and funding were limited , visual outcomes were poorer and did not reflect the changes seen in clinical studies . further research into optimal treatment regimens and alternative therapeutic options may help to further improve patient outcomes in real - life settings .
approval from the country - specific independent ethics committees or institutional review boards was received based on the requirements of local law and/or regulations , as follows : uk : nres committee north west greater manchester west ; france : the authors advise that in france only a data privacy committee is involved , as no institutional review board or ethics committee is involved for observational studies ; canada : the st .
michael s hospital research ethics board , institutional review board services ( irb services ) , university health network research ethics board , the university of western ontario research ethics board for health sciences research involving human subjects ( hsreb ) ; germany : ethik kommission , medizinische fakultt bonn , biomedizinisches zentrum , ( first approval in germany ) ; netherlands : ethical committee of catharina hospital in eindhoven ; venezuela : centro nacional de biotica ( cenabi ) , iec del hospital militar , iec del centro mdico de ojos ; italy : comitato etico per la sperimentazione , ospedale luigi sacco , azienda ospedaliera polo universitario ;
ireland : research ethics committee , health service executive , south east area ( this was the bigger site ) , mater misericordiae university hospital and mater private hospital research ethics committee .
timeline for study design . abbreviations : fpfv , first patient , first visit ; lpfv , last patient , first visit ; lplv , last patient , last visit ; vegf , vascular endothelial growth factor . | purposeaura was an international , retrospective , observational study that monitored the real - life use and effectiveness of ranibizumab injections in patients with neovascular age - related macular degeneration ( namd ) .
this paper reports the findings from the uk.methodspatients who started treatment with ranibizumab between january 1 , 2009 , and august 31 , 2009 , and had documented follow - up to the end of their treatment and/or monitoring or until august 31 , 2011 , were retrospectively monitored ; the diagnosis and subsequent decision to treat was made by the patient s own physician .
assessments included the change in visual acuity ( standardized letter count ) during the first and second years after start of ranibizumab therapy and resource utilization.resultsfour hundred and ten patients from 13 uk centers were analyzed .
the mean ( standard deviation [ sd ] ) letter score at baseline was 55.0 ( 17.8 ) . the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 and + 4.1 ( 16.9 ) at year 2 .
most of the patients ( 86.6% ) completed a 3-month loading phase ; the visual improvements were numerically higher in these patients .
over 2 years , the mean ( sd ) number of clinic visits and injections was 18.4 ( 5.0 ) and 9.0 ( 4.7 ) , respectively .
resource use and visual acuity gains were greater than those observed in the global population , which included other countries enrolled in aura ( canada , france , germany , ireland , italy , the netherlands , and venezuela ) .
when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was also higher for the uk than for the global population across all subgroups.conclusionmonitoring and treatment rates were high in the uk , resulting in better visual acuity outcomes compared with other included countries .
this suggests that translation of clinical study outcomes into real - life settings is achievable , but at the expense of higher resource utilization than is currently the norm in most developed countries . | Introduction
Materials and methods
Study design and participants
Assessments
Statistical analysis
Results
Participants
Change in visual acuity over time
Resource utilization
Discontinuations (UK cohort)
Discussion
Ethical approval
Supplementary material |
implant - supported oral rehabilitation has provided predictable treatment outcomes and elevated the success rate of selected clinical cases in the anterior maxilla.1 however , implant rehabilitation is a complex , challenging , and meticulous procedure for oral surgeons in patients with an unfavorable periodontal biotype associated with extensive bone defects and skeletal anteroposterior discrepancies.23 in this context , facial profile , dental occlusion , esthetic arrangement of the teeth , systemic alterations , and qualitative and quantitative assessments of available soft and hard tissues are important aspects that must be evaluated to achieve successful implant rehabilitation.4567 from an esthetic standpoint , interdisciplinary treatment planning plays an important role in the clinical success of dental implants.189 under normal conditions , osseointegrated implants must be properly sized and correctly positioned by following the rules of restorative - driven three - dimensional ( 3d ) placement into the receptor areas.10 this enables an optimal gingival contour , the presence of papillae , adequate gap dimensions , and decreased buccal bone resorption.18910 improper implant placement may result in an undesirable loss of bone tissue , leading to mucogingival , biological , and biomechanical problems.1112131415 slow orthodontic forces along both the mesiodistal and vertical teeth axes are viable alternatives to 3d reconstructive procedures instead using bone grafts.16171819 orthodontic movement , especially forced orthodontic eruption ( foe ) , increases the amount of attached gingiva and buccal bone , improving the 3d topography of the periodontal apparatus.161718192021 moreover , in patients with an unfavorable facial pattern caused by anteroposterior involvement , orthodontic movement is limited by the lack of a natural anterior protrusion on the alveolar ridge .
one alternative for the correction of such sagittal discrepancies is orthognathic surgery.22 when attempting to improve skeletal malocclusion , increase patient satisfaction , and enhance esthetic outcomes , an interdisciplinary approach is essential for a more stable , predictable , and successful result .
thus , the aim of this clinical report is to present a complex oral rehabilitation procedure designed to meet the patient 's functional and esthetic expectations that involved orthodontic movement , foe , implant placement , orthognathic surgery , and prosthetic rehabilitation .
a 25-year - old caucasian woman was referred to the department of oral implantology for treatment of her anterior maxillary region .
when she was 12 years old , she suffered trauma leading to the avulsion of her central incisors with no possibility of replantation .
five years later , she underwent orthodontic compensation followed by implant replacement of her central incisors .
eight years after implant placement and prosthetic rehabilitation , the patient expressed dissatisfaction with the esthetic outcome of her previous rehabilitation because of gingival recession and marginal bone loss .
she had no significant medical history , was a nonsmoker , and denied consuming alcohol .
the initial facial examination revealed a class iii skeletal pattern with moderate maxillary deficiency ( figure 1 ) .
the occlusal relationship was class i bilaterally , and markedly compensated with an end - to - end bite and reduced overjet and overbite .
a clinical examination showed 2 osseointegrated implants in the central incisor region with a marked collapse of peri - implant tissues , extensive gingival recession , absence of keratinized gingiva , and 5 mm of probing depth .
moreover , prosthetic abutments and the cervical portions of the implants were exposed to the oral environment ( figure 2 ) .
panoramic ( figure 3a ) and periapical radiographs ( figure 3b - e ) showed advanced vertical bone resorption in the implant region , with the first 3 threads exposed on the left implant .
furthermore , the left implant was in intimate contact with the left lateral incisor , violating the minimum distances established to optimize esthetics , and thus hindering the implant 's maintenance .
an analysis of lateral cephalometric radiography confirmed an increased maxillomandibular discrepancy with marked compensation of the anterior teeth and increased labial angulation of the implants ( figure 3f ) .
the following objectives were established : removal of the dental implants from the central incisor area ; orthodontic decompensation and foe of the lateral incisors followed by implant placement ; establishment of a class ii molar relationship on both sides where the mesiobuccal cusp of the upper first molar is anterior to the mesiobuccal groove of the lower first molar , with the mesiobuccal cusp resting between the first mandibular molars and second premolars ; facial skeletal profile improvement with orthognathic surgery ; and definitive prosthetic rehabilitation .
based on clinical and radiographic examinations , we suggested two treatment plans : the first included removal of the implants , autogenous block bone graft procedure to increase the buccal bone width , lateral incisors extraction and implant placement followed by orthognathic surgery .
the second included a comprehensive treatment protocol with 5 operatory stages : ( 1 ) removal of the osseointegrated implants ; ( 2 ) combined orthodontic decompensation with mesial displacement and foe of the lateral incisors in an attempt to induce soft and hard tissue formation , thus reducing the edentulous space and favoring the placement of 2 new implants without the need for bone grafting ; ( 3 ) extraction of the lateral incisors and placement of 2 new implants corresponding to the central incisors , which received provisional crowns ; ( 4 ) orthognathic surgery for maxillary advancement to improve occlusal and facial relationships ; and finally , ( 5 ) orthodontic refinement followed by definitive prosthetic rehabilitation and reshaping of the adjacent teeth .
the first plan required an extensive surgical procedure to graft removal , which imposes heavy physical burdens on the patient , while second could facilitate achievement of treatment objectives with minimum interventions and post - operative discomfort .
the second treatment plan was selected by the patient and written informed consent was obtained prior to initial treatment .
the initial stage of treatment involved removal of the osseointegrated implants and immediate placement of an upper arch fixed appliance . initial alignment and leveling of the upper arch was commenced using 0.4 mm nickel - titanium wires ; brackets were bonded to prefabricated crowns and tied to the arch wire for acceptable esthetics . during the following month , a lower arch fixed appliance ( capelozza prescription , standard i , 0.022 0.028 ; abzil 3 m , so jos do rio preto , brazil ) was set and a leveling wire placed to induce protrusion .
orthodontic therapy involved decompensation as a bridge to orthognathic surgery . within this context , the alveolar defect was partially solved by transposition of the lateral incisors and foe for soft and hard tissue augmentation prior to implant placement .
progressive mesial displacement reduces the upper arch perimeter , which was the goal of orthodontic decompensation of the maxillary arch .
once this goal was achieved , the molars would be in a class ii relationship with the canines taking the places of the lateral incisors , thus requiring reshaping for anatomical acceptability . in the mandible , alignment and leveling
were performed to increase the protrusion of the incisors and the perimeter of the arch , deliberately worsening existing occlusal relationships to make them more consistent with the patient 's facial discrepancies .
orthodontic mesial movement of the lateral incisors was started with 0.45 mm stainless steel wire , with a consequent reduction in the upper arch perimeter . upon completion of this process
, foe was initiated by placing step - down bends , which were gradually increased at each appointment ( figure 4 ) .
the patient was seen every 2 weeks to reduce the incisal surface of the teeth to prevent occlusal interference during extrusive movement .
once orthodontic extrusion was complete after 12 weeks ( figure 5 ) , a cone - beam computed tomography ( cbct ) scan was acquired ( figure 6 ) and acceptable outcomes were achieved in the superior alveolar region with successful canine migration and appropriate prosthetic space , allowing adequate implant installation in a 3d position . at this time ,
the lateral incisors were extracted and 2 osseointegrated implants ( 3.25 11 mm ; biomet 3i nt , palm beach gardens , fl , usa ) were placed ; they remained submerged during the osseointegration period ( figure 7a - c ) .
foe is considered a less invasive approach to restore vertical soft and hard tissues lost as a consequence of dental disease or trauma . in this case ,
foe allowed immediate implant placement with sufficient implant length ( 11 mm ) ; implant threads were sufficiently engaged in the remaining bone , favoring adequate primary implant stability . because of advanced bone resorption in the buccal - palatal direction , we decided to install an implant with a narrow diameter ( 3.25 mm ) .
a narrow diameter implant plays an important role in reducing the rate of vertical bone resorption at the buccal aspect of implants placed in fresh extraction sockets .
after implant placement , a particulate anorganic bovine bone graft ( bio - oss ; geistlich , wolhusen , switzerland ) was applied to the buccal side of the alveolar crest and over the implant to increase buccal bone thickness and reduce the chances of vertical bone resorption postoperatively .
cbct showed correct positioning of the implants in relation to the remaining alveolar bone ( figure 7d ) .
the upper and lower alignment and leveling stage was continued with up to 0.48 0.63 mm stainless steel wires . a dynamic analysis of diagnostic study models with the upper model in the predicted position after maxillary advancement confirmed that the patient was a candidate for orthognathic surgery ( figure 8) .
preoperative lateral cephalometric radiography showed that the labial angulation of the implants was reduced , and the positions of the mandibular incisors were attenuated ( figure 9 ) .
four months postoperatively , the implants received provisional prosthetic crowns fabricated in accordance with the arch morphology created during orthodontic decompensation until she underwent skeletal malocclusion surgery .
orthognathic surgery was performed with advancement of the maxilla to create an appropriate facial profile and occlusal relationship .
after the conclusion of orthodontic treatment , final panoramic , periapical , and lateral cephalometric radiographs were obtained ( figure 10 ) and the wires were bent to enable good occlusal contact ( figure 11 ) . at this time ,
a black triangle was present between the central incisors from interproximal bone loss , which resulted in a papillary height defect .
according to tarnow et al.,23 when the distance from the base of the contact point to the bone crest was > 6 mm , interdental papillae were usually absent .
both appliances were removed after 3 years and 8 months of treatment ( the patient became pregnant , thus increasing the total treatment time ) .
the patient underwent esthetic procedures to correct dental discrepancies i.e. , reshaping of the canines into lateral incisors and the premolars into canines that occurred as a consequence of the tooth migration process ( figure 12 ) .
another approach to improve esthetic outcomes in this case involved decreasing the distance from the prosthesis contact point close to the gingival margin to the bone crest , allowing the papilla to fill almost the entire space between the implants . at the three - year follow - up , facial and occlusal esthetic improvements were achieved with the proposed treatment and the results remained stable , meeting the functional and esthetic expectations of the patient ( figures 13 and 14 ) .
cephalometric descriptions are shown in table 1 for the pre - treatment phase ( t1 ) , after tooth decompensation ( t2 ) , and posttreatment ( t3 ) . in the proposed treatment , osteotomy
was accomplished only in the maxilla with anterior movement and clockwise rotation without any surgical intervention in the mandible . however , rotation of the maxillary plane induced a rotation in the mandibular condyle axis , which resulted in a slight anterior movement of the mandible and concomitant alteration of the mandibular plane .
furthermore , occlusal plane alterations have much greater effects in the anteroposterior region ( incisal region ) than in the symphysis region , as shown in the post - treatment cephalometric tracing ( figure 15 ) only on the right side of the mandible .
pre- and post - treatment cephalometric tracings ( figure 15 ) show maxilla advancement and minimal changes in mandibular position .
implant - based rehabilitation of the anterior region has always been a challenge in dental practice .
the esthetic and functional maintenance of implants with adjacent natural teeth can be particularly difficult.342425 in these cases , appropriate interdisciplinary treatment planning is essential.13 the manipulation of alveolar bone defects in the esthetic zone is another of the major difficulties in implant dentistry , especially when vertical defects are present , which is a particularly common finding in adult patients who have experienced periodontal tissue losses over time.2627 limitations inherent to complex oral rehabilitation include high morbidity associated with reconstructive procedures such as autogenous block bone grafts and osteopromotive techniques.28 these procedures involve major surgical manipulations ( including donor site surgery ) , numerous invasive interventions , and the need for subsequent soft tissue enhancement .
furthermore , final volumes and esthetic outcomes are often unpredictable , despite satisfactory graft progress.2930 in this case , a vertical bone defect was associated with extensive bone resorption in the neighboring teeth , hindering bone graft placement at the original cervical level .
another aspect related to defect etiology that must be taken into account is potentially limited mesiodistal space for implant placement . in the present case , we decided to use mesial orthodontic displacement and foe of the lateral incisors after implant removal to appropriately augment soft and hard tissue prior to implant placement .
these procedures led to the activation of angiogenic growth factors stimulated by mechanical stresses exerted in the alveolar bone that contribute to the formation of new periodontal support tissue , i.e. , gingiva , periodontal fibers , and new bone deposition via osteoblastic activity , favoring implant placement in an appropriate 3d position.1618 the only prerequisite for the adequate application of these procedures is that the apical third of the root should provide an intact fiber apparatus.31 therefore , in an attempt to reduce the edentulous area of the incisors , displacement of the lateral incisors toward the defect and migration of the canines to take the places of the lateral incisors were performed before foe .
moreover , due to considerable periodontal attachment loss in the lateral incisal area , foe alone would not have provided the necessary gain in bone height in the anterior region .
for this reason , we opted to increase the amount of bone by placing particulate bone graft material over the buccal bone , decreasing the amount of vertical bone resorption that occurs after implant placement . after accomplishing these procedures , a substantial reduction in upper arch perimeter
was achieved , favoring implant rehabilitation following all the necessary steps for esthetic restoration , which conserved biologic width and supported implant maintenance .
this change also improved the relationship between the upper and lower arch perimeters , facilitating orthognathic surgery treatment planning .
at the end of this stage , there was a significant sagittal discrepancy , which is an unquestionable indication for surgical correction in patients with a class iii skeletal pattern for improved esthetics and quality of life.32 the maxillary advancement procedure created a divergence between the maxillary and mandibular occlusal planes with intrusion of the anterior segment , which helped alveolar ridge leveling . as the skeletal discrepancy created was insignificant in relation to the patient 's expectations , there was no indication for bimaxillary surgery .
once the provisional crowns were placed , we identified a lack of exposure of the maxillary incisors , which enabled clockwise rotation of the maxilla with anterior extrusion rather than posterior intrusion to correct the lip - teeth relationship .
finally , multiple coronoplasty procedures ( grinding and composite resin augmentation ) were required to optimize the volume and intercuspation .
in conclusion , oral rehabilitation in adult patients requires well - planned , interdisciplinary treatment to ensure better esthetic and functional outcomes . in patients with missing teeth and alveolar bone loss in the anterior region , foe of the teeth adjacent to the edentulous space
may be considered a primary treatment option to induce soft tissue and bone formation and to create more favorable bone and periodontal conditions .
changes in arch perimeter as a result of such orthodontic displacement can cause skeletal discrepancies , which in turn may require surgical correction to improve facial esthetics and the occlusal profile . | the aim of this clinical report is to describe the complex treatment of an adult class iii malocclusion patient who was disappointed with the outcome of a previous oral rehabilitation .
interdisciplinary treatment planning was performed with a primary indication for implant removal because of marginal bone loss and gingival recession , followed by orthodontic and surgical procedures to correct the esthetics and skeletal malocclusion .
the comprehensive treatment approach included : ( 1 ) implant removal in the area of the central incisors ; ( 2 ) combined orthodontic decompensation with mesial displacement and forced extrusion of the lateral incisors ; ( 3 ) extraction of the lateral incisors and placement of new implants corresponding to the central incisors , which received provisional crowns ; ( 4 ) orthognathic surgery for maxillary advancement to improve occlusal and facial relationships ; and finally , ( 5 ) orthodontic refinement followed by definitive prosthetic rehabilitation of the maxillary central incisors and reshaping of the adjacent teeth .
at the three - year follow - up , clinical and radiographic examinations showed successful replacement of the central incisors and improved skeletal and esthetic appearances .
moreover , a class ii molar relationship was obtained with an ideal overbite , overjet , and intercuspation . in conclusion , we report the successful esthetic anterior rehabilitation of a complex case in which interdisciplinary treatment planning improved facial harmony , provided gingival architecture with sufficient width and thickness , and improved smile esthetics , resulting in enhanced patient comfort and satisfaction .
this clinical case report might be useful to improve facial esthetics and occlusion in patients with dentoalveolar and skeletal defects . | INTRODUCTION
DIAGNOSIS AND ETIOLOGY
TREATMENT OBJECTIVES
TREATMENT ALTERNATIVES
TREATMENT PROGRESS
RESULTS
DISCUSSION
CONCLUSION |
ondansetron hcl is a 5-ht3 receptor antagonist indicated for the treatment and/or prophylaxis of postoperative or chemotherapy- or radiotherapy - induced emesis .
the oral bioavailability of ondansetron hcl is almost 59% , and peak plasma about 0.030.04 g / ml is obtained after 1.5 to 2 h of administration . as administering drug through the transdermal route avoids hepatic first - pass metabolism , thus , the delivery of ondansetron hcl to the systemic circulation via the transdermal route would improve its bioavailability . in the previous literature , various attempts of transdermal delivery of ondansetron hcl
transdermal delivery has many advantages over conventional modes of drug administration as it avoids hepatic first - pass metabolism and improves patient compliance .
in addition , it is simple to terminate the therapy , if any adverse or undesired effect occurs .
however , skin is a natural barrier , and only a few drugs can penetrate the skin easily and in sufficient quantities to be effective .
in recent years , numerous drug penetration enhancement techniques were studied through the transdermal route [ 9 , 10 ] . among them , one of the most promising techniques for enhancement of transdermal drug permeation is the microemulsion technique [ 1116 ] .
microemulsions as colloidal carriers are one of the promising systems that have nowadays attracted the main interest in penetration enhancement .
they are optically isotropic , transparent , and thermodynamically stable homogeneous solutions of oil and water , stabilized by addition of a surfactant and usually a cosurfactant [ 13 , 14 ] . due to their special features
, microemulsions offer several advantages for the pharmaceutical use , such as enhanced drug solubilisation , good thermodynamic stability , ease of preparation , low viscosity , high drug loading capacity , and small droplet size , which make them promising as a drug delivery tool [ 17 , 18 ] . as a vehicle for transdermal systems
, microemulsions can increase the local or systemic delivery of drugs by different mechanisms [ 15 , 18 , 19 ] .
first , their composition and structure enable them to incorporate a greater amount of drug due to the high solubilizing capacity with increased thermodynamic property toward the skin than various conventional topical formulations .
second , the diffusional barrier of the skin may be modified depending on the composition of microemulsion system .
third , the surfactants and cosurfactants used in microemulsions may reduce diffusional barriers by acting as penetration enhancers . in the current investigation ,
ondansetron hcl microemulsions for transdermal delivery , containing isopropyl myristate or oleic acid as the oil phase , tween 80 as the surfactant , and isopropyl alcohol as the cosurfactant , were formulated and evaluated .
india ) , oleic acid ( qualigens fine chemicals , india ) , isopropyl alcohol ( qualigens fine chemicals , india ) , and tween 80 ( merck specialties pvt . ltd .
the oil ( oleic acid or isopropyl myristate ) and aqueous phases were first combined with the surfactant ( tween 80 ) .
cosurfactant ( isopropyl alcohol ) was added gradually with magnetic stirring at room temperature until the system was transparent .
, these were stirred vigorously for a sufficient length of time and end point ( onset of turbidity or phase separation ) was visually monitored against a dark background by illuminating the samples with a white light .
the experiments were performed in triplicate to check reproducibility . from the end point composition of titrated samples ,
the mass percent composition of the components like oil , surfactant , and water was calculated and plotted on triangular coordinates to construct a pseudoternary phase diagram .
the pseudoternary phase diagrams were constructed to determine the composition of an aqueous phase , an oil phase , and a surfactant : cosurfactant ( 3 : 1 ) phase that will yield microemulsions ( transparent solutions ) at room temperature which were represented in the nondarkened area of the following diagrams ( figure 1 ) . from the microemulsion regions in the pseudoternary phase diagram , the eight different formulas for the development ondansetron hcl w / o microemulsions were selected as shown in table 1 .
ondansetron hcl w / o microemulsions were prepared by mixing candesartan cilexetil to the mixture of oleic acid , tween 80 , and isopropyl alcohol .
an appropriate amount of distilled water was added to the mixture drop by drop while with vigorous stirring using a magnetic stirrer ( remi motors , india ) until the transparent microemulsions were produced .
these microemulsions were allowed to equilibrate with gentle magnetic stirring for 15 minutes , and then these were passed through whatman filter paper ( no . 40 ) .
then the hair was removed from the upper portion of the ear skin using an animal hair clipper , and subsequently full thickness of the skin was harvested .
the fatty layer , adhering to the dermis side , was removed by surgical scalpel .
finally , these excised skins were thoroughly rinsed with distilled water and packed in aluminum foils .
the permeation of formulated all ondansetron hcl microemulsions was carried out using franz diffusion cell .
the cell consists of two chambers , the donor and the receptor compartment with a diffusion area of 0.785 cm .
the excised porcine skin was mounted between the compartments of the diffusion cell with stratum corneum facing the donor compartment and clamped into position .
magnetic stirrer bars were added to the receptor chambers and filled with the receptor phase .
phosphate buffer saline ( pbs ) , ph 7.4 was used as the receptor medium .
the small concentration of sodium azide ( 0.0025% w / v ) was added to prevent any microbial growth .
the entire setup was placed over magnetic stirrer , and the temperature was maintained at 37 0.5c .
the skin sections were initially left in the franz cells for 2 hours in order to facilitate hydration of the skin samples .
after this period , 5 ml of the appropriate formulation was applied onto the surface of the skin . 1 ml of medium was collected from the receptor compartment at predetermined intervals over study period and replaced with the same amount of fresh buffer .
the amount of permeated drug was measured using a uv - visible spectrophotometer ( thermo spectronic uv-1 , usa ) by measuring absorbance at max 248 nm .
the amount of ondansetron hcl from various microemulsions was permeated while porcine skin was plotted against the function of time .
the permeation fluxes for each microemulsions were calculated as the slope divided by the skin surface area [ 15 , 21 ] .
j
ss = ( dq / dt)ss 1/a , where jss is the steady - state permeation flux ( g / cm / hour ) , a is the area of skin tissue ( cm ) through which drug permeation takes place , and ( dq / dt)ss is the amount of drug passing through the skin per unit time at a steady state ( g / hour ) . the data of in vitro ondansetron hcl permeation from various ondansetron hcl microemulsions through porcine skins were evaluated kinetically using various mathematical models like zero order , first order , higuchi , and korsmeyer - peppas model equations .
zero order kinetics : f = kot , where f represents the fraction of drug released in time t and ko is the zero order release constant .
first order kinetics : ln ( 1 f ) = k1 t , where f represents the fraction of drug released in time t and k1 is the first - order release constant .
higuchi model : f = kht , where f represents the fraction of drug released in time t and kh is the higuchi dissolution constant .
korsmeyer - peppas model : f = kpt , where f represents the fraction of drug released in time t and kp is the korsmeyer - peppas release rate constant , and n is the diffusion exponent .
various ondansetron hcl microemulsions were evaluated by centrifugation ( remi motors , india ) at 1250 rpm for a period of 5 hours and then , they were examined for any phase separation .
various ondansetron hcl microemulsions were submitted to a total of 3 complete cycles ; each cycle consisting of 24 hours at 25c followed by 24 hours at 5c .
droplet size , polydispersity index and zeta potential of the best formulation were determined using a laser scattering particle size analyzer ( malvern zetasizer , mal500999 ) .
0.1 ml of the microemulsion was diluted to 10 ml of doubled distilled water to prepare samples for study .
a phase behaviour investigation to develop microemulsion systems is the suitable approach of determining the water phase , oil phase , surfactant / cosurfactant concentrations .
therefore , pseudoternary phase diagrams of investigated microemulsion systems were constructed to determine compositions of microemulsions .
the microemulsion regions in pseudoternary phase diagrams to determine the composition of an oil phase containing oleic acid or isopropyl myristate , an aqueous phase , and a surfactant / cosurfactant ( 3 : 1 ) phase containing tween 80 as surfactant and isopropyl alcohol as cosurfactant for the formulation of hcl microemulsions at room temperature , which were represented in figures 1(a ) and 1(b ) , as non - darkened area . from the microemulsion regions in the pseudoternary phase diagrams ,
eight formulas were selected for the development of ondansetron hcl microemulsions ( table 1 ) . using the composition of selected microemulsion formulas , ondansetron hcl microemulsions were formulated and investigated .
these ondansetron hcl microemulsions were investigated for in vitro skin permeation through excised porcine skin .
the in vitro skin permeation from these formulated microemulsions was sustained over 24 hours ( figure 2 ) .
the amount of ondansetron hcl permeated over 24 hours period was plotted against the function of time .
the permeation fluxes ( g / cm / hour ) for all these microemulsions through the excised porcine skin were determined and presented in table 1 .
this result showed higher permeation profile ( with the highest permeation flux of 0.284 0.003 g / cm / hour ) for microemulsion f-8 , which contained 10% of isopropyl myristate as oil phase , 8% of aqueous phase , and 82% of surfactant phase containing tween 80 and isopropyl alcohol , 3 : 1 .
the permeation flux of ondansetron hcl microemulsions containing oleic acid as oil phase was comparatively lower than that of microemulsions containing isopropyl myristate as oil phase , when all other excipients were the same .
it was also apparent that the ondansetron hcl permeation was increased with the increase in the amount of surfactant phase and aqueous phase in their composition .
surfactants can loosen or fluidize the lipid matrix of the stratum corneum the principal diffusional barrier of the skin and act as skin permeation enhancer .
in addition , other components such as isopropyl myristate or oleic acid , which were used as oil phase in these formulated microemulsions , have the capacity as skin permeation enhancers and they could add the skin permeation enhancement of ondansetron hcl from formulated microemulsions . in order to predict and correlate the in vitro ondansetron hcl permeation behavior from ondansetron hcl microemulsions through excised porcine skin , the in vitro permeation data were evaluated kinetically using various mathematical models like zero - order , first - order , higuchi , and korsmeyer - peppas model .
the results of the curve fitting into these above - mentioned models indicate the in vitro ondansetron hcl permeation behavior of ondansetron hcl microemulsions ( f-1 to f-8 ) ( table 2 ) .
when respective correlation coefficients were compared , it was found that all these microemulsions followed the korsmeyer - peppas model ( r = 0.971 to 0.998 ) , over a period of 24 hours as a best fit amongst all other models investigated .
again , the korsmeyer - peppas model was employed in the in vitro ondansetron hcl permeation behavior analysis of these formulations to find out permeation mechanisms : fickian ( nonsteady ) diffusional release when n 0.5 , case - ii transport ( zero - order ) when n 1 , and non - fickian anomalous release when the value of n is between 0.5 and 1
. the determined values of diffusion exponent ( n ) ranged between 0.529 and 0.602 ( table 2 ) .
these results indicated that the drug permeation from these ondansetron hcl microemulsions followed the non - fickian , anomalous mechanism .
the stability of all these formulated ondansetron hcl microemulsions was studied by performing centrifuge stress test , and freeze - thaw cycles ( ftc ) test .
after 5 hours of centrifugation at 1250 rpm and 3 complete freeze thaw cycles ( each cycle consisting of 24 hours at 25c followed by 24 hours at 5c ) , all ondansetron hcl microemulsions were found stable as there were no sign of phase separation . from the above results , ondansetron hcl microemulsion formulation f-8 ( 10% of isopropyl myristate as oil phase , 8% of aqueous phase , and 82% of surfactant phase containing tween 80 and isopropyl alcohol , 3 : 1 ) was selected as best formulation based on its higher permeation flux through the excised porcine skin than other microemulsions formulated in this investigation .
the ondansetron hcl microemulsion f-8 was further studied for determination of droplet size , polydispersity index , and zeta potential .
the droplet size of the ondansetron hcl microemulsion f-8 was measured by a laser scattering particle analyzer malvern zetasizer ( mal 500999 ) .
the average droplet diameter ( z - average diameter ) of best microemulsion formulation was 427.53 nm .
the droplet volume distribution curves and number distribution curves of ondansetron hcl microemulsion f-8 were presented in figures 3 and 4 , respectively . due to its small droplet size , its surface areas were assumed high .
therefore , droplets of microemulsion settled down to close contact with the skin providing high concentration gradient and improved permeation of drug from microemulsions .
therefore , the negative zeta potential of microemulsion f-8 might cause an influence in improved drug permeation through the skin due to electrostatic repulsion between the same charge of the skin surface and the microemulsion . the polydispersity index has been found to be 0.35 , which means that the globules are homogeneously distributed .
ondansetron hcl microemulsions were developed using oleic acid or isopropyl myristate as the oil phase , tween 80 as the surfactant , and isopropyl alcohol as the cosurfactant .
all these microemulsions showed sustained drug permeations through the excised porcine skin and followed the korsmeyer - peppas model with non - fickian , anomalous mechanism over a period of 24 hours . among them , this highest permeation flux of 0.284 0.003 g / cm / hour was measured in case of ondansetron hcl microemulsion f-8 ( contained 10% of isopropyl myristate as oil phase , 8% of aqueous phase , and 82% of surfactant phase containing tween 80 and isopropyl alcohol , 3 : 1 ) .
the average droplet diameter , zeta potential , and polydispersity index of the ondansetron hcl microemulsion f-8 were found as 427.53 nm , 11.80 mv , and 0.35 , respectively .
overall , these results showed the promise of ondansetron hcl microemulsions for transdermal delivery in the treatment and/or prophylaxis of postoperative or chemotherapy- or radiotherapy - induced emesis with improved patient compliance . | ondansetron hcl delivery through oral route suffers due to its low bioavailability due to first - pass metabolism .
therefore , the microemulsion - based transdermal delivery may be a better substitute for it .
the pseudoternary phase diagrams were constructed to determine compositions of microemulsions , and ondansetron hcl microemulsions for transdermal delivery were developed using isopropyl myristate or oleic acid as the oil phase , tween 80 as the surfactant , and isopropyl alcohol as the cosurfactant evaluated for in vitro skin permeation through excised porcine skin .
the in vitro skin permeation from these formulated microemulsions was sustained over 24 hours .
the microemulsion f-8 ( contained 10% of isopropyl myristate as oil phase , 8% of aqueous phase , and 82% of surfactant phase containing tween 80 and isopropyl alcohol , 3 : 1 ) showed the highest permeation flux of 0.284 0.003 g / cm2/hour .
all these microemulsions followed the korsmeyer - peppas model ( r2 = 0.971 to 0.998 )
with non - fickian , anomalous mechanism over a period of 24 hours . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion |
a large number of patients with depression have incorrect and negative views not only toward antidepressants but depression itself.16 the most common misperception among these patients is that depression is caused only by nonbiological or environmental factors , such as stress or family problems.79 patients also believe that antidepressants are addictive,2,10,11 that they can alter the patient s personality,12,13 that fewer tablets can be taken on days when one feels better , and that extra tablets can be taken on days when they feel depressed.12 a significantly negative correlation has been found between patients attitudes and beliefs toward depression and antidepressants and the percentage of days medication was missed .
therefore , subjects with negative attitudes missed their medications more frequently.11,12,14 in fact , research has shown that compared to side effects and demographics , patients beliefs about a disease and its perceived controllability or consequences had a greater influence on patients adherence rates.15,16 indeed , it is also one of the few alterable characteristics that can be targeted by clinicians in programs to increase adherence rates.12,15,1719 cultural differences in beliefs about diseases have resulted in different perceptions and ways in which people from different cultures seek treatment.8,2022 givens et al conducted a cross - sectional internet survey where responses were rated using a 5-point likert scale .
the study involved 68,319 caucasians , 3,596 african americans , 2,794 asians / pacific islanders , and 3,203 hispanics .
results revealed that african americans , asians / pacific islanders , and hispanics were less likely than caucasians to attribute depression to biological factors or to believe that medicines were effective in treating depression , believing instead in nonpharmacological options , such as counseling and prayer .
other than that , a higher percentage believed antidepressants were addictive compared to caucasians.23 in a cross - sectional study utilizing the beliefs about medication questionnaire general scale and sensitive soma scale involving 83 asians and 417 europeans , horne et al found that asians had more negative views about medication compared to the western population , and believed that prolonged use could lead to harm and addiction.21 a telephone survey by cooper et al involving 659 caucasians , 97 african americans , and 73 hispanics found that hispanics may be more concerned about the side effects of antidepressants than caucasians.24 it is therefore important to determine the cultural aspects that influence the beliefs and attitudes of the patients , so that these erroneous beliefs can be addressed in an appropriate manner.3,12,15 most studies however usually involve the western population , which makes generalizability to the asian population inaccurate ; particularly those in the southeast asian region .
there seems to be a false assumption that individuals on one end of the world would share similar health beliefs with individuals on the other end of the world simply because of a shared sex.25 as such , this study sought to elicit the attitudes and beliefs of patients toward antidepressants and depression , and to assess the influence of ethnicity on patients attitudes and beliefs .
this study was part of the pharmacy - managed adherence program study , a 6-month randomized prospective study designed to investigate the effect of the program on adherence levels .
the study involved patients with chronic depression being followed up at the outpatient clinic of the psychiatric department of a government - run hospital in malaysia.26 results reported here represent baseline data from the study .
all patients who were diagnosed with major depressive disorder according to the fourth edition of the diagnostic and statistical manual of mental disorders , regardless of severity , and who had been on antidepressants for a minimum of 6 months were included in the study .
sample size was calculated for a type i error of 0.05 and a power of 0.80 . in order to detect an absolute difference of 25% in the proportion of patients judged to have adequate adherence and taking into account a predicted dropout rate of 30% , the sample size needed was 160 patients .
the following patients were excluded : patients with a comorbid psychiatric diagnosis , such as schizophrenia or bipolar disorder during the study period , patients < 18 years of age , patients who were pregnant or breast - feeding , patients with current suicidal ideation or with a terminal illness , patients with dementia , cognitive disabilities , mental retardation , alzheimer s , or parkinson s , patients who did not understand / speak / read english or the national language ( bahasa malaysia [ bm ] ) , and patients who had no telephone . patients who agreed to participate and who signed informed consent forms were asked to fill in the antidepressant compliance questionnaire ( adcq ) , which was used to measure patients attitudes and beliefs toward depression and antidepressants .
demographic data as well as medical and psychiatric history were also obtained from each patient .
the madrs is a clinician - rated scale that consists of ten items , namely apparent sadness , reported sadness , inner tension , reduced sleep , reduced appetite , concentration difficulties , lassitude , inability to feel , pessimistic thoughts , and suicidal thoughts . for each item ,
the rating can range from 0 to 6 , where 0 equals no symptoms and 6 equals severe symptoms .
the total score is then tabulated , and the following scoring system used to determine the patient s severity : 06 indicates recovery , 719 indicates mild depression , 2034 indicates moderate depression , and 35 and above signifies severe depression.28 the time period covered by the scale is the clinical condition of the patient at the time of the interview.29 the scale is available in both the english and bm versions .
the bm version was obtained from the mapi research institute.30 the adcq is a 33-item questionnaire that is divided into four components , namely : perceived doctor
patient relationship ( component 1 ) , preserved autonomy ( component 2 ) , positive beliefs on antidepressants ( component 3 ) , and partner agreement ( component 4 ) .
scoring is as follows : components 1 and 4 : 1 mostly disagree , 2 rather disagree , 3 rather agree , and 4 mostly agree ; component 2 : 1 mostly agree , 2 rather agree , 3 rather disagree , and 4 mostly disagree . in component 3 ,
the items 8 , 23 , 26 , and 31 are scored as follows : 1 mostly agree , 2 rather agree , 3 rather disagree , and 4 mostly disagree , while the other items are scored as follows : 1 mostly disagree , 2 rather disagree , 3 rather agree , and 4 mostly agree .
the higher the score , the more positive the patient s beliefs and attitudes toward antidepressants and depression.31 the scale was translated to bm using brislin s back - translation method , as proposed by the world health organization.32,33 both the english and the bm scales were made available to the patients . face and content validation of the bm version
was conducted by the principal investigator and four psychiatric specialists from the hospital , while a test retest longitudinal design was used to analyze reliability .
continuous variables are expressed by means and standard deviations , whereas categorical / nominal data are presented using frequency and percentage . for translation of the adcq
, internal consistency was analyzed using cronbach s . a measure of 0.70 and above was considered highly related.34 test retest reliability was explored at two time points , 2 weeks apart .
simple linear regression analyses were performed to determine independent influential factors for total adcq scores .
the a priori level of significance was 0.05 , and all analyses were performed using spss 18.0 statistical software ( spss inc , chicago , il , usa ) .
ethical approval was sought and received from the medical research ethics committee ( mrec ) of malaysia ( dlm .
the madrs is a clinician - rated scale that consists of ten items , namely apparent sadness , reported sadness , inner tension , reduced sleep , reduced appetite , concentration difficulties , lassitude , inability to feel , pessimistic thoughts , and suicidal thoughts . for each item ,
the rating can range from 0 to 6 , where 0 equals no symptoms and 6 equals severe symptoms .
the total score is then tabulated , and the following scoring system used to determine the patient s severity : 06 indicates recovery , 719 indicates mild depression , 2034 indicates moderate depression , and 35 and above signifies severe depression.28 the time period covered by the scale is the clinical condition of the patient at the time of the interview.29 the scale is available in both the english and bm versions .
the bm version was obtained from the mapi research institute.30 the adcq is a 33-item questionnaire that is divided into four components , namely : perceived doctor patient relationship ( component 1 ) , preserved autonomy ( component 2 ) , positive beliefs on antidepressants ( component 3 ) , and partner agreement ( component 4 ) .
scoring is as follows : components 1 and 4 : 1 mostly disagree , 2 rather disagree , 3 rather agree , and 4 mostly agree ; component 2 : 1 mostly agree , 2 rather agree , 3 rather disagree , and 4 mostly disagree . in component 3 ,
the items 8 , 23 , 26 , and 31 are scored as follows : 1 mostly agree , 2 rather agree , 3 rather disagree , and 4 mostly disagree , while the other items are scored as follows : 1 mostly disagree , 2 rather disagree , 3 rather agree , and 4 mostly agree .
the higher the score , the more positive the patient s beliefs and attitudes toward antidepressants and depression.31 the scale was translated to bm using brislin s back - translation method , as proposed by the world health organization.32,33 both the english and the bm scales were made available to the patients . face and content validation of the bm version
was conducted by the principal investigator and four psychiatric specialists from the hospital , while a test retest longitudinal design was used to analyze reliability .
the madrs is a clinician - rated scale that consists of ten items , namely apparent sadness , reported sadness , inner tension , reduced sleep , reduced appetite , concentration difficulties , lassitude , inability to feel , pessimistic thoughts , and suicidal thoughts . for each item ,
the rating can range from 0 to 6 , where 0 equals no symptoms and 6 equals severe symptoms .
the total score is then tabulated , and the following scoring system used to determine the patient s severity : 06 indicates recovery , 719 indicates mild depression , 2034 indicates moderate depression , and 35 and above signifies severe depression.28 the time period covered by the scale is the clinical condition of the patient at the time of the interview.29 the scale is available in both the english and bm versions .
the adcq is a 33-item questionnaire that is divided into four components , namely : perceived doctor
patient relationship ( component 1 ) , preserved autonomy ( component 2 ) , positive beliefs on antidepressants ( component 3 ) , and partner agreement ( component 4 ) .
scoring is as follows : components 1 and 4 : 1 mostly disagree , 2 rather disagree , 3 rather agree , and 4 mostly agree ; component 2 : 1 mostly agree , 2 rather agree , 3 rather disagree , and 4 mostly disagree . in component 3 ,
the items 8 , 23 , 26 , and 31 are scored as follows : 1 mostly agree , 2 rather agree , 3 rather disagree , and 4 mostly disagree , while the other items are scored as follows : 1 mostly disagree , 2 rather disagree , 3 rather agree , and 4 mostly agree .
the higher the score , the more positive the patient s beliefs and attitudes toward antidepressants and depression.31 the scale was translated to bm using brislin s back - translation method , as proposed by the world health organization.32,33 both the english and the bm scales were made available to the patients .
face and content validation of the bm version was conducted by the principal investigator and four psychiatric specialists from the hospital , while a test retest longitudinal design was used to analyze reliability .
continuous variables are expressed by means and standard deviations , whereas categorical / nominal data are presented using frequency and percentage . for translation of the adcq
, internal consistency was analyzed using cronbach s . a measure of 0.70 and above was considered highly related.34 test retest reliability was explored at two time points , 2 weeks apart .
simple linear regression analyses were performed to determine independent influential factors for total adcq scores .
the a priori level of significance was 0.05 , and all analyses were performed using spss 18.0 statistical software ( spss inc , chicago , il , usa ) .
ethical approval was sought and received from the medical research ethics committee ( mrec ) of malaysia ( dlm . kkm / nihsec / o8/0804/p10 - 79 ) .
a total of 104 patients met the selection criteria and were administered the study instruments ( table 1 ) .
all patients , who were on once - daily dosing , were on only one antidepressant , except for two patients who were on two antidepressants .
retest analysis found a significant correlation for the bm version of the adcq ( r=0.75 , p=0.013 ) .
simple linear regression analyses revealed that total adcq scores were significantly predicted by chinese ethnicity ( table 3 ) .
scores for the first two columns ( mostly disagree and rather disagree ) were summed up to give a composite picture of what patients disagreed on , while the scores for the last two columns ( rather agree and mostly agree ) were totaled to give a composite picture of what patients agreed on , as was done in a previous study.31 on component 1 ( perceived doctor
patient relationship ) , more than 80% felt that their doctor listened properly to what they thought about antidepressants and stressed the importance of taking their antidepressants regularly , while 77% agreed that their doctor provided sufficient psychological support and encouragement . on component 2 ( preserved autonomy ) ,
more than 40% of patients believed that antidepressants were addictive , that the body could become immune to antidepressants , and that skipping a day now and again could prevent the body from becoming immune to them ; 59% believed it would be difficult to stop taking antidepressants when they have been taken over a long period of time , and a further 67% believed that antidepressants could alter one s personality . on component 3 ( positive beliefs on antidepressants ) ,
more than 30% of patients believed it was appropriate to take more tablets than prescribed on days when they felt more depressed , while 60% believed it was okay to take fewer tablets on days when they felt better .
sixty six percent believed that antidepressants helped solve their emotional problems , and helped them worry less .
meanwhile , 52% felt that depression was only due to factors associated with their personality . on component 4 ( partner agreement )
, approximately 90% believed that antidepressants could correct the changes that occurred in their brain due to stress or problems .
a total of 104 patients met the selection criteria and were administered the study instruments ( table 1 ) .
all patients , who were on once - daily dosing , were on only one antidepressant , except for two patients who were on two antidepressants .
retest analysis found a significant correlation for the bm version of the adcq ( r=0.75 , p=0.013 ) .
simple linear regression analyses revealed that total adcq scores were significantly predicted by chinese ethnicity ( table 3 ) .
scores for the first two columns ( mostly disagree and rather disagree ) were summed up to give a composite picture of what patients disagreed on , while the scores for the last two columns ( rather agree and mostly agree ) were totaled to give a composite picture of what patients agreed on , as was done in a previous study.31 on component 1 ( perceived doctor
patient relationship ) , more than 80% felt that their doctor listened properly to what they thought about antidepressants and stressed the importance of taking their antidepressants regularly , while 77% agreed that their doctor provided sufficient psychological support and encouragement . on component 2 ( preserved autonomy )
, more than 40% of patients believed that antidepressants were addictive , that the body could become immune to antidepressants , and that skipping a day now and again could prevent the body from becoming immune to them ; 59% believed it would be difficult to stop taking antidepressants when they have been taken over a long period of time , and a further 67% believed that antidepressants could alter one s personality . on component 3 ( positive beliefs on antidepressants ) ,
more than 30% of patients believed it was appropriate to take more tablets than prescribed on days when they felt more depressed , while 60% believed it was okay to take fewer tablets on days when they felt better .
sixty six percent believed that antidepressants helped solve their emotional problems , and helped them worry less .
meanwhile , 52% felt that depression was only due to factors associated with their personality . on component 4 ( partner agreement )
, approximately 90% believed that antidepressants could correct the changes that occurred in their brain due to stress or problems .
the mean total adcq score was 96.95 , which was slightly less than that reported by chakraborty et al who found a total mean score of 105.06.12 as there is no official scale for comparison proposed by the scale designers , we can surmise that the overall attitudes and beliefs of our patients toward depression and antidepressants were quite positive , given that the total score for all 33 items of the adcq would amount to 132 .
the majority of our respondents were female and young adults , and approximately 70% of those unemployed were housewives .
these three characteristics could somewhat explain the more positive attitude based on results from previous studies.3,12,21 simple linear regression analyses found that chinese patients had significantly more negative attitudes and beliefs toward depression and antidepressants compared to malay and indian patients . in the chinese culture , similar to that seen in south koreans , mental illnesses are viewed as shameful not only to the individual but to the family as well .
jang et al noted that asians of chinese descent tend to follow confucian ethics , which state that self - concealment of emotional trouble is a virtue.3 therefore , patients tend to tolerate the disease internally and suppress their emotions , instead of seeking treatment . in our study , it was also observed that malay patients had more positive attitudes and beliefs toward depression and antidepressants .
malaysia is a multiracial country made up of 60% malays , more than 20% chinese , and less than 10% indians . by constitutional law ,
the more religious patients had significantly lower suicide ideation scores , and this was most pronounced in muslims.35 therefore , we can postulate that greater religiosity in malay patients compels them to seek treatment , as suicide is prohibited in islam.36 studies have also shown that chinese and indian patients prefer traditional therapy , such as ayurveda , or sin sehs , compared to western medication ; which could explain their more negative attitudes and beliefs toward antidepressants as compared to malay patients.3,7,37 in keeping with results found by demyttenaere et al,31 chakraborty et al12 and other studies,38,39 no significant relationship was found between symptom severity and adcq scores .
there was no significant correlation between adcq scores and sex , age , and attending counseling sessions either . on component 1 , the majority of patients gave positive feedback as to the care received from their doctors .
it must however be taken into account that asians tend to have very high respect for doctors and view them almost as gods.12 in answering this section , a few patients expressed concern , as they were worried whether the doctors could view their answers and were concerned about the repercussions ; therefore , results could have been compromised . with regard to component 2
, roughly 60% of respondents believed that as long as they were taking antidepressants , they were not sure if they were actually necessary , and that the medication made them have less control over their thoughts and feelings . in previous studies , patients expressed negative views about antidepressants and felt they reduced their alertness.8,13,31 a large number of patients in our study also believed that antidepressants could alter one s personality , and that one could become immune to the drugs .
indeed , studies have revealed that patients were worried that taking antidepressants on a daily basis could be harmful to the body.6,40 approximately 60% of patients felt that it was difficult to stop taking antidepressants once one had started taking them , and this is true especially with regard to certain antidepressants , such as mirtazapine and venlafaxine , which can not be stopped abruptly , and instead have to be tapered off over several days to weeks .
many patients in our study also believed that antidepressants could cause addiction , echoing several studies or reviews where patients either expressed a concern over the potential for addiction with antidepressants or simply stopped their medication completely due to fear of addiction.2,8,13,18,20,41 this is because most people tend to confuse antidepressants with tranquilizers , which have the potential for addiction , since most antidepressants also cause drowsiness.42 in component 3 , the majority of our patients had positive beliefs regarding antidepressants , with more than 80% believing they helped them worry less about their problems and 70% believing they solved their problems .
this is contrary to studies conducted in western populations , as well as studies involving asians living in western countries , where the majority of patients had negative views toward antidepressants.18,21,23,31 western cultures are more individualistic , and place importance on asserting individual independence and autonomy .
eastern cultures , on the other hand , stress that responsibility to the community is greater than duty to oneself .
this could somewhat explain why easterners have more favorable views , as they feel antidepressants will help them maintain the status quo in the community.25 in a paper by horne et al , asians reported less experience with prescribed medication compared to the western population.21 this is not surprising , given that many asians tend to resort first to traditional medicine.3,7,37 however , having more experience with medication could also mean that the western population are more aware of the side effects associated with these medications , and this perhaps has elicited more negative views toward antidepressants .
our results also show that we can not assume similarity with asians living in western countries , as there is the issue of cross - culture influence from their adoptive country .
therefore results obtained from those studies can not necessarily be applied to our population.21,23 a substantial number of patients in this study however seem to have erroneous views with regard to the dosing of antidepressants .
more than 30% believed extra doses could be taken on days when they felt low , and 60% believed it was acceptable to take fewer doses on days when they felt better .
patients tend to go on drug holidays or alter their doses when they feel better , and a study found a significant correlation between patients who stopped their medication when they felt better with perceived stigma toward depression .
this could explain our findings with regard to the cultural stigma , whereby patients want to stop the medication as soon as possible.43 finally , more than 50% of respondents believed that depression was caused by their own personality .
this is similar to other studies , where the majority believed that depression was mainly a psychological problem and not a medical problem.4,13,20,44 this points to the nature of patients who prefer to support only a nonbiological cause of depression , which then causes them to reject biological interventions , ie , antidepressants in moderate - to - severe cases , as they feel they can heal themselves by simply changing their behavior or personality.8 we were unable to reach the target sample size due to the strict inclusion and exclusion criteria with regard to language requirements . as mentioned earlier , patients were rather hesitant to answer the questionnaires for fear that the doctors would see their responses .
given that this study only involved one center where the majority of patients came from low to middle socioeconomic backgrounds , this might somewhat limit the generalizability of the results .
on component 1 , the majority of patients gave positive feedback as to the care received from their doctors .
it must however be taken into account that asians tend to have very high respect for doctors and view them almost as gods.12 in answering this section , a few patients expressed concern , as they were worried whether the doctors could view their answers and were concerned about the repercussions ; therefore , results could have been compromised . with regard to component 2
, roughly 60% of respondents believed that as long as they were taking antidepressants , they were not sure if they were actually necessary , and that the medication made them have less control over their thoughts and feelings . in previous studies , patients expressed negative views about antidepressants and felt they reduced their alertness.8,13,31 a large number of patients in our study also believed that antidepressants could alter one s personality , and that one could become immune to the drugs .
indeed , studies have revealed that patients were worried that taking antidepressants on a daily basis could be harmful to the body.6,40 approximately 60% of patients felt that it was difficult to stop taking antidepressants once one had started taking them , and this is true especially with regard to certain antidepressants , such as mirtazapine and venlafaxine , which can not be stopped abruptly , and instead have to be tapered off over several days to weeks .
many patients in our study also believed that antidepressants could cause addiction , echoing several studies or reviews where patients either expressed a concern over the potential for addiction with antidepressants or simply stopped their medication completely due to fear of addiction.2,8,13,18,20,41 this is because most people tend to confuse antidepressants with tranquilizers , which have the potential for addiction , since most antidepressants also cause drowsiness.42 in component 3 , the majority of our patients had positive beliefs regarding antidepressants , with more than 80% believing they helped them worry less about their problems and 70% believing they solved their problems .
this is contrary to studies conducted in western populations , as well as studies involving asians living in western countries , where the majority of patients had negative views toward antidepressants.18,21,23,31 western cultures are more individualistic , and place importance on asserting individual independence and autonomy .
eastern cultures , on the other hand , stress that responsibility to the community is greater than duty to oneself .
this could somewhat explain why easterners have more favorable views , as they feel antidepressants will help them maintain the status quo in the community.25 in a paper by horne et al , asians reported less experience with prescribed medication compared to the western population.21 this is not surprising , given that many asians tend to resort first to traditional medicine.3,7,37 however , having more experience with medication could also mean that the western population are more aware of the side effects associated with these medications , and this perhaps has elicited more negative views toward antidepressants .
our results also show that we can not assume similarity with asians living in western countries , as there is the issue of cross - culture influence from their adoptive country .
therefore results obtained from those studies can not necessarily be applied to our population.21,23 a substantial number of patients in this study however seem to have erroneous views with regard to the dosing of antidepressants .
more than 30% believed extra doses could be taken on days when they felt low , and 60% believed it was acceptable to take fewer doses on days when they felt better .
patients tend to go on drug holidays or alter their doses when they feel better , and a study found a significant correlation between patients who stopped their medication when they felt better with perceived stigma toward depression .
this could explain our findings with regard to the cultural stigma , whereby patients want to stop the medication as soon as possible.43 finally , more than 50% of respondents believed that depression was caused by their own personality .
this is similar to other studies , where the majority believed that depression was mainly a psychological problem and not a medical problem.4,13,20,44 this points to the nature of patients who prefer to support only a nonbiological cause of depression , which then causes them to reject biological interventions , ie , antidepressants in moderate - to - severe cases , as they feel they can heal themselves by simply changing their behavior or personality.8
we were unable to reach the target sample size due to the strict inclusion and exclusion criteria with regard to language requirements .
as mentioned earlier , patients were rather hesitant to answer the questionnaires for fear that the doctors would see their responses .
this too could have affected the final results obtained . given that this study only involved one center where the majority of patients came from low to middle socioeconomic backgrounds , this might somewhat limit the generalizability of the results .
the majority of patients gave very positive feedback with regard to the care received from the physicians , and a substantial amount also believed in the effectiveness of antidepressants .
however , most patients had erroneous views with regard to the perceived harmful effects of antidepressants .
patients beliefs and attitudes influence their adherence , outcome , and preference for treatment . by understanding patients beliefs about their treatment , as well as the impact of their respective cultures
, clinicians can alter treatment accordingly , either through the dissemination of information or through improved patient prescriber relationships .
more detailed research should also be done to assess the influence of ethnicity on patients attitudes and beliefs , so as to ensure the generalizability of research outcomes . | backgroundmany patients have erroneous views with regard to depression and its management , and it was noted that these attitudes and beliefs significantly affected their adherence rates.objectivesthe primary aim of this study was to determine the attitudes and beliefs of patients with depression toward depression and antidepressants .
a secondary aim was to assess the influence of ethnicity on patients attitudes and beliefs.patients and methodsthe study involved patients with chronic depression being followed up at an outpatient clinic at a government - run hospital in malaysia .
patients attitudes and beliefs were assessed using the antidepressant compliance questionnaire.resultsa total of 104 patients of malay , chinese , and indian ethnic groups met the selection criteria .
chinese patients had significantly negative attitudes and beliefs toward depression and antidepressants compared to malays and indians ( b=-8.96 , t103=-3.22 ; p<0.05 ) .
component analysis revealed that 59% of patients believed that antidepressants can cause a person to have less control over their thoughts and feelings , while 67% believed that antidepressants could alter one s personality ; 60% believed it was okay to take fewer tablets on days when they felt better , while 66% believed that antidepressants helped solve their emotional problems and helped them worry less.conclusionpatients had an overall positive view as to the benefits of antidepressants , but the majority had incorrect views as to the acceptable dosing of antidepressants and had concerns about the safety of the medication . assessing patients attitudes and beliefs , as well as the impact of their respective cultures ,
can be used in tailoring psychoeducation sessions accordingly . | Introduction
Patients and methods
Study instruments
Montgomerysberg Depression Rating Scale
Antidepressant Compliance Questionnaire
Statistical analysis
Ethical approval
Results
Characteristics of study participants
Reliability analysis of ADCQ
Patients attitudes and beliefs
Discussion
Component analysis
Limitations
Conclusion |
oral melanin pigmentation is a frequently encountered clinical condition and has multifactorial etiology which includes genetic factors , tobacco use , drugs such as antimalarials and tricyclic antidepressants , systemic disorders like addison 's disease , endocrine disturbances , albright 's syndrome , malignant melanoma , peutz - jeghers syndrome , trauma and other causes .
smoking tobacco also induces the activation of melanocytes to produce melanin via noxious agents present in the smoke .
the colour of gingiva is determined by a number of factors , including the amount of keratinization , thickness of epithelium , size and number of blood vessels and the presence of various pigments .
melanin pigmentation often occurs on the gingiva as a result of excessive deposition of melanin .
active melanocytes convert tyrosine to melanin and is stored in the melanosomes and is transferred to the keratinocytes via them .
the colour of pigmentation may vary from light brown to black depending upon the localization in the tissues and is dictated mainly by melanoblastic activity .
gingival hyperpigmentation is seen as a genetic trait in some populations and is more appropriately termed as physiologic or racial gingival pigmentation .
high levels of oral pigmentation are normally seen in african , east asian , mediterranean , and hispanic populations .
although melanin pigmentation of the gingiva does not cause any medical problems , it is of esthetic concern for the conscious patient .
demand for cosmetic therapy is made , especially by fair skinned people with moderate or severe gingival pigmentation .
the colour of the gingiva that may cause esthetic concerns is an essential part of overall esthetics parallel to today 's high cosmetic expectations .
numerous techniques have been devised and used for the treatment of gingival hyperpigmentation such as gingivectomy , free gingival grafts , acellular dermal matrix , laser therapy , and electrosurgery and gas expansion cryosurgery .
laser surgery has become the treatment of choice as it improves hemostasis and causes less postoperative discomfort over scalpel surgery .
although cryosurgery does not require suturing or application of dressing as well as having the advantages of less / no post - operative bleeding and no scarring , it has not been used widely due to the expense of cryosurgery instruments and the safety concerns regarding storage of liquid gases .
hence , a new material , a colourless , non inflammable gas , 1,1,1,2 tetrafluoroethane ( tfe ) , used as a coolant for refrigerating systems and electronic circuits and not having the disadvantages of frequently used cryogenic gases has been employed in this study . with a melting point of 101c and a boiling point of 26c
, it is available in a pressurized spray can and immediately evaporate without residue following spraying .
several human and animal toxicology studies have proved the material to be biocompatible with no oncogenic or genotoxic effect on animals .
since hypopigmentation has been reported as a side effect of cryosurgery , the aim of this study is to evaluate the effectiveness of tfe in the treatment of gingival hyperpigmentation .
five systemically and periodontally healthy patients with chief complaint of hyperpigmented gingiva were recruited from the department of periodontology , dr . r. ahmed dental college and hospital , kolkata , for the study . the whole procedure and its effect
were explained to the patients and a written informed consent was obtained from each of them prior to the study .
prior permission from the institutional ethical committee was also obtained . a detailed medical history including smoking habit , systemic diseases associated or not associated with gingival melanin pigmentation , any malignancy , and medications was taken .
evaluation of any skin or perioral pigmentation was done and the gingival hyperpigmentation was carefully assessed . before application of tfe ,
the pigmented area was isolated and air dried immediately after spraying , the temperature of the cotton applicator ranged from 46.7c to 48c .
topical anesthesia with 2% lignocaine spray ( lignocaine hydrochloride and adrenaline bitartarate injection , 2% with adrenaline 1 in 80,000 , icpa health products ltd , 233a , adarsh indl .
estate , sahar road , chakala , andheri ( east ) , mumbai - 400 099 , maharashtra , india ) was used to minimize discomfort [ figures 1 and 2 ] .
tfe ( 134a , refrigerant for ac systems , contains 1.1.1.2-tetrafluoroethane , dupont suva , dupont fluorochemicals , louisville packaging site , 7745 , national turnpike , suite 190 , louisville , ky , usa 40214 ) was sprayed on a cotton swab and immediately rolled gently over the pigmented area and a freezing zone was continuously maintained in each area for about 30 - 40 s [ figures 3 and 4 ] .
after the cryosurgical procedure , patients were examined at 1 , 2 , and 4 weeks [ figures 5 and 6 ] and subsequently after 1 year [ figure 7 ] .
digital photographs of the pigmented area were taken preoperatively and at each subsequent postoperative visit .
[ figures 7 and 8 ] pre - operative photograph freezing zones being maintained for 30 - 40s in the maxillary arch freezing zones being maintained for 30 - 40s in the mandibular arch one week post - operative 4 weeks post - operative post - operative after 1 year pre and post - operative photographs of case 2
slight erythema of the gingiva developed immediately after the procedure . during the next 3 - 4 days
, superficial necrosis became apparent and a whitish slough could be separated from the underlying tissue leaving a clean pink ulcer bed .
the gingiva appeared normal within 1 week and epithelization was complete within 3 - 4 weeks of cryosurgical treatment .
the patients did not suffer from any hemorrhage , infection , or any scar tissue formation .
the initial healing was uneventful following tfe application , patient acceptance of the procedure was good , and only one patient complained of mild pain and discomfort .
the follow - up period was up to 1 year including clinical examination and photographic comparisons .
melanin hyperpigmented gingiva can be an esthetic problem in many individuals especially if it is present on the facial aspect which can be seen during speech and smile , more so if gummy smile is present . in the present study ,
as shown previously , following epithelial destruction by cryosurgery , epithelial migration covered the denuded connective tissue and regenerated rapidly .
cryosurgery is a technique that transfers heat from tissues and is commonly used in dermatological practice .
the melanin is found in basal keratinocytes and subjacent macrophages , and destruction of subjacent cells is sufficient for depigmentation .
minimum temperature needed for cell damage is cell specific , and melanocytes are very sensitive to low temperatures at 4c to 7c where cell death can occur .
physical and chemical changes induced by freezing lead to cell destruction and cell death . with tfe ,
the presence of any residual pigmentation may be diagnosed as early as 1 week postoperatively , and the procedure can be repeated to eliminate any residual pigmentation .
arikan followed the patients through 30 months , including clinical examination and photographs , and reported no recurrence , with the exception of two patients who were heavy smokers ( 20 cigarettes / day ) , who had partial recurrence in small areas near the border of freezing zone at 24 and 30 months , respectively .
of all the techniques used for depigmentation of gingiva , laser surgery has become the treatment of choice but , it may not be used efficiently at the gingival margins and interdental papillary region due to close proximity to the tooth .
this limitation may result in incomplete treatment and recurrence due to migration of melanocytes from the left - over area .
however , yeh reported no repigmentation following cryosurgical treatment during a 2-year follow - up period . in the present study ,
tfe showed successful depigmentation with good patient compliance as the procedure was simple and effective and was well tolerated by all the patients .
in addition , there was no post - operative bleeding and no scar formation . in this procedure
, there is no need of anesthesia , sutures , and dressing which makes it superior to scalpel , laser , and other conventional procedures .
cryosurgical depigmentation of gingiva by tfe is a novel method and can be used with minimum armamentarium and complications . however , to allow for its usage more commonly in routine dental practice , more studies with a large number of patients are warranted .
it can be concluded within the limitations of this study that it is a safe , cost effective , and non - invasive method for the treatment of gingival melanin pigmentation . | to highlight the effect of 1,1,1,2 tetrafluoroethane ( tfe ) , a new material for cryosurgery of gingival melanin pigmentation ( gmp ) .
five patients were treated using a tfe - cooled swab and the pre- and post - treatment photographs were compared .
complete keratinization took place in 3 - 4 weeks after application without any trace of pigmentation . during the follow - up period ,
no side effects were observed and the improved esthetics were maintained upto 1 year . | INTRODUCTION
CASE REPORT
RESULTS
DISCUSSION
CONCLUSION |
rhabdomyolysis is defined as a clinical and biochemical syndrome resulting from the lysis of skeletal muscle cells .
it usually affects healthy individuals following excessive physical activity , trauma , infections , use of medications or illicit drugs and other toxic agent s consumption .
they result due to an inability of muscle cells to produce an adequate amount of energy for their needs because of underlying defects in glucose , glycogen , lipid or nucleoside metabolism .
this heterogeneous group of inherited diseases include disorders of fatty acid oxidation ( fao ) such as very long - chain acyl - coa dehydrogenase ( vlcad ) deficiency .
vlcad is a key enzyme catalyzing the first reaction in the mitochondrial beta - oxidation of long - chain fatty acids with a chain length of 14 to 20 carbons .
this autosomal recessive disorder has a prevalence of about 1:85,000 , and was first identified in 1992 by izai et al .
the human vlcad gene has been identified and located on the short arm of chromosome 17 between bands p11.2 and p11.13105 . since 2004 , portuguese newborn screening program includes this disorder in its panel . until the end of 2012 a total of 738,816 neonates were screened and eight vlcad deficiency cases were identified ( 1:92,352 ) . in this cohort , vlcad deficiency is the second more prevalent fao after medium - chain acyl - coa dehydrogenase deficiency .
there are three phenotypes described , according to the age at onset of clinical manifestations : i ) early infantile - onset cardiac type , a severe form that involves hyperthrophic cardiomyopathy and sometimes leads to sudden death in the early infantile period ; ii ) childhood - onset hypoglycemia type , of moderate - severity childhood onset , that usually presents as a hypoketotic hypoglycemia eventually with hepatomegaly which is induced by fasting or preceding infections ; and iii ) adolescent / adult - onset myopathic type , of mild severity and adolescent or adult onset , that occurs during situations of increased energy demand , e.g. , after physical exercise or fasting and mostly results in muscle weakness or muscle pain that can proceed to severe rhabdomyolysis with highly elevated creatine phosphokinase ( cpk ) concentrations . here
, we describe the clinical , biochemical and molecular findings of a female teen with the late - onset type of vlcad deficiency .
a 13-year old female was admitted for severe , generalized muscle pain and dark urine lasting less than one day . the day before
no history of recent infection or trauma neither intake of herbs , alcohol or other drugs .
no history of previously similar episodes or other relevant medical history , such hypoglycemia , recurrent vomiting , liver disease or coma .
her 19-year old brother had a history of repeated similar episodes of muscle pain and dark urine after physical exertion of moderate intensity since the age of 17 and a hospitalization , one year ago , for rhabdomyolysis , but without further etiologic diagnosis . on admission her temperature was normal . except for tenderness in her muscles with mobilization difficulties ,
215,800 u / l ( normal<167 ) , lactate dehydrogenase 6000 u / l ( normal 240 - 480 ) , myoglobin 30,950 ng / ml ( normal 25 - 58 ) ; and aspartate aminotransferase 4773 u / l ( normal 10 - 50 ) . glucose , urea , creatinine , sodium , potassium , calcium and phosphorous levels were normal .
urinalysis was positive for blood by reagent testing and had 5 - 10 red blood cells per high - power field by microscopy .
influenza a and b , epstein - barr , herpes simplex , parainfluenza , coxsackie , echovirus , adenovirus , human immunodeficiency virus or cytomegalovirus were excluded based on serological studies . based on clinical presentation , family history and laboratory results , the diagnosis of metabolic myopathy with rhabdomyolysis precipitated by exercise was suspected . for further clarification
mmol / l ( normal 0.63 - 2.44 ) , pyruvate 77 mol / l ( normal 54.1 - 119.9 ) , ammonia 77 g / dl ( normal 45 - 80 ) and free carnitine 15
acylcarnitine analysis by tandem mass spectrometry of the patient s dried blood spot disclosed elevated tetradecanoyl carnitine ( c14 ) ( 0.26 m , normal < 0.24 ) , tetradecenoyl carnitine ( c14:1 ) ( 0.26 m , normal<0.18 ) and tetradecadienoyl carnitine ( c14:2 ) ( 0.13 m , normal<0.08 ) , suggesting vlcad deficiency .
this was confirmed on acyl - coa dehydrogenase very longchain ( acadvl ) gene analysis , using standard polymerase chain reaction procedures , with in - house designed primers , to amplify from genomic dna all exons and exon - intron boundaries of this gene .
sequence analysis was performed in an abi prism 3100xl genetic analyzer using the big dye terminator v3.1 cycle sequencing kit ( applied biosystems , inc .
, foster city , ca , usa ) and allowed the identification of a homozygous mutation , p.l500del ( c.1500_1502delcct ) , already described .
analgesic treatment with acetaminophen was started as well as aggressive intravenous hydration in order to avoid acute renal injury .
favorable clinical and biochemical evolution was noted , always with preserved renal function and no electrolytic abnormalities , and she was discharged after five days . despite
having received advice on lifestyle and diet , a second episode of rhabdomyolysis occurred two months later , following a prolonged physical effort , a two hour long soccer game without hydration or food intake during the exercise . between the two episodes she was asymptomatic with no laboratory abnormalities . since the second episode
, patient compliance has improved , and she remains asymptomatic when following a dietary regimen avoiding fasting for more than eight hours overnight , prolonged exercise and a high - carbohydrate , low - fat diet .
her younger brother , though asymptomatic , was studied through acylcarnitine analysis by tandem mass spectrometry of a dried blood spot specimen collected during a period of fasting that showed no alterations .
episodes of exercise intolerance followed by dark urine as a result of rhabdomyolysis , and increased serum cpk activity are symptoms of skeletal muscle disease that can be acquired or inherited
. recognized heritable causes of rhabdomyolysis are defects in the glycogen metabolism and glycolysis , in the respiratory chain or in fatty acid oxidation . within the group of disorders of fatty acid metabolism ,
carnitine palmitoyl - transferase ii deficiency is the most frequently reported condition , but other defects , like vlcad deficiency , have also been described . for the present patient , common causes of rhabdomyolysis
a metabolic etiology , namely a fatty acid metabolism disorder , was suspected given the onset of symptoms after prolonged physical exercise and the suggestive family history , such as parent s consanguinity and brother s similar symptoms .
studies were conducted to confirm this diagnosis and exclude other above mentioned metabolic disorders , also associated to this presentation . the key biochemical test which allowed an accurate diagnosis of vlcad deficiency was the measurement of blood spotted onto filter paper acylcarnitines by tandem mass spectometry that revealed accumulation of tetradecenoyl carnitine ( c14:1 ) .
these biochemical results were sufficiently characteristic to proceed directly to mutation analysis of acadvl , the only gene known to be associated with this disorder .
the molecular basis of vlcad deficiency is very diverse with more than 80 mutations reported in the literature .
we report a known mutation , first described in 2006 which results in deletion of leucine at position 501 of the mature vlcad protein , and are associated with the same phenotype
several studies have addressed the question whether or not it is possible to correlate genotype and phenotype in vlcad deficiency .
in fact the only established relations are the presence of null mutations in severe and early phenotypes and missense mutations in milder and later phenotypes . in patients with two null mutations ,
the complete absence of vlcad activity will affect many tissues , including the heart and liver , resulting in cardiomyopathy , hepatomegaly , and recurrent episodes of metabolic decompensation .
patients with missense mutations or single - amino acid deletion mutations may have sufficient residual vlcad activity , when receiving adequate nourishment , to avoid liver and cardiac symptoms , and may not undertake sufficient sustained exercise , in childhood , to precipitate severe muscle symptoms .
the late onset of disease in adolescent / adult patients could simply be the result of their avoidance to metabolic stress during childhood .
alternatively , it is possible that they have mutations with higher residual enzyme activity than that observed in patients with the mild childhood phenotype , resulting in a difference in tolerance of metabolic stress between the two groups of patients .
although vlcad deficiency may present with severe cardiac involvement during childhood , almost all patients with later presentation , including the case presented here , did not have symptomatic cardiac abnormalities .
treatment of vlcad deficiency is based on dietary modification , with avoidance of longchain fatty acids and eventually supplementation with medium chain triglycerides ( mct ) , so that the enzyme - deficient step can be bypassed .
such diet should reverse most symptoms , although during periods of stress , like exercise , the mct dosage may need to be raised in order to supply the extra energy , thus helping to prevent mobilization of long - chain fatty acids from the adipose tissue .
the diagnosis of the older brother through family screening , stresses the need to consider this group of disorders in similar cases of extreme exercise intolerance and rhabdomyolysis .
in a patient with rhabdomyolysis , and after more common causes are excluded , it is important to consider the possibility of a metabolic disease , in order to provide a quick and adequate diagnosis . in this particular case ,
the history of parent s consanguinity and a brother with similar symptoms help us to have a high index of suspicion of this disorder . after the diagnosis ,
adjustments in life - style and diet are crucial in the prevention of recurrent episodes of metabolic decompensation . | very long - chain acyl - coenzyme a dehydrogenase ( vlcad ) deficiency ( mim 201475 ) is a rare inherited disorder with three forms of clinical presentation : a severe early - onset form ; an intermediate form with childhood onset ; and an adult - onset form , of mild severity . during adolescence and adulthood ,
exercise intolerance , myalgia and recurrent episodes of rhabdomyolysis are the main clinical features .
the authors present a case of a 13-year old female , with severe myalgia and dark urine after prolonged exercise .
analytical evaluation showed marked elevation plasma creatine kinase and myoglobin .
the increased levels of tetradecenoyl carnitine in patient s dried blood spot suggested a vlcad deficiency , which was confirmed by molecular study .
family history is remarkable for first grade consanguinity of parents and a 19-year old brother with records of repeated similar episodes after moderate intensity physical efforts which was subsequently also diagnosed with vlcad deficiency .
this is one of the first cases of late - onset of disease diagnosed in portugal . | Introduction
Case Report
Discussion
Conclusions |
pseudoaneurysm is a rare complication reported after any surgery such as orif of fracture , plate removal , ankle or knee arthroscopy or arthroplasty . to our knowledge ,
we report a case of a 18-year - old male patient who had pseudoaneurysm of posterior tibial artery following plate removal .
a 18-year - old male was referred to our outpatient clinic because of mass - like lesion in his left calf .
he had a history of car accident and left isolated tibia fracture 30 months ago that was treated by open reduction and internal fixation with dcp plate .
after union in radiograph , the plate was removed 7 months ago on the patient request .
finally , pseudoaneurysm was diagnosed and was repaired in a common surgery with a vascular surgeon without any difficulty .
pseudoaneurysms of the posterior tibial artery after tibial plate removal are an extremely rare phenomenon .
this complication in most cases follows penetrating injuries but it has been reported following blunt trauma .
in fact , in most of these patients the diagnosis was initially missed because the large hematoma concealed the underlying pseudoaneurysm and its pulsations .
hence , the surgeon should be suspicious to this complication following plate removal when he / she is doubtful about the sign and symptoms .
implant removal in orthopaedic procedures have more frequently been a subject of controversy [ 1 , 2 ] . some surgeons prefer to remove the implant routinely in skeletally immature patients and also in adults , others do this by patient s request or when complications such as infection , pain , breakage , malunion , delayed or nonunion occurs .
we present a 18-year - old male patient with isolated pseudoaneurysm of the posterior tibial artery ( proximal third ) after plate removal in tibia .
the patient presented to our outpatient department with complaints of a firm , pulse less , non - tender swelling on the posterior aspect of the leg which gradually increased in size by overtime .
a 18-year - old male patient was referred to our outpatient clinic because of masslike lesion in his left calf .
he had a history of car accident and left isolated and closed tibia fracture 30 months ago that was treated by open reduction and internal fixation with 4.5 mm broad dcp plate ( fig . 1 , 2 ) .
the surgical approach was through 1 cm lateral to tibial crest and plate was in lateral aspect of tibia . in this period ,
after 2 years , union was obvious in ap and lateral radiographs , and removal of tibial plate was decided .
the plate had been removed 7 months ago on patient s request . at the time of surgery
plate and screw removal was done without any complication ( eg , plate or screw break down ) .
, the bone in radiograph was completely united and the patient was discharged when we find near 50 - 100 cc collection in the drain at the second day post operation.(fig .
post - operation radiograph after plate removal shows the bone was completely united . at the first post operation visit
about 4 weeks after plate removal , he came back with mild paresthesia in the left foot at the plantar surface .
also , he had firm , pulse less and non - tender swelling with unclear border on the posterior aspect of the leg which gradually increased over time . position of ankle was plantigrade . in vascular examination , we found the dorsalis pedis and the posterior tibialis pulses were equal to the opposite leg .
we followed this patient without further investigation . at about 3 months after plate removal ,
thus , we thought that paresthesia was due to iatrogenic tibial nerve injury ( eg , bennet tip ) .
approximately 5 months after surgery , he returned with calf muscle firmness and occasional spasm like pain .
paresthesia remained mild and distal pulses were normal and swelling was more firm and fixed . in physical exam , the skin over the mass was intact and we did nt see ecchymosis or discoloration in this area .
we found no tenderness at the fracture site and patient did nt compliant from pain similar the previous fracture .
mri was requested for this patient for calf muscle evaluation . at 1 month after mri request ,
the patient came back and discovered a large ( 5 x 7 cm ) soft tissue mass that was enhanced with probable diagnosis of hemotoma or pseudoaneurysm ( fig .
after the patient was systemically heparinized with 5000 units of unfractionated heparin , angiography showed the widening between the anterior and posterior tibial artery which confirmed the pseudoaneurysm of the posterior tibial artery ( fig.7 , 8) .
coronal magnetic resonance imaging ( mri ) of the left leg reveals the pseudoaneurysm of the posterior tibial artery .
sagittal mri of the left leg confirmed the diagnos is of the pseudoaneurysm arising from the posterior tibial artery in this patient .
, clots and hemotoma was evacuated and the posterior tibial artery perforation was repaired ( fig . 9 , 10 ) . at the operation time , clots and hemotoma was evacuated .
this photograph that was taken at the operation time shows pseudoaneurysms of the posterior tibial artery .
to our knowledge , pseudoaneurysms of the posterior tibial artery after tibial plate removal are too rare phenomenon [ 4 , 5 ] .
this branch of popliteal artery begins at the lower border of the popliteus , descends obliquely and supplies the posterior compartment of the leg and plantar surface of the foot .
along its course , it accompanied by the posterior tibial vein [ 6 , 7 ] .
pseudoaneurysms in most cases follow penetrating injuries but in our case it has been reported following blunt trauma [ 8 , 9 ] . indeed , the literature showed that in most of these patients the diagnosis was initially missed because the large hematoma concealed the underlying pseudoaneurysm and its pulsations .
thus , delay in the diagnosis is usual in this phenomenon [ 8 , 10 ] .
maxwell - armstrong and his co - author reported pseudoaneurysm of the anterior tibial artery following removal of tibial plate .
fung and his coauthors also reported pseudoaneurysm of the radial artery following plate removal in radius .
although this complication can be seen after fixation of leg , distal radius , clavicle and femur fracture with plate , total knee arthroplasty , the overall pseudoaneurysm incidence after plate removal is unknown but in our knowledge it is too rare [ 13 - 17 ] . in this manuscript
, we present a 18-year - old male patient with a mass in his left calf that was diagnosed pseudoaneurysm of the posterior tibial artery .
the time for the diagnosis of pseudoaneurysm in the literature is seen to vary from hours to years , depending on the location of pseudoaneurysm and clinical signs and symptoms .
the signs and symptoms include a pulsatile mass , pain , swelling , bruit , blood and neurologic deficit .
repair of tibial artery pseudoaneurysm can be challenging and a different surgical operation was described in the recent manuscripts such as direct arterial repair , interposition grafting or arterial ligation , duplex - guided compression and finally endovascular intervention [ 17 - 19 ] . during surgery ,
left leg is elevated for 2 minutes and then the tourniquet turned on and inflated to 280 mm hg .
the present surgery was done through the incision over the line that had greatest firmness .
a perforation was found in posterior tibial artery at the proximal third which was repaired with prolin 5 - 0 ( fig.9,10 ) . in this patient , it was not obvious whether this injury was caused by direct tearing of the artery or due to plate removal , but it s important to note that we should turn off the tourniquet and carefully find bleeding origin in any operation in the proximity of major vessels .
another point that we should say is this complication may be a pulsless mass , thus pulslessness could nt rule out the pseudoaneurysm .
we suggest having this complication in mind and being familiar with the presentation of the signs and symptoms .
it seemed vital to release the tourniquet before closing the wound to prevent this complication rate .
incidence of this complication is unknown , but has been reported after any surgery such as orif of fracture , plate removal , ankle or knee arthroscopy , or following arthroplasty .
although in most cases we find a pulsatile soft tissue mass , but we should know that the mass may be pulseless which may cause misunderstanding and delay in the diagnosis . in most cases , the diagnosis is delayed initially owing to slow progression in sign and symptoms .
an important key in the diagnosis is that the surgeon should have a high degree of suspicion when the patient compliants of a mass , with paresthesia and pain that was inappropriate to the operation and soft tissue swelling .
further assessment should be carried out carefully . when the diagnosis is confirmed , patient should be referred to a vascular surgeon . at a final result
, we should release the tourniquet before wound closure for accurate hemostasis and decrease this complication rate .
orthopaedic surgeon should consider pseudoaneurysm in his / her mind even if this complication is too rare after plate removal . | introduction : pseudoaneurysm is a rare complication reported after any surgery such as orif of fracture , plate removal , ankle or knee arthroscopy or arthroplasty . to our knowledge ,
pseudoaneurysms of the posterior tibial artery after tibial plate removal are a rare phenomenon .
we report a case of a 18-year - old male patient who had pseudoaneurysm of posterior tibial artery following plate removal.case presentation : a 18-year - old male was referred to our outpatient clinic because of mass - like lesion in his left calf .
he had a history of car accident and left isolated tibia fracture 30 months ago that was treated by open reduction and internal fixation with dcp plate .
after union in radiograph , the plate was removed 7 months ago on the patient request .
four weeks after surgery , the patient had complaints of pain and swelling . finally , pseudoaneurysm was diagnosed and was repaired in a common surgery with a vascular surgeon without any difficulty.conclusion:pseudoaneurysms of the posterior tibial artery after tibial plate removal are an extremely rare phenomenon .
this complication in most cases follows penetrating injuries but it has been reported following blunt trauma .
in fact , in most of these patients the diagnosis was initially missed because the large hematoma concealed the underlying pseudoaneurysm and its pulsations .
thus , a delay in the diagnosis is common in this phenomenon .
hence , the surgeon should be suspicious to this complication following plate removal when he / she is doubtful about the sign and symptoms . | Introduction:
Case Presentation:
Conclusion:
Introduction
Case report
Discussion
Conclusion |
exercise - induced muscle damage occurs after high - intensity resistance and endurance exercise ( santos et al . , 2004 ; veggi et al . ,
exercise - induced muscle damage is classified into primary and the secondary damage ( howatson and van someren , 2008 ) .
primary muscle damage is related to morphological changes , including sarcomere ( z - disc , i , and a band ) , sarcolemma , sarcoplasmic reticulum , and cytoskeletal elements ( clarkson and hubal , 2002 ) .
secondary muscle damage occurs due to impaired calcium homeostasis and the inflammatory response ( beaton et al . , 2002 ;
impaired calcium homeostasis due to sarcoplasmic reticulum dysfunction is activated by calpain-3 , which is a calcium - activated neutral protease ( beaton et al . , 2002 ) , that increases muscle damage and protein degradation ( murphy , 2010 ) .
neutrophils and macrophages invade the damaged site , facilitate phagocytosis , and secrete substances that induce oxidative stress ( tidball , 2005 ) .
this phenomenon may lead to a decrease in maximal strength and increase delayed - onset muscle soreness and muscle proteins , such as creatine kinase ( ck ) and lactate dehydrogenase ( ldh ) in the blood ( clarkson and hubal , 2002 ) .
use of dietary supplements is a recommended scheme to attenuate exercise - induced muscle damage ( sousa et al . , 2014 ) .
creatine has been used as a dietary supplement for a long time by many athletes and others ( bird , 2003 ) .
creatine ( n - aminoiminomethyl - n - methylglycine ) is a naturally generated endogenous guanidine compound synthesized in the kidneys , pancreas , and liver from methionine , glycine , and arginine ( bemben and lamont , 2005 ) and released into the blood ( dantona et al . , 2014 ) .
most creatine is localized in skeletal muscle and stored as creatine phosphate ( pcr ) .
ck and pcr play a pivotal role in short - term ( only a few seconds ) exercise ( bird , 2003 ) .
high creatine levels are found naturally in meat and fish ( dantona et al . , 2014 ) and
intramuscular pcr levels can be increased by approximately 20% by using a creatine supplement ( harris et al . , 1992 ) .
the ergogenic effect of creatine is well - known to improve exercise performance such as explosive muscle power ( claudino et al . , 2014 ;
zuniga et al . , 2012 ) and increased lean body mass after resistance exercise ( candow et al . , 2014 ; chilibeck et al . ,
several studies have reported that creatine promotes recovery by attenuating muscle damage after eccentric exercise ( cooke et al . , 2009 ; rosene et al . , 2009
( 2009 ) indicated that creatine supplementation may help rescue maximal strength and inhibit ck release due to high intensity exercise .
in contrast , other studies have reported that creatine does not reduce muscle damage after eccentric exercise ( mckinnon et al . , 2012 ; rawson et al . , 2001 ) . despite that creatine
potentially reduces muscle damage , it has not generally been used in the sports rehabilitation field .
the aim of this review was to introduce the effects of taking creatine on exercise - induced muscle damage .
( 2000 ) verified the effect of creatine on exercise - induced muscle damage . in this study mice performed 150 eccentric muscle contractions in response to electric stimulation after ingesting 0.5 or 1% creatine for 14 days .
as shown in tables 1 and 2 , several studies have reported that creatine attenuates exercise - induced muscle damage ( bassit et al . , 2010 ; cooke et al . , 2009 ; rosene et al . , 2009 ; veggi et al . ,
g / kg / day , four servings / day , 5 days ; maintenance period : 0.1 g / kg / day , one serving / day , 14 days ) beginning 5 days before exercise until 14 days after exercise improved maximal isometric strength and decreased ck compared with those who consumed a carbohydrate placebo only .
( 2010 ) also reported that ingesting 20 g / day creatine over 5 days decreases ck and ldh after a triathlon competition .
2009 ) reported the acute ( 20 g / day , 7 days ) and chronic ( 6 g / day , after 7 days followed for 23 days ) effects of creatine on exercise - induced muscle damage .
this study demonstrated that chronic ingestion of creatine effectively increased maximal isometric strength after resistance exercise .
( 2013 ) suggested that taking 20 g / day creatine for 6 days between the first and the second exercise phase contributed to decreased muscle soreness , inhibited the elevation in ck , and enhanced of range of motion .
2013 ) , suggested that taking creatine may increase the repeated bout effect after initial exercise - induced muscle damage .
the repeated bout effect is protective against subsequent muscle damage through neural , mechanical , and cellular adaptations after exercise ( mchugh , 2003 ) .
however , several studies suggested that creatine had no benefit on exercise - induced muscle damage ( mckinnon et al . , 2012 ; rawson et al .
rawson et al . ( 2001 ) demonstrated that creatine ( 20 g / day ) taken for 5 days before and after exercise does not change the levels of muscle damage markers after exercise between subjects taking creatine and a placebo .
rawson et al . ( 2007 ) reported that creatine ( loading period : 0.3 g / kg / day , three servings / day , 5 days ; maintenance period : 0.03
g / kg / day , one serving / day , 5 days ) does not change muscle damage marker levels after exercise .
( 2012 ) reported that taking creatine ( loading dose : 40 g , two servings / day , 5 days ; maintenance period : 10 g , two servings / day , 5 days ) had no effect on exercise - induced muscle damage .
these conflicting results may be partly explained by differences in exercise protocols used in the studies .
a number of potential mechanisms explain the effect of creatine on exercise - induced muscle damage , including the inflammatory response , oxidative stress , calcium homeostasis , and satellite cells activities in damaged muscle ( fig .
the first potential mechanism of creatine is that it reduces the inflammatory response after exercise - induced muscle damage .
santos et al . ( 2004 ) demonstrated that 20 g / day creatine for 5 days before 34 male marathon runners raced significant reduced ldh , prostaglandin e2 ( pge2 ) , and tumor necrosis factor - a ( tnf- ) after the 30 km race .
( 2008 ) reported that 11 male triathletes who ingested 20 g / day creatine for 5 days prior to a half - ironman competition had significant decreases in tnf- , interferon- ( inf- ) , interleukin-1 ( il-1 ) , and pge2 after the competition compared to those in the placebo group .
( 2013 ) also reported that ingesting 0.3 g / kg creatine for 7 days abolishes the increase in tnf- after a repeated running - based anaerobic test . pge2 and
tnf- facilitate the inflammatory response and pain sensation after exercise - induced muscle damage ( tidball , 2005 ) .
interestingly , all three studies showed a decrease in the inflammatory response ( bassit et al .
santos et al . ( 2004 ) particularly showed a decrease in ldh and inflammation .
the inflammatory response is associated with markers of sarcolemma damage ( kanda et al . ,
( 2013 ) reported a positive correlation between neutrophil migratory activity and myoglobin after exercise .
these results indicate that the reduction of inflammatory response factors by creatine may decrease disruption of sarcolemma due to exercise - induced muscle damage .
( 2010 ) reported that taking creatine ( 5 g / kg / day , 5 days ) significant decreases outflux of intracellular enzymes after continuous muscle contraction .
2013 ) found that ingesting creatine ( 300 mg / kg / day , 15 days ) does not significantly reduce inflammatory response markers , such as tnf- , il-1 , and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) in mice .
the second potential creatine mechanism is diminished oxidative stress ( lawler et al . , 2002 ; rahimi , 2011 ) .
lawler et al . ( 2002 ) reported the first evidence for the antioxidant capacity of creatine .
deminice and jordao ( 2012 ) found that ingesting 2% creatine during the 28 days before acute exercise decreases thiobarbituric acid - reactive substances ( tbars ) and lipid hydroperoxides but increases the glutathione ( gsh ) and glutathione disulfide ( gssg ) ratio and total antioxidant capacity .
however , these studies were limited to cultured cells models , and animals . according to a human study by rahimi ( 2011 ) , taking 20 g / day creatine for 7 days decreases malonyldialdehyde ( mda ) and 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) levels after resistance exercise compared to those taking a placebo .
in contrast , several studies have reported that creatine does not decrease oxidative stress after exercise - induced muscle damage ( deminice et al . , 2013 ; silva et al . , 2013 ) .
impaired sarcoplasmic reticulum due to muscle damage may increase calcium concentrations in the cytosol , causing secondary muscle damage ( beaton et al .
creatine assists in maintaining the sarcoplasmic reticulum calcium pump function by phosphorylating adp to atp , which decreases cytosolic calcium levels ( cooke et al .
1996 ) suggested that increasing muscle pcr accelerates atp homeostasis , leading to reduced secondary damage due to an increase in calcium concentration .
finally , creatine has been associated with satellite cells or so - called muscle stem cells
( olsen et al . , 2006 ; safdar et al . , 2008 ) .
satellite cells play a critical regenerating role after muscle damage ( paulsen et al . , 2012 ) .
olsen et al . ( 2006 ) demonstrated that ingesting creatine ( loading period : 24 g / day , 6 g / serving , four servings / day , 7 days ; maintenance period : 6 g / day , one serving / day , 15 weeks ) and performing resistance exercise increases the number of satellite cells and myonuclei concentration in human muscle .
( 2008 ) reported that taking creatine ( loading period : 20 g / day , 10 g / serving , two servings / day , 3 days ; maintenance period : 5 g / day , one serving / day , 7 days ) promotes proliferation and differentiation of satellite cells and activate cytoskeletal remodeling genes .
creatine may be a useful dietary supplement for preventing muscle damage and facilitating recovery from high - intensity exercise , which is applicable to the sports rehabilitation field .
however , several mechanisms of how creatine prevents exercise - induced muscle damage need to be examined in future well - designed studies . | muscle damage is induced by both high - intensity resistance and endurance exercise .
creatine is a widely used dietary supplement to improve exercise performance by reducing exercise - induced muscle damage .
many researchers have suggested that taking creatine reduces muscle damage by decreasing the inflammatory response and oxidative stress , regulating calcium homeostasis , and activating satellite cells .
however , the underlying mechanisms of creatine and muscle damage have not been clarified .
therefore , this review discusses the regulatory effects of creatine on muscle damage by compiling the information collected from basic science and sports science research . | INTRODUCTION
CREATINE AND EXERCISE-INDUCED MUSCLE DAMAGE
POTENTIAL MECHANISMS OF CREATINE ON EXERCISE-INDUCED MUSCLE DAMAGE
CONCLUSIONS |
in economically developed countries , the human immunodeficiency virus ( hiv ) infection is now considered a chronic disease , with life expectancy approaching that of the general population .
management of the hiv - positive pregnant patient should focus on both decreasing the risk of mother to child transmission ( mtct ) and minimizing maternal and neonatal complications .
the society of obstetricians and gynaecologists of canada ( sogc ) and american college of obstetricians and gynecologists ( acog ) recommend that elective cesarean section ( cesarean section before labor or rupture of membranes ( roms ) be performed for delivery when viral load is detectable or greater than 1000 copies / ml as there is a 12-fold increased risk of mtct [ 3 , 5 ] .
this is based on several studies that showed that the combination of intrapartum zidovudine ( zdv ) and elective cesarean section significantly decreased vertical transmission compared to other delivery modes [ 68 ] . with the addition of highly active antiretroviral therapy ( haart ) , the risk of vertical transmission has continued to decrease .
rom increases fetal exposure to maternal blood and vaginal fluids , and prolonged duration of rom has been shown to be a significant risk factor for vertical transmission [ 6 , 911 ] .
evidence exists that after 4 hours of rom the risk of mtct rises and the protective effect of a cesarean section is lost [ 9 , 10 ] .
however , these conclusions were based on studies in which only intrapartum monotherapy with zdv was used and maternal vl was not known . since the addition of haart , subsequent research has been performed to determine if prolonged duration of rom remains an important risk factor for vertical transmission .
these studies have demonstrated that there is no increased risk of transmission with rom longer than 4 hours and no protective effect of cesarean section [ 5 , 12 ] .
given that postpartum morbidity from cesarean section is potentially higher in hiv - positive women , achieving a vaginal birth in this population is beneficial .
given the paucity of literature addressing this question , the objective of this study was to review our experience and evaluate whether the effect of duration of rom or mode of delivery on mtct still exists among optimally managed hiv - positive women on haart .
following ethical approval , we performed a retrospective chart review of all hiv - positive women who delivered at mount sinai and st .
women were defined as optimally managed if they were taking antepartum haart and had a vl less than 1000 copies / ml at the time of delivery .
thus , eligibility criteria included pregnant women with a predelivery diagnosis of hiv , adherence to antepartum haart , vl less than 1000 copies / ml at the time of delivery , and delivery at either of the two sites .
if a woman had had multiple deliveries during the time period , the most recent delivery data was collected .
women not on haart or with viral loads greater than 1000 copies / ml were excluded .
information regarding the patient 's age , ethnic background , gravidity , parity , administration of intravenous zdv , time of rom , time of birth , mode of delivery , and intrapartum procedures ( artificial rupture of membranes ( aroms ) , fetal scalp electrodes ) were all recorded from the antenatal charts and/or from the medical records .
duration of rom was expressed as total length of time in minutes from time of rom to time of birth .
the proportion of women receiving intravenous zdv , with vaginal birth or cesarean section , and with any invasive procedures was determined .
all children born to hiv - positive mothers were prescribed a 6-week course of oral zidovudine and referred for follow - up care in the pediatric hiv clinic , at the hospital for sick children , in toronto .
during the study period , 213 hiv - positive women delivered at the participating centers . of these
, 3 were excluded due to a viral load > 1000 copies / m therefore the final study cohort consisted of 210 women . during the 10-year study period ,
eighty ( 38% ) women gave birth to their first child . a high rate ( 16% ) of preterm birth , defined as delivery
the average gestational age at birth was 38 weeks and 2 days with a range of 24 weeks and 6 days to 41 weeks and 3 days .
of those 200 patients with known vl , the majority of the group ( n = 167 , 84% ) had an undetectable vl ( less than 50 copies / ml ) at the time of delivery .
the majority of women ( n = 179 , 85% ) received adequate intrapartum zdv , defined as having received a loading dose followed by 3 hours of maintenance infusion prior to delivery . in the entire cohort , 107 women ( 51% ) had a vaginal birth and 103 ( 49% ) had a cesarean birth . among women with cesarean birth , 75 ( 73% )
were performed electively ( prior to labor ) and 28 ( 27% ) were performed in labor . among women with undetectable vl , 90 ( 54% ) had a vaginal birth , and 77 ( 46% ) had a cesarean birth . in this cohort ,
of those , 20 women had dilation between 0 and 4 cm , 20 had greater than 4 cm but less than 10 cm , and 6 were ruptured just prior to delivery . among the 107 women with a vaginal birth ,
six had a vacuum - assisted vaginal delivery and one had a fetal scalp electrode placed .
the median length of time of rupture of membranes for the entire cohort was 0.63 hours ( 0.0077.87 ) .
the median length of time of rupture of membranes for the vaginal birth group was 2.56 hours ( 0.0053.90 ) and cesarean birth group was 0.02 hours ( 0.0077.87 ) ( p < 0.0001 ) .
for those women with an undetectable vl , the median length of time of rupture of membranes was 0.62 hours ( 0.0077.87 ) and for those with a detectable vl was 0.57 hours ( 0.0033.63 ) ( p > 0.92 ) . when removing those who were ruptured less than 0.03 hours ( elective cesareans , precipitous vaginal deliveries , etc . ) , there were 131 patients whose membranes were ruptured for 0.05 hours or longer .
their median length of time of rupture was 3.53 hours ( 0.0577.87 ) . in total , 59 ( 28% )
women had rupture of membranes for 4 hours or longer . for women who were less than 37 weeks at the time of delivery , the median length of time of rupture was 0.63 hours ( 0.0077.87 ) , and 0.66 hours ( 0.0053.90 ) for those greater than 37 weeks .
the median lengths of time of rupture and gestational ages are shown in figure 1 .
there were no cases of mtct in this cohort of hiv - positive pregnant women .
this study suggests that , in a group of 210 optimally managed hiv - positive women who gave birth over a 10-year period , increasing the duration of rom did not increase the likelihood of mtct . since the use of haart , little has been published specifically exploring the role of duration of rom and mtct in optimally managed women [ 11 , 12 ] . in this cohort of women ,
the median length of time of rom for the entire group was 0.63 hours with a range of 0 to 77.87 hours .
a large proportion of the group was ruptured for minutes only , and when removing those women , the median increases to 3.53 hours , with a range from 0.05 hours to 77.87 hours .
the median for the undetectable vl women was similar to the entire group , at 0.65 hours , which follows since the majority of the group had undetectable vl .
it is clear that some women certainly had rom longer than the previously recommended time limit of 4 hours [ 9 , 10 ] , but given that this recommendation is based on studies where women were not on haart and maternal vl was not known , this recommendation may not apply to women who are optimally managed . in all of our subgroups
previous literature has stated that even in women with undetectable vl , elective cesarean birth reduces mtct ; however when adjusted for haart , the effect was no longer significant . in our study
the median times of rom for the vaginal ( 2.56 hours ) and cesarean groups ( 0.02 hours ) were statistically different , but neither group had a case of mtct .
this is consistent with previous literature that for women on haart , mode of delivery does not influence risk of mtct , even if the length of time of rom increased .
the adherence rate for iv zdv prior to delivery in this study was 85% , which is similar to rates in other studies .
all women who did not receive adequate iv zdv prior to delivery either had precipitous deliveries or operative deliveries for emergency indications ( cord prolapse , footling breech in labor ) . in the majority of cases , the zdv was started on admission using preprinted orders , but delivery occurred before three hours of maintenance infusion could be completed .
our cesarean birth rate was 49% , which is well above the national average of approximately 26% .
first , the majority of the group was multiparous , accounting for two - thirds of the women who underwent cesarean .
many of these women chose to undergo a repeat cesarean , similar to hiv - negative women in canada .
second , some hiv - positive women choose to undergo an elective cesarean regardless of previous delivery status or vl . a large proportion of women in this study originate from resource - poor countries where different strategies are employed , such as cesarean birth , to prevent mtct . in our cohort of hiv - positive women , the preterm birth rate was 16% , which is double the average preterm birth rate in canada of 8% .
this increased preterm birth rate has been observed in other studies of hiv - positive women on haart [ 5 , 16 ] . in previous work
done at one of our institutions , the preterm delivery rate in a cohort of hiv - positive women was similar to the control group ( in press ) .
possible reasons for the increased rate in the current cohort could include earlier induction of labor for abnormal liver function tests or preeclampsia , or spontaneous preterm labor . these factors
could also be a contributor to the high cesarean birth rate observed in this study .
preterm premature rupture of membranes ( pproms ) in optimally managed hiv - positive women poses a clinical dilemma for management when weighing the risk of prematurity over the risk of mtct .
our study did not specifically address the issue of pprom ; however there was a small number of preterm patients who were ruptured for greater than the 4-hour recommendation without a case of mtct .
overall though , the median lengths of time of rupture for both the preterm and term patients were similar .
more studies with a larger number of optimally managed patients with pprom are required to further explore this complex issue .
traditionally , the use of invasive procedures during labor in hiv - positive women has been discouraged because of the potential for increasing mtct [ 510 ] . in this study ,
arom was employed for labor induction and during the active phase of labor in 46 women without increasing the likelihood of vertical transmission .
one patient did have a fetal scalp electrode placed , although this is not typical practice even among optimally managed women and is not recommended . during the study period of ten years , we observed a trend of an increasing number of deliveries .
there are many possible reasons for this , including an increase in the proportion of women among hiv - positive persons , changing immigration patterns , and better long - term management of hiv resulting in better overall health and an increased desire for childbearing .
since the introduction of haart in 1998 , there has been a steady decline in antenatal monotherapy use in ontario , with an increasing acceptance of and adherence to haart regimens .
this also may have resulted in an increasing number of women who met inclusion criteria of antenatal haart use and low vl over time .
given the number of cases from january to november 2010 , the number for 2011 is projected to be similar to 2008 and 2009 .
we had a large cohort of hiv - positive women managed by a small number of practitioners at only two geographic sites .
this resulted in a cohort of optimally managed and virally suppressed women , with the majority having undetectable vl and all having vl less than 1,000 copies / ml . physicians and nurses at these two sites have experience in the antenatal and intrapartum care of these women , which increases the likelihood of appropriate decision - making regarding medication use and labor management .
finally , complete follow - up of children born to women in this study allows us to evaluate mtct rates over time .
there are also some limitations . due to the relatively small proportion of hiv - positive women delivering in canada
, our ability to examine larger cohorts of women is limited without employing a multicentre approach .
the size of our cohort prevents us from determining small differences in transmission rate . to more accurately investigate the relationship of duration of rom and mtct of hiv in optimally managed women , a larger multicenter study would be required .
with no cases of mtct , we were unable to evaluate patient - specific or labor and delivery characteristics predictive of transmission .
however , we found no association with increasing duration of rom or mode of delivery and mtct .
it is reassuring that , in the current era and among women with low vl , allowing length of time of rom to increase beyond four hours does not appear to increase the risk of mtct .
in a cohort of over 200 optimally managed hiv - positive women on haart with low vl , there were no cases of mtct .
allowing optimally managed women with low vl to continue in labor beyond four hours of rom is acceptable .
additional studies with larger numbers of women are important to further evaluate the impact of duration of rom and mode of delivery on mtct among hiv - positive women in the current era . |
objective . to evaluate whether the length of time of rupture of membranes ( rom ) in optimally managed hiv - positive women on highly active antiretroviral therapy ( haart ) with low viral loads ( vl ) is predictive of the risk of mother to child transmission ( mtct ) of the human immunodeficiency virus ( hiv ) .
study methods .
a retrospective case series of all hiv - positive women who delivered at two academic tertiary centers in toronto , canada from january 2000 to november 2010 was completed .
results .
two hundred and ten hiv - positive women with viral loads < 1,000 copies / ml delivered during the study period .
vl was undetectable ( < 50 copies / ml ) for the majority of the women ( 167 , 80% ) , and < 1,000 copies / ml for all women .
mode of delivery was vaginal in 107 ( 51% ) and cesarean in 103 ( 49% ) .
the median length of time of rom was 0.63 hours ( range 0 to 77.87 hours ) for the entire group and 2.56 hours ( range 0 to 53.90 hours ) for those who had a vaginal birth . among women with undetectable vl , 90 ( 54% )
had a vaginal birth and 77 ( 46% ) had a cesarean birth . among the women in this cohort
there were no cases of mtct of hiv .
conclusions .
there was no association between duration of rom or mode of delivery and mtct in this cohort of 210 virally suppressed hiv - positive pregnant women . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion |
the importance of ethical principles guiding research involving human beings have been evident since the code of nuremberg1 ( 1947 ) , which was created with the intention of preventing the repetition of the atrocities committed in illegal experiments conducted by nazi physicians in concentration camp prisoners . since then , several other national and international regulations , such as the declaration of helsinki2 ( 1964 ) , the belmont report3 ( 1978 ) and the resolution 196/964 ( 1996 ) , provide guidance concerning ethical issues related to research involving human beings . currently , research involving human subjects must be reviewed and approved by a research ethics committee ( rec ) before its start .
the assessment by a rec is needed in order to guarantee respect to the autonomy of the research volunteers , as well as to guarantee beneficence , nonmaleficence and justice5 .
thus , the recs main functions are to evaluate research projects based on their scientific relevance , technical and operational feasibility , in addition to reviewing ethics and morality of the research.6 at the hospital das clnicas , faculdade de medicina , universidade de so paulo ( hcfmusp ) , the projects are reviewed by the rec , named comisso para anlise de projetos de pesquisa ( cappesq ) , which consists of 59 members who meet every two weeks . in 2007 , the committee reviewed 1,256 new research projects .
some of these projects were reviewed over the course of two or more meetings for several reasons , such as lack of documentation , incomplete information provided at forms , and problems with either the research project or the informed consent form ( icf ) .
these issues compelled the researcher to resubmit the project with the necessary corrections , adjustments and inclusions , resulting in extension of deadlines and consequent delay in issuing the final decision by the cappesq . in light of such obstacles in the research projects review processes , it is fundamentally important for the community of researchers linked to the institution to know the profile of the research proposals reviewed by the committee in two or more meetings as well as the main reasons why some research projects must be resubmitted for analyses . with access to this information , the professionals will be able to better prepare their research projects , as well as the documentation needed for the project to be properly submitted and , thus , speed up the review and the ethical approval procedures for such research projects . thus , the objectives of the study were : to characterize the research projects submitted for review to the rec with relation to subject area , academic purpose , specific thematic areas , sponsorship , number of meetings necessary to analyze the research project , deliberation and issuing of final opinion , and average time it takes for the final decision to be made . to identify the reasons why the research projects needed to be resubmitted for analysis to the rec .
this is a tertiary - level university hospital , located in the city of so paulo .
all research projects involving human subjects that were submitted to the cappesq during 2007 and that went through two or more reviews by this committee were included in this study .
a set of documents are required by the cappesq in order to analyze research projects . for all research projects ,
the committee prepares a binder containing all the documentation submitted , such as an institutional registration form for the investigator and for the research project , outline of the research project , informed consent form ( icf ) , as well correspondences between the researchers and the committee and opinions of the rec .
the following data were collected : subject or field of research , research in special thematic areas , academic purpose , sponsorship , number of sessions needed for issuing the committee s final decision , time elapsed between the start of the process and the final decision and the number of sessions in which the research project was analyzed , reasons for returning documents to the researchers , and details of the reasons for subsequent resubmission of research projects to the cappesq .
specifically , with regard to the reasons for returning research documents , the data were extracted from the opinions given by the committee . because these data are not described in a standardized manner , all data were fully transcribed and later categorized in an attempt to have homogenous results .
statistical analyses were performed using statistical package for social sciences ( spss , version 11.0 ) and minitab ( version 15.0 ) . for the quantitative variables ,
the qualitative variables were analyzed through the calculation of absolute and relative frequencies.7 this project was approved by the cappesq ( 0680/08 ) .
the average time for evaluating the research projects and related documents until a final decision was reached was 49.95 days .
most of the projects , 857 projects ( 68% ) , were reviewed in a single meeting , and their average review time was 39 days . among the other projects , 342 ( 27.2% ) were reviewed in two meetings , in an average time of 68.78 days ; 53 projects ( 4.2% ) were reviewed in three meetings , in an average time of 99.62 days ; and 4 projects ( 0.3% ) went through four meetings in an average time of 127.30 days .
of the 399 projects that were reviewed in two or more sessions , 392 binders were retrieved and included in this study .
the rest were unavailable during data collection period since they were being analyzed by the reviewers . from the 392 projects , 357 ( 91.1% ) referred to research involving human subjects and 35 ( 8.9% ) involved animal studies ,
table 1 shows the areas of research , as registered by the researchers themselves in a specific cappesq form .
this table shows that cardiology / pulmonology had the highest number of projects submitted ( 45 , 12.6% ) , followed by general medicine ( 35 , 9.8% ) , and psychiatry ( 26 , 7.3% ) . with regard to special thematic research , investigators considered 120 research projects related to special thematic areas : 51 ( 42.5% ) research projects were related to new drugs , vaccines and diagnostic tests , 26 ( 21.7% ) were related to new procedures , and 23 ( 19.2% ) were related to human genetics .
the classification of the research project as special thematic areas is determined by the researcher at the time the specific cappesq form is completed ( table 2 ) . concerning academic purpose , 173 ( 48.5% ) research projects
were related to undergraduate students ( predominantly those in scientific initiation modality ) , 156 ( 43.7% ) research projects were linked to graduate students ( master and phd students ) , and 28 ( 7.8% ) studies had other purposes .
one hundred and eleven researches ( 31.1% ) were sponsored by industry or governmental research funding agencies .
of the 357 projects that were included in this study , the vast majority ( 348 or 97.5% ) were approved .
five ( 1.4% ) projects were rejected , and four ( 1.1% ) were submitted to the commission only for committee awareness .
the reasons why the research projects were returned to the researchers for corrections , adjustments or clarifications and subsequent resubmitting to the cappesq are described next .
eight hundred reasons for project document return were listed , with an average of 2.2 reasons for each returned research project .
the two main reasons for returning projects to the researchers were problems with the icf ( 422 ( 52.7% ) occurrences ) and inadequacies at the research protocol ( 205 ( 25.6% ) occurrences ) .
both of these reasons represent 78.3% ( 627 ) of the reasons for returning research projects to the researchers .
other reported reasons for return were : incomplete or incorrect documentation ( 68 cases , 8.5% ) , incomplete or incorrect registration form of the research and/or of the researcher ( 65 ; 8.1% ) , absence of research execution schedule ( 21 ; 2.6% ) , doubts regarding financial support ( 15 ; 1.8% ) and issues related to the cover page of the comisso nacional de tica em pesquisa ( conep ) ( 4 ; 0.5% ) .
details were listed for every reason of each project that was returned . regarding the icf ( table 3 ) , main problems identified were the use of inadequate language and/or difficulty in understanding the icf s content ( 136 cases , 32.2% ) .
insufficient information related to the protocol in the icf ( 109 ; 25.8% ) was also reported . in four cases
, it was not possible to identify the problems identified at the icf . in regard to the 205 project returned due to research protocol issues ( table 4 ) , the main reason concerned doubts about methodology and/or statistics ( 158 cases , 77.1% ) .
as for the documentation related to the project , the major issues were need for clarification about and/or agreement to the participation of external institutions ( 24 ; 35.3% ) such as diagnostic and imaging services , health services and educational institutions . there were also 11 ( 16.2% ) cases in which lack of researcher s or research team s documentation ( such as curriculum vitae ) ( table 5 ) . pertaining to the registration forms of the researcher or the research project , the main problem that led to the return of research projects was incomplete / inaccurate data ( 32 cases , 49.2% ) . in one case , it was not possible to determine the reason for the return . regarding the research schedule ,
issues related to the study financing also caused 15 ( 4.2% ) research projects to be returned , the main reason being absence of information on financial support ( 6 cases , 40.0% ) .
improper filling out of the cover page ( conep ) resulted in the return of four ( 1.1% ) documents ( table 6 ) .
several research projects and related documents submitted to the recs for review show common errors that may be corrected in a quick and simple way , if appropriate attention is paid while preparing the research project as well as the necessary documentation.5 moreover , investigators who are familiar with the relevant ethical information , which is generally debated in literature , show greater knowledge and give better arguments in discussions and negotiations with the rec in regard to the ethical analysis of their research projects.8 data presented demonstrate that the issues raised by the cappesq usually involve the content of the icf and/or methodological or statistical issues of the research project .
therefore , the researcher and his or her team must dedicate special attention to developing both .
icf is currently considered to be the cornerstone of research ethics9 and , therefore , has become one of the main areas to which attention is paid in the ethical review of research projects . moreover ,
mandatory signature of the icf by the volunteer and by the researcher before the inclusion at the research may minimize the possibilities of including volunteers who are not aware about the study purposes and procedures or even who have not given consent to be included in a research.10 in this study , the major problem associated with icf were the use of inadequate language and/or of difficulty in understanding the content of these forms .
the analysis of 12 icf indicated that the mean readability index was 41.8% , and only one form had an adequate level of understanding .
moreover , the results indicated that the level of education needed for understanding 91.7% of icf was 11 years of education.11 another study evaluated 48 forms for research and medical procedures and results indicated that the level of education necessary for understanding the text was 16.4 years of education.12 one of the major responsibilities of the recs is to ensure that the icf is written in such a way that the research volunteer understands the research , especially the potential risks and benefits.5 the icf content and language might be directed to the research volunteer.8 thus , it is essential that the language used in the forms is adapted to the type of population that will be included in the study , which is the researcher s responsibility .
poor information about the protocol in the icfs was also a frequent reason for returning the research projects to the researchers .
the icf must contain information regarding background , justification and objectives of the study in addition to the procedures that will be carried out during the study.4 it ensures that the volunteer is aware of all the events and procedures to which he or she will be subjected during the research . with regard to the protocol analysis
, the scientific merit of the research project must be reviewed along with the ethical issues involved9 .
it is also necessary for the recs to review the methods used in the research project .
in addition , the need for obtaining previous ethical approval to develop studies involving human subjects actually motivates investigators to write research projects,10 instead of conducting researches without any protocol or previous planning .
the current study data indicate that the main protocol related reasons for returning research protocols are doubts about either study design or statistical planning .
comments accompanying returned protocols mainly address clarification of the type of study ( retrospective or prospective ) , the need for better description of study procedures , justifications for sample size , and description of statistical tests to be used , among others .
investigators must pay attention to the structure of the research project in order to enable the scientific and operational aspects of the study to be understood not only by review experts in that field of research , but also by reviewers from other research fields , and even reviewers who are not linked to the research but are members of the community.5 items such as research schedule , detailed financial budget , and origin of available resources are also important pieces of information that should be described.4 if they are not duly attached to the research project , the recs must request clarifications about such issues . concerning documentation , it is important to know which documents and forms must be filled out and sent to each specific committee
. there are models and specific guidelines for the rec that must be followed by the researchers and their research teams , with the objective of adjusting the material to be sent and , thus , facilitating a speedy review and reducing procedure time .
the ethical analysis of research projects must be viewed as an important and fundamental step in conducting studies involving human subjects .
the researchers expectations related to the recs analysis may include learning , based on the considerations made by the rec in regard to the research project , and also may include potential benefits to their research.8
in 2007 , the research projects analyzed by the cappesq took an average of 49.95 days to get through all the procedural steps . among the research projects involving human subjects that were submitted for two or more reviews by the cappesq
, the majority , 12.6% , belonged to cardiology / pulmonology ; 48.5% of the research involved undergraduate students ; 42.6% of the research projects were related to new drugs , vaccines and diagnostic tests ; 68.9% did not involve any sponsorship .
the main reason for returning documents to the researchers for subsequent resubmission to the cappesq was issues with the icf , specifically regarding inaccurate use of language and poor information related to the research protocol .
the second most common reason for returning the documents to the researchers was inadequacy of the protocol itself .
based on the results obtained , this study will contribute to the appropriate preparation and structuring of research projects and related documents to be submitted to the rec .
the reasons presented for return reveal that , when the research projects are prepared , the researcher shows insufficient knowledge and pays little attention to the guidelines . the ethical and methodological issues reported by this study need to be considered by researchers , so the process of submission to the rec can cease to be a painful ordeal , as it is currently considered , and can instead become a simple and quick procedure . | introductionit is important to know the reasons for resubmitting research projects to the research ethics committee in order to help researchers to prepare their research projects , informed consent forms and needed research documentation.objectivesto verify the reasons for resubmitting projects that were previously rejected by the ethics committee.methodthis is a cross - sectional study that evaluated research projects involving human beings .
research projects were submitted in 2007 to the research ethics committee of the hospital das clnicas , faculdade de medicina da universidade de so paulo.resultsone thousand two hundred and fifty six research projects were submitted to the ethics committee and the average time for evaluating the research projects and related documents until a final decision was reached was 49.95 days . from the total ,
399 projects were reviewed in 2 or more meetings until a final decision was reached .
of these , 392 research projects were included in the study ; 35 projects were subsequently excluded for involving animals . among the research projects
included , 42.5% concerned research with new drugs , vaccines and diagnostic tests , 48.5% consisted of undergraduate students research projects , 68.9% of the research had no sponsorship , and 97.5% were eventually approved .
the main reasons for returning the projects to the researchers were the use of inadequate language and/or difficulty of understanding the informed consent form ( 32.2% ) , lack of information about the protocol at the informed consent form ( 25.8% ) , as well as doubts regarding methodological and statistical issues of the protocol ( 77.1% ) .
other reasons for returning the research projects involved lack of , inaccuracy on or incomplete documentation , need of clarification or approval for participation of external entities on the research , lack of information on financial support.conclusionamong the research projects that were returned to the researchers for additional clarification , the main reasons were inadequacies or doubts about the terms used in the informed consent form as well as lack of information regarding the research at the informed consent form and methodological and statistical issues regarding the protocol . | INTRODUCTION
METHOD
RESULTS
DISCUSSION
CONCLUSION
FINAL CONSIDERATIONS |
over the past 10 years , the world organisation of family doctors ( wonca ) has benefited from and supported the development of young doctor movements ( ydms ) comprised of individuals within five years of residency completion and family medicine ( fm ) trainees .
increasingly , these groups are reaching out to medical doctors and students as potential participants .
though each is slightly different , all have a regional chair as well as representatives for each fm academy / college .
the budgets are minimal and are supported by national academies , fundraising , conference attendances , and donations .
the first ydm , vasco da gama ( vdgm ) , was conceived as a grassroots movement by junior family physicians ( fps ) and general practitioners ( gps ) throughout europe in 2005 .
after several years of success and rapid growth , this concept spread rapidly throughout the world .
first came rajakumar ( asia pacific ) in 2009 , then the spice route ( south asia ) and waynakay ( latin america ) in 2010 , al razi ( east mediterranean region ) as well as afriwon renaissance ( africa ) in 2013 , and finally wonca polaris in 2014 . ydms promote the discipline by increasing opportunities to acquire new skills , creating a forum for support and information , establishing a communication network , improving the quality of fm training programs by creating a central information database , promoting mentoring , sharing best practices , publishing news about the movements for national colleges and associations , providing access to wonca international and regional conferences , developing junior doctors conferences , contributing to the development of new and future fp representation and encouraging foreign exchanges among young fps .
innovation , especially when intertwined with positive social interactions , proves integral and manifests itself through the events performed by their leadership and members .
the most popular activities are the pre - conferences , novel adjuncts to standard fp conferences coordinated by ydms and have occurred in various countries around the globe .
exchanges are another popular item with ydms collaborating to increase availability and a global standard of exchanges .
though ydms have gained prominence over the past decade , minimal inquiry into members motives exists .
this is necessary to determine if there is a proper alignment between the expectations of members , would - be members and the stated ydms role in family medicine promotion and development .
this study 's objectives include : ( 1 ) determining major reasons why young fps ( those < 5 years of residency completion ) and aspiring fps joined their regional ydm and are involved in their activities , ( 2 ) determining the main factors that will make non - members want to join a ydm , ( 3 ) assessing for differences in the responses between ydm members and non - members and ( 4 ) studying differences in the responses between the ydm members .
this study 's objectives include : ( 1 ) determining major reasons why young fps ( those < 5 years of residency completion ) and aspiring fps joined their regional ydm and are involved in their activities , ( 2 ) determining the main factors that will make non - members want to join a ydm , ( 3 ) assessing for differences in the responses between ydm members and non - members and ( 4 ) studying differences in the responses between the ydm members .
the study population included young fps ( 5 years after residency completion ) and aspiring fps ( fp trainees , medical degree holders and medical students who have chosen family medicine as a career ) . using open epi version 2.2.1 , a 95% confidence interval ( ci )
was selected on the assumption that 50% of an infinite population of young and aspiring fps would agree to the listed reasons for joining a ydm .
a convenience sampling method was used by posting the survey link to the online ydm platforms , departments of family medicine websites and online platforms of young doctors in the various wonca regions .
responses of all those who opted into the survey and met the inclusion criteria were included .
face and content validity for the survey tool was ascertained following two discussion and feedback sessions with a group of nine young fps ( five fp trainees and three fps in nigeria with input from a fp from the usa ) .
five cycles of the survey were sent . using a five - point likert scale (
i.e. strongly agree , agree , neutral , disagree and strongly disagree ) , respondents indicated to what extent they agreed to each item as being the possible reason they joined ( or may want to join ) a ydm .
whitney u test was used for the distribution of responses and to test for statistically significant difference . the kruskal
wallis test was used to describe the distribution of responses by the various ydms as well as to test for statistically significant difference .
the chi - square test was used to determine association between age groups , gender , professional status and agreement with the listed reasons for joining ydm .
the mean age ( sd ) was 32.3 years ( 4.9 ) and the largest group by age ( 41.5% ) was between 25 and 30 years .
female respondents were 86 ( 43.0% ) while the male respondents were 84 ( 42% ) ; 30 ( 15% ) of the respondents did not state their gender .
young fps who were < 5 years post - residency were 80 out of the 200 respondents ( 40% ) , fp trainees were 109 ( 54.5% ) , medical degree holders about to start family medicine residency were 4 ( 2% ) , medical students who had chosen a career in family medicine were 6 ( 3% ) ; 1 respondent did not state his / her current status . further classification of the respondents showed that 102 ( 51.0% ) were current ydm members , 97 ( 48.5% ) were non - members and 1 ( 0.5% ) respondent did not state his / her status . out of the 102 current ydm members , 49 ( 48% )
were fps while 53 ( 52% ) were aspiring fps . among the 97 respondents who were not yet members of ydm
, there were more aspiring fps ( 66 out of 97 ; 68% ) than fps ( 31 out of 97 ; 31.9% ) , which was statistically significant ( = 4.69 , df = 1 , p = 0.03 ) .
of the 102 ydm members : 38 ( 39.2% ) belonged to vasco da gama , 23 ( 23.7% ) to afriwon renaissance , 13 ( 13.4% ) to waynakay , 11 ( 11.3% ) to wonca polaris , 5 ( 5.2% ) to spice route movement , 5 ( 5.2 ) to al razi movement , 2 ( 2.1% ) to rajakumar movement and 5 did not state their ydm .
with regards the annotated reasons for joining a ydm , ydm members had the highest mean rank score on item 6 ( i.e. , the opportunity to socialise with other fps abroad ) followed by the desire to socialise with fps within their country .
non - ydm members had the highest mean rank score on item 4 ( i.e. , the need for mentorship ) followed by the desire to improve their practice .
their least mean rank score was item 6 , the need to socialise with other fps in their country [ table 1 ] .
statistically significant difference in the mean rank score for ydm members and non - members was noted for item 2 ( joining to improve their practice ) , item 4 ( opportunity for mentorship ) and item 9 ( meeting academic needs beyond what their institutions could ) [ table 2 ] . comparing ydm and non - ydm members for each annotated reason for membership difference in the sum of rank scores of the responses from ydm and non - ydm members
respondents from vasco da gama had the highest mean rank scores for items 2 , 3 , 5 , 7 , 8 and 10 ; while those from spice route had the highest mean rank scores for items 1,4,9 and 11 .
respondents from waynakay had the highest mean rank score for item 6 [ figure 1 ] .
significant differences for the mean rank scores were seen for items 2 , 5 , 9 and 10 [ table 3 ] . comparing the various ydms for each annotated reason for membership difference in mean rank scores of the responses from the various ydms irrespective of the type of ydm , being an aspiring fp
was associated with joining / wanting to join a ydm for recognition as a fp ( = 4.72 , df = 1 , p = 0.03 ) and the desire to improve practice ( = 5.89 , df = 1 , p = 0.02 ) , while respondents younger or equal to 32 years of age were associated with joining / wanting to join a ydm for foreign exchange ( = 4.83 , df = 1 , p = 0.03 ) [ tables 46 ] . a cross tabulation of participant 's status and their response to gaining recognition as a reason for joining a ydm a cross tabulation of participant 's status and their response to improvement of their practice as a reason for joining a ydm a cross tabulation of participants ' age group and their response to foreign exchanges being a reason for joining a ydm
this survey has provided insight into young / aspiring fps motives for current or desired ydm participation . to the authors knowledge , this is the first study addressing the objectives enumerated above ; hence comparisons with similar studies are limited .
socialising with other fps abroad and in their country were the two foremost reasons stated by ydm members for their membership .
this is not surprising considering the role of the social media in connecting young professionals locally and globally and suggests that ydm members are interested in social networks with other young fps . these findings support observations made in an article describing young doctors as generation x and generation y and saying that being a doctor is interesting and good and i hope i am good at it , but there are a lot of other things in my life that are equally important .
this description may well fit these respondents and thus suggests that young fps / aspiring fps who are ydm members look beyond work to building relationships with their kind .
anecdotal findings at recent ydm activities have shown that young fps interact with their cohort across the globe , travel and attend conferences with interactions transcending academics or research in order to socialise and create bonds .
this is in contrast to findings suggesting that physicians tend to work in isolation with 50% of their professional interactions being patient referrals and the remaining 50% consisting of collegial interactions with friends / co - workers and associations within practice groups or hospitals .
these findings do not negate the professional outlook of ydms where respondents appear to have invested in social networks from which academic / professional ideals are developed .
this is in contrast with non - ydm members who had a predominant academic / professional motive for wanting to join a ydm .
the reason may not be unconnected with the fact that most of the non - ydm respondents were aspiring fps seeing ydms as a means to gain recognition and improve their practice . perhaps a predominant social motive for ydm membership is the young fp 's way of coping with the demands of the profession . looking for differences among different ydms , vasco da
gama participants expressed the greatest interest in : improving their practice , collaborative research , foreign exchange , socialising with young fps abroad , global opportunities and social needs beyond what their institution could offer .
this is not surprising considering that this was the first movement with the opportunity to grow into a larger organisation compared to other ydms .
it has been successful in its ability to create a brand and market fm in unique ways that appeal to young fps across europe .
on the other hand , spice route respondents indicated their greatest interest in : gaining recognition as fps , opportunities for mentorship , academic / professional needs beyond what their institution could offer and opportunities for medical leadership .
the first 2 of 10 items contained in the mission / vision statement of the spice route movement reads thus :
foster mentoring between current and future leaders in family medicine in the south asia region and promote representation by young family medicine leaders in the south asia region .
if these are taken as their top priorities , then these findings reflect the unique needs of young fps in this region .
interestingly , despite the perceived benefits of mentoring as described by the national college of physicians , the least number of ydm members indicated the need for mentoring as reason for joining .
the spice route movement may have employed a successful model in helping bridge the generation gap .
this may not be unconnected to the societal influence and the respect for elders displayed in this region .
perhaps other regions may benefit from this model or allowing for societal differences , they may choose to add a peer - based mentoring program to existing models of mentoring .
respondents from waynakay expressed that their main reason for participation was to socialise within the country .
this suggests that young fps from this ydm preferred a national social network probably as a result of issues of trust and an aversion for risk or a preference for already established identity , practices and norms .
it brings to the fore an existing academic discourse on social capital in which bonding social capital was referred to as inward looking bonds , focusing on relationships and networks of trust and reciprocity that reinforce ties within groups .
this is in contrast to bridging social capital which is concerned with outward looking connections amongst heterogeneous groups .
this study 's strengths include being able to obtain responses from young / aspiring fps across all wonca regions and as such achieved good coverage despite being unable to attain the minimum sample size and equal representation of ydm respondents .
it also provides useful suggestions on the predominant needs of young and aspiring fps as well as their motives for ydm membership .
this will no doubt guide current and future efforts in providing leadership for this unique group
while recognition as a fp and improving one 's practice may be the ultimate goal of an aspiring fp ; socialising within a network of like - minded professionals , maybe the young fp 's way of coping with the demands of the discipline . | background : over the past decade , young doctor movements ( ydms ) have gained recognition for their efforts in promoting the discipline of family medicine . with growth and expansion comes the need for an inquiry into the membership motives of current / intending members.aim and objectives : this study was aimed at determining the main reasons why young and aspiring family physicians ( fps ) joined their regional ydm .
it was also concerned with determining the main factors that will make non - members want to join a ydm as well as assessing for differences in the responses within ydm members on the one hand , and between ydm members and non - members on the other.materials and methods : this was a cross - sectional web - based study . using a list of 11 items generated following a series of discussions and feedback among selected fps and fp trainees , respondents annotated levels of agreement on reasons for current or desired ydm membership .
the mann
whitney u test was used to determine the distribution and differences in the mean of rank scores of the responses from ydm and non - ydm members while the kruskal
wallis test was used to describe same for the various ydms.results:the total number of respondents was 200 , out of which 102 ( 51.0% ) were current ydm members , 97 ( 48.5% ) were non - members and 1 ( 0.5% ) respondent did not state his / her membership status .
non - ydm members indicated a predominantly academic / professional motive for membership while ydm members indicated the opportunity to socialise with fps abroad and in their country as their foremost reasons for membership .
a mixture of academic , professional and social motives was observed for respondents from vasco da gama ; predominantly academic and professional motives for respondents from spice route.conclusions:while gaining recognition and improving one 's practice may be the ultimate goal of an aspiring fp , socialising within a network of like - minded professionals maybe the young fp 's way of coping with demands of the discipline . | Introduction
Objectives
Materials and Methods
Results
Discussion
Conclusion |
though implant dentistry is very successful and predictable in treatment of patients with destroyed dentition , there are some cases with limitations to implant therapy . in these cases ,
a patient with destroyed dentition was rehabilitated with a lateral rotational path removable partial denture . according to the diagnosis
the edentulous patients with compromised esthetics and functions can be successfully treated with a rotational path removable partial denture through adequate treatment planning and precise laboratory procedure .
patients with destroyed dentition have several characteristics such as loss of posterior occlusal support , severe occlusal wear of remained teeth , missing of multiple teeth , etc .
the loss of posterior occlusal support may cause following problems : the attrition of anterior teeth , the reduction of vertical dimension ( vd ) , and the alteration of normal occlusal plane .
these problems will eventually cause the unesthetic facial appearance , the pathologic changes of the temporomandibular joint ( tmj ) and masticatory muscles , and the inability of mastication.1 patients with destroyed dentition require extensive restorative treatment.1 in such cases the restoration with implant - supported crowns and fixed partial dentures is preferred .
however , in patients with financial , anatomical , and/or medical limitations alternative treatment modalities , such as removable partial denture ( rpd ) , should be considered.2 the edentulous patients with compromised esthetic zone can be successfully treated with a rotational path rpd.3 - 9 rotational path rpd has been often overlooked by the dental profession due to its complex prosthetic design and sensitive laboratory techniques.10 with better understanding of the principles of rotational path rpd , the dental clinician can deliver excellent esthetic outcome in compromised areas in which other treatment options may often be limited .
the rotational path design concept uses a rigid retainer portion of the framework as the retention component.11 the proximal plate provides retention through its intimate contact with the proximal tooth surface below the height of contour at a zero - degree tilt .
these rigid retentive components have dual paths : the first path to engage into the undercuts and the second rotational path to fully seat the prosthesis.3,10,12 there are three basic types of rotational path rpds : anterior - posterior ( ap ) , posterior - anterior ( pa ) , and lateral .
jacobson and krol narrowed the rotational paths to two categories.11 category i includes all prosthesis designs that first seat the rest that is associated with the rigid retainer .
after the first rest is seated , the second segment rotates into place and conventional clasps are engaged.9 the rotational centers are located at the end of long rests .
category i includes all pa and ap paths of insertion that replace posterior teeth.10 category ii includes all lateral paths and ap paths that replace anterior teeth .
the centers of rotation and the rigid retainers are located at the gingival extension of minor connectors.9,11 these prostheses use a dual path of insertion : at first rpd is inserted straightly from an incisal or occlusal direction to the rotational centers and then the conventional posterior or contralateral retainer is rotated into position .
it is important that the initial straight path of insertion must be parallel with the cingulum rests to permit complete seating .
the rotational path rpd offers the advantages of improved esthetics by eliminating anterior clasps , shortened treatment duration , cleanliness , and lower treatment cost over implants or fixed partial dentures.5,6,11
the patient was a 70 year - old female complaining of inability of mastication and unesthetic facial appearance .
typical features of destroyed dentition were observed in the clinical and radiographic examinations ( fig .
1 ) . however , she had several problems : severe attrition of almost remained teeth , # 14 horizontal root fracture , # 13 periapical lesion and buccal fistula formation , insufficient interocclusal distance , loss of anterior guidance .
she had fear of surgery , financial limitations and severe ridge resorption of esthetic zone .
for these reasons , we decided that not implant prosthesis but removable prosthesis was indicated for her . in this case , the extremely worn anterior dentition was planned to be restored with fixed prosthesis .
all attrited teeth were restored with single porcelain - fused gold ( pfg ) crowns except four lower incisors , which were restored with 4-unit splinted pfg fixed dentures due to their very short crown lengths .
the rotational path rpd is the excellent treatment modality of restoring missing incisors when patients have contraindications for implants or when patients are financially limited .
preliminary impression was made and then recording base and wax rim were fabricated on the diagnostic casts for vd determination and centric relation ( cr ) registration .
anatomic landmarks , facial measurements and the resting positions of mandibular jaw were used to determine appropriate vertical dimension for the patient . as the result
after that , cr registration was taken using bilateral manipulation1 , and diagnostic wax up was performed to confirm the need of increasing vd for esthetic and functional rehabilitation .
prior to teeth preparation , composite resin was filled in the incisal and occlusal surfaces of all attrited teeth .
this conservative correction was for conservation of destroyed tooth structure and ideal tooth preparation.1 teeth were prepared using a omni - vac as the preparation guide . to compensate for short clinical crown lengths ,
2 ) . while the patient was wearing the provisional restorations for the 4 months , occlusal stability and tmj were periodically checked .
thus , it was decided to reproduce of temporary vd and cr state on the final restoration .
it was performed using the recording base , waxrim and two pattern resin ( dura lay , reliance , illinois , usa ) coping crowns .
pfg crown copings were tried in the mouth to check for marginal fit . before glazing ,
all pfg prostheses were cemented with resin modified glass ionomer cement ( fuji - cem , gc , tokyo , japan ) . to fabricate rpd framework , upper and lower master casts were made .
1 rpd and was designed in lateral rotational path rpd with no clasp in the anterior region . lateral or category ii rotational path rpd in this report was surveyed in the next two steps ( fig .
the first surveying was performed at a zero - degree tilt to identify the mesial surface undercut of the anterior abutments ( at least 0.010 inch ) and buccal undercuts of the posterior abutments .
the diagnostic cast was then tilled upward until the mesial undercuts on the anterior abutments were eliminated .
the cast was again surveyed to determine if the anterior rest seats are accessible during the initial straight path of insertion .
both rpd frameworks were tried in the mouth and adapted with silicone material ( fit - checker , gc , tokyo , japan ) .
a face - bow transfer was completed , and interocclusal registration was made with wax rim and bite material ( o - bite , dmg , hamburg , germany ) .
denture teeth were set up to establish the stable and harmonious occlusion as such13 : ( 1 ) simultaneous bilateral contacts of opposing posterior teeth in the centric occlusion ( co ) ( 2 ) unilateral balanced occlusion by denture teeth for left working side contacts ( 3 ) canine guidance by natural teeth for right working side contacts ( 4 ) contacts of opposing anterior teeth in the co. after obtaining the patient 's approval , rpds were processed using pink denture resin ( rapidsimplified , vertex , zeist , netherlands ) .
the finished rpds were placed in the mouth , and the following criteria were evaluated : adaptation of the clasps and rests , retention of the rpd , esthetics , and occlusion ( fig .
6 ) . finally the patient was instructed in placing the prosthesis and maintaining oral hygiene .
treatment of patients with destroyed dentition is very difficult clinical procedure and challenging for dental profession . in this case ,
proper diagnosis and treatment plans are the necessity for not only esthetic and functional restorations but also the stability and adaptation of the neuromuscular system and tmj . for compromised patients to implant dentistry ,
it improves esthetics to replace missing teeth without placing a conventional clasp in the esthetic region .
it is important that dentists evaluate each patient carefully and survey casts meticulously to ensure success . | backgroundthough implant dentistry is very successful and predictable in treatment of patients with destroyed dentition , there are some cases with limitations to implant therapy . in these cases ,
alternative treatment modality should be considered.case descriptiona patient with destroyed dentition was rehabilitated with a lateral rotational path removable partial denture . according to the diagnosis , we determined to raise vertical dimension for esthetic and functional restoration .
the final restoration was performed after four months of provisional period.clinical implicationthe edentulous patients with compromised esthetics and functions can be successfully treated with a rotational path removable partial denture through adequate treatment planning and precise laboratory procedure . | BACKGROUND
CASE DESCRIPTION
CLINICAL IMPLICATION
INTRODUCTION
CASE STUDY
CONCLUSION |
diabetes therapies can negatively affect health status / health - related quality of life ( hrqol ) because of regimen complexity or rigidity , fear of injections , hypoglycaemia and fear of hypoglycaemia 1,2 .
thus , reducing hypoglycaemia and providing a more predictable treatment regimen might be expected to improve hrqol .
insulin degludec ( ideg ) is a new basal insulin that offers advantages over previous basal insulins .
action profile , an ultra - long duration of action lasting beyond 42 h , and low within - patient variability 3 .
seven randomised , open - label , controlled , phase 3 trials of 26- or 52-weeks duration using a treat - to - target non - inferiority design , all confirmed similar glycaemic control for ideg compared to insulin glargine ( iglar ) as expected in treat - to - target trials .
however , individual trials , as well as a prospectively planned meta - analysis , showed advantages for ideg over iglar with respect to hypoglycaemia 4 .
hrqol , another important treatment outcome , was measured in all of these trials using the sf-36 v2 questionnaire 5 and additional meta - analyses have shown that hrqol 6 , health utility 7 and mental health 8 are improved with ideg compared to iglar .
the analysis presented here includes previously unreported data from insulin - nave patients with type 2 diabetes who were studied for a total of 2 years in a 52-week trial with a 52-week extension ; this was the largest of the phase 3 trials in the ideg clinical development programme 9 . as previously reported 10 , at 105 weeks
, hba1c was similar for ideg and iglar ( estimated treatment difference [ etd ] 0.12%-points [ 0.01 to 0.25]95% ci , p = 0.078 ) .
observed mean reductions in laboratory - measured fasting plasma glucose ( fpg ) were greater with ideg ( etd 0.38
mmol / l [ 0.70 to 0.06]95% ci , p = 0.019 ) .
importantly , these improvements in glycaemic control were accompanied by a lower risk of hypoglycaemia over the entire treatment period . nocturnal confirmed hypoglycaemia was 43% lower with ideg than with iglar ( estimated rate ratio [ err ] , ideg / iglar : 0.57 [ 0.40 ; 0.81]95%ci , p = 0.002 ) and rate of severe hypoglycaemia was 69% lower with ideg ( err 0.31 [ 0.11 ; 0.85]95%ci , p = 0.023 ) . overall confirmed hypoglycaemia ( plasma glucose < 3.1 mmol / l or severe ) was similar between ideg and iglar ; estimated rate ratio ( ideg / iglar ) : err 0.84 [ 0.68 ; 1.04]95%ci , p = 0.115 10 .
given that the clinical benefit of reduced hypoglycaemia occurred in conjunction with improved glucose control after 2 years of treatment , improvements in hrqol might also be expected . in the original 52-week trial , participants treated with ideg showed improvements in clinical measures and reported greater improvements in
overall physical and physical functioning short form 36 ( sf-36 ) scores compared with iglar 9 .
the aim of this subanalysis at 105 weeks was to compare ideg versus iglar with respect to change in health status from baseline ( initiation of basal insulin therapy ) in patients with type 2 diabetes , using the sf-36 v2 questionnaire .
subjects provided informed consent and re - started treatment with ideg or iglar at the same dose levels at end - of - treatment in the main trial 9 with optional adjustment at the discretion of the investigator . at the end of the core trial , patients were transferred to neutral protamine hagedorn insulin for 7 days to allow for insulin antibody evaluation .
the insulin dose at the end of the core trial was used as a starting point , and was titrated to a target fpg of 3.94.9 mmol / l based upon the mean pre - breakfast self - measured blood glucose for the preceding 23 consecutive days .
all assessments for efficacy and safety in this extension trial were as described for the core trial 9 .
health status was measured at baseline ( time of randomisation of the core trial ) , at 52 weeks , and at 105 weeks using the sf-36 v2 questionnaire .
the sf-36 scale is a validated multi - purpose questionnaire that has raw , norm - based scores with a mean ( standard deviation ) of 50 ( 10 ) .
the sf-36 questions are grouped into eight domains ( figure 1 ) , including a physical component summary ( pcs ) score and a mental component summary score .
patients were provided with instructions and given privacy to complete the questionnaires , which were sealed in envelopes and not reviewed by local investigators or study nurses .
scores from the intent - to - treat population were analysed using anova , with treatment , therapy at screening , sex and region as fixed factors , and age and relevant baseline values as covariates .
the long study duration was considered sufficient to ensure that any initial patient reporting bias was minimal .
as previously reported , 607 ( 79% ) of 773 patients randomised to ideg and 197 ( 77% ) of 257 randomised to iglar completed the core trial of 52 weeks 10 .
a total of 551 ( 71% ) of those initially randomised to ideg and 174 ( 68% ) of those initially randomised to iglar continued into the extension study on the core phase treatment allocation , with 505 ( 65% ) of those continuing on ideg and 154 ( 60% ) continuing on iglar completing the entire 104 weeks of treatment .
baseline characteristics have previously been reported , and were well - matched between treatment groups 10 .
health status results for the full analysis set are summarised in table1 . at 105 weeks ,
the changes from baseline in overall pcs score , physical functioning score and bodily pain score were statistically significantly ( p < 0.05 ) in favour of ideg compared with iglar , with etds ( ideg / iglar ) of 1.1 [ 0.1 ; 2.1]95%ci , 1.1 [ 0.0 ; 2.3]95%ci and 1.5 [ 0.2 ; 2.9]95%ci , respectively .
when only completers were used , scores tended to further improve in favour of ideg compared to the full analysis set , but because of the lower number of respondents and loss of statistical power , only the bodily pain domain remained statistically significant .
short form 36 ( sf-36 ) scores at end - of - trial ci , confidence interval ; ideg , insulin degludec ; iglar , insulin glargine ; od , once daily . significantly better ( p < 0.05 )
. data are estimated mean standard error of the mean , full analysis set .
the score and change from baseline in the score at 105 weeks were analysed using an ancova model .
the statistically significant difference in pcs , physical functioning score in favour of ideg was maintained after 2 years treatment as also reported in the core 52 weeks of the trial 9 .
in addition to sustaining the significant difference in pcs and physical functioning , the bodily pain domain also was significantly in favour of ideg after 2 years .
these results are consistent with meta - analysis results of previous studies of ideg reporting improvements in sf-36 scores 6 and improvements in health utility versus iglar 7 .
similarly , earlier studies have reported improvements in various patient - reported outcomes when iglar was compared against oral therapy or other basal insulins 11 .
with respect to the clinical implications of these results , in diabetes , it has been estimated that a 1-point difference on pcs and physical functioning scores , for example , is associated with a 59% increase in mortality risk , a 24% increased risk of hospitalisation within 6 months and a 712% increased risk of being unable to work 12 .
although the sf-36 does not have an established minimal important difference in diabetes , the sf-36 v2 user manual suggests that even small differences in these compressed scores ( < 1 ) are relevant in other chronic diseases .
the precise reason for the improvement in sf-36 scores has not been determined . however , as mentioned earlier , ideg has been shown to have a beneficial effect on hypoglycaemia compared to iglar 4 .
hypoglycaemic events , both non - severe and severe , are known to be a key marker for reduced hrqol in diabetes 1 .
also , the sf-36 physical functioning score includes measures of vigorous activity , which may be enhanced if hypoglycaemia is less of a concern .
given that clinical trials exclude people at high risk of hypoglycaemia , the benefit may be even greater in real - world settings .
although an open - label design could have affected results of this study , the baseline values for sf-36 were collected prior to randomisation , and the long duration of the main trial plus extension study should have allowed any bias associated with initiation of a particular therapy to be minimised .
this study emphasises that patient - reported outcomes , which are an important measure of any diabetes treatment , can be improved with ideg .
h. w. r. has received grants / research support from amylin , eli lilly , merck and sanofi - aventis ; served as a consultant for biodel ; served on advisory panels of amylin pharmaceuticals , roche diagnostics , astrazeneca , biodel , novartis , novo nordisk , janssen , bristol - myers squibb and sanofi - aventis ; served on the speakers bureau for amylin pharmaceuticals , astrazeneca , bristol - myers squibb , boehringer ingelheim , eli lilly , merck and co. , and novo nordisk .
b. c. has served on the advisory panel for eli lilly and company , genfit , novo nordisk and sanofi - aventis and received grants / research support from novo nordisk and sanofi - aventis .
b. z. has served on the advisory panel for eli lilly and company , novo nordisk , and sanofi - aventis and received research support from novo nordisk .
y. h. has received grants / research support from boehringer ingelheim , conjuchem , daiichi sankyo , glaxosmithkline , lexicon , novo nordisk , takeda , sanofi - aventis , xoma , and tolerx ; served as consultant for amylin pharmaceuticals , daiichi - sankyo , gilead , genetech , glaxosmithkline , novo nordisk , merck , xoma , tolerx , janssen , halozyme , amarin , liposcience and santarus ; served on speakers bureau for amylin , astrazeneca , boehringer ingelheim , bristol - myers squibb , daiichi - sankyo , eli lilly ,
glaxosmithkline , novo nordisk , and santarus ; and is president of the american association of clinical endocrinologists . m. l. w. and a. r. are employees of novo nordisk and own stock in the company .
c. m. has served on the advisory panel for novo nordisk , sanofi - aventis , merck sharp and dohme ltd .
, eli lilly and company , novartis , bristol - myers squibb , astrazeneca lp , pfizer , johnson and johnson , and mannkind ; has received research support from novo nordisk , sanofi - aventis , merck sharp and dohme ltd . ,
eli lilly and company , and novartis ; and has served on the speakers bureau for novo nordisk , sanofi - aventis , merck sharp and dohme , eli lilly and company , and novartis .
all authors ( h. w. r. , b. c. , b. z. , y. h. , m. l. w. , a. r. and c. m. ) were involved in critical analysis and interpretation of the data , drafting / critically revising the article and shared in the final responsibility for the content of the manuscript and the decision to submit it for publication . | insulin degludec ( ideg ) is a new basal insulin with an ultra - long and stable glucose - lowering effect .
we compared once - daily ideg and insulin glargine ( iglar ) , both in combination with metformin dipeptidyl peptidase-4 inhibitors , in a 52-week , open - label , treat - to - target trial in patients with type 2 diabetes followed by a 52-week extension trial in which subjects [ n = 725/1030 ( 70.4% ) ] maintained their initial randomised treatment . health status was assessed at baseline and 105 weeks using the short form-36 ( sf-36 v2 ) questionnaire .
sf-36 scores were analysed ( itt population ) using anova , with adjustments for covariates . at 105 weeks , the overall physical component score was significantly better with ideg versus iglar [ treatment contrast ( tc ) : 1.1 ( 0.1 ; 2.1)95%ci , p < 0.05 ] .
this was largely because of significantly better physical functioning [ tc : 1.1 ( 0.0 ; 2.3)95%ci , p < 0.05 ] and bodily pain sub - domain scores [ tc : 1.5 ( 0.2 ; 2.9)95%ci , p < 0.05 ] .
improvements in health status with ideg compared to iglar were maintained after 2 years . | Introduction
Methods
Results
Discussion
Conflict of Interest
Author declaration |
preterm birth , defined as birth prior to 37 weeks of gestation , is a leading cause of infant morbidity and mortality worldwide and has been increasing [ 2 , 3 ] . since 1990 , the number of preterm births has risen by 20% and is even more significant when the increased costs associated with preterm pregnancies are considered . however , despite advances in identifying some of the causes of preterm birth , our understanding of the physiologic process leading to preterm labor is poorly understood
. a growing body of literature has been examining possible proteomic markers for preterm labor and for intrauterine infection , a leading cause of preterm labor , in maternal serum [ 8 , 9 ] and in amniotic fluid [ 9 , 10 ] .
on the other hand , metabolomics , which is the summation of ongoing cellular activity and downstream of protein metabolism [ 9 , 11 ] , may provide another interesting approach .
these small molecules are the products of protein metabolism and cellular function within an organism .
when examined as a whole , these metabolites can be viewed as biomarkers of a functional phenotype . in the case of preterm labor ,
differences in metabolomic profiles found in amniotic fluid are thought to be possible since biological processes of the fetus and the mother both impact on its biochemical composition
. one successful approach to measure metabolomic profiles uses near - infrared ( nir ) spectroscopy .
the use of nir vibrational spectroscopy preserves the matrix of constituent metabolites and provides important information about the interactions among the various constituents in situ
. this can provide insight into metabolism , based on relational properties that can not be captured when individual components are measured .
metabolite profiling using nir spectroscopy has been used to detect disease in different scenarios where discrimination between groups is an objective .
specifically , the application of nir spectroscopy to amniotic fluid has been used to predict fetal lung maturity [ 14 , 15 ] .
differences in these metabolomic profiles obtained by nir spectroscopy also employ multivariate regression models and optimization functions [ 16 , 17 ] .
this pilot study was undertaken to test the hypothesis , using nir spectroscopy , that differences in the metabolomic profile exist in second trimester amniotic fluid samples for term ( 37 weeks ) compared to preterm births ( 35 weeks ) .
we propose that identifying the existence of a metabolic fingerprint for preterm labor early in pregnancy could be of major importance in the appropriate ongoing monitoring of at - risk pregnancies and the development of a better understanding of the biologic basis of preterm labor .
this was a retrospective cohort study , approved by both the mcgill institutional review board and st .
the population included 227 subjects recruited between 2000 and 2003 who provided a small volume of amniotic fluid for spectral profiling using nir .
inclusion criteria for term births ( n = 216 ) included age - related amniocentesis for genetic testing and a singleton pregnancy with no fetal complications .
the preterm group included only patients with premature rupture of members ( prom ) and/or preterm labour and a spontaneous vaginal delivery ; patients who were induced or had a c - section were excluded .
the two study groups were similar with respect to both maternal age and maternal bmi .
the af samples were obtained following genetic testing and stored at 80c ; there is minimal source of biochemical error resulting from repeated freezing and thawing of amniotic fluid [ 18 , 19 ] . to determine the feasibility of estimating true premature births using spectral analysis of 2nd trimester amniotic fluid collected at 1220 wks gestation
, a calibration model was constructed using a set of af samples with known birth outcomes in the nir region of the spectra ( 7001050 nm ) .
this spectral region is known to contain functional group information on overtone bands of ch , nh , and oh moieties .
glucose , proteins , fatty acids , oils , and myoglobin have been identified as contributing to the absorbance in this nir region [ 2123 ] .
prior to analysis , frozen af samples were thawed at room temperature ( 25c ) for 30 minutes .
nir profiles were analyzed using a reflective spectrograph with a ccd detector ( b&w tek , newark , de ) in randomized order . a flow sample cell with 10 mm path length was filled with 15 l of sample media .
spectra were recorded from 7001050 nm at room temperature ( 25c 1c ) .
the spectrophotometer was set to measure absorbance relative to air , and a signal average of 200 measurements with an integration time of 100 ms for each measurement was used . this measurement procedure
involved rinsing the sample cell with 1 ml of 0.1 m naoh followed by 5 ml of distilled , deionized water .
quantification of the sample properties from nir spectra consisted of determining the most parsimonious combination of variables in selected wavelength domains using a genetic algorithm optimization . in this method
preprocessing based on haar wavelets , which is similar to jpeg compression of images , was used to objectively select wavelength regions . a combination of wavelength regions that most parsimoniously estimated prematurity index was determined by inverse least - squares regression , using a genetic algorithm optimization .
the model investigated is of the form
( 1)y=0+1x1+2x2++nxn ,
where y is the dependent variable or prematurity index ( pi ) , x1 , x2 ,
, xn are independent variables ( i.e. , integrated wavelength region ) , and 0 , 1 , , n are the coefficients determined from a set of calibration x 's .
many models were screened using the ga which is based on genetic principles such as mating , crossover , and mutation , to select the wavelength region that best separates the term and preterm groups .
each sample was estimated independently using a leave - one - out cross - validation approach in a continuous multilinear model .
for each individual in the population , the coefficients 1 to n of ( 1 ) were calculated by inverse least - squares regression using an independent calibration .
estimates of prematurity index were obtained by applying ( 1 ) with the determined n parameters and the x - values of a monitoring set .
fitness of the model for each wavelength region selected was calculated as the squared difference between the mean of the term and preterm groups divided by the sum of the pooled variance .
a higher fitness corresponds to better separation between the two groups as calculated by a student 's t - test .
subsequent to the estimated prematurity index ( pi ) optimization , notched box plots , student 's t - test results , and a receiver operator curve ( roc ) were used to separately determine the statistical characteristics of the groups .
the optimum value for the sensitivity and specificity was determined from the roc [ 24 , 25 ] .
additionally , spectra were examined using the major chemical groups present in the 7001050 nm region of the nir spectra . molecular absorbance regions in the nir related to h2o , roh , ch2 , and
means and standard deviations were then calculated for normal ( 37 wks ) and preterm ( 35 wks ) using these integrated regions .
likewise , ratios of pairs of selected functional groups ( nh3/ch2 , nh3/roh , ch2/roh ch2/roh , nh3/h2o , and ch2/h2o ) were determined to characterize concentration shifts in glucose , proteins fats and oils relative to water as well as polyols , which may play important roles in maintaining atp concentrations , cellular redox potential , and in drawing water and solutes across the placenta .
all statistical analysis was done using the matlab ( the math works inc . , ma ) programming package .
figure 1 describes nir spectral differences for each individual in the range of 7001050 nm . of all of the possible wavelet combinations , only two wavelet regions were needed to develop the best model to distinguish preterm from the term births .
those wavelet regions selected by the genetic algorithm were at 872879 nm ( region a ) and 943954 nm ( region b ) , respectively .
the first wavelength region corresponded to third overtone ch3 and second overtone nh group vibrations .
wavelet b selected an absorbance region characterized by aliphatic alcohol functional groups and third overtones from ch vibrations in the 943 to 954 nm wavelength range .
a parsimonious calibration model was constructed with the 2 wavelets selected by the genetic algorithm . using these regions , the prematurity index ( pi )
each data point represented a blind estimation of the optimal separation value using the rest of the data for calibration .
results are represented in figure 2 as notch box plots showing the statistical distribution for each group of classified samples . a relative prematurity index ( rpi )
the size of each box was determined by the quartile distribution about the median of the data .
the median of the term group was 1.00 0.02 and was 0.78 0.08 for the preterm group .
notches of box plots , which do not overlap , have different medians at the 5% confidence level .
in addition , the mean and standard deviations of preterm and term prematurity indices were 0.77 0.08 and 1.00 0.02 , respectively ( p < 0.001 ) . table 2 summarizes the results of the model 's validation . using only the 2 components for the calibration model
, af samples were classified into preterm and term groups with 100% sensitivity and specificity determined by a roc curve .
positive and negative predictive values were 100% ( table 2 ) . to further understand the relationship of the measured spectra to prematurity , differences in the amniotic fluid metabolomic profile between preterm and term infants arising from changes in selected spectral regions were examined .
regions selected for integration of the various functional groups as well as normalized spectra for the means for both the normal and preterm groups are shown in figure 3 . there were substantial metabolomic differences in the spectra between term and preterm infants .
as well , ratios of the integrated signals from the different functional groups ( table 3 ) suggested nonsignificant trends in the concentrations of functional groups relative to water and/or polyols .
there was a relative increase in concentration of protein as compared to carbohydrates and fats ( nh / ch ratio ) even though both decrease in total concentration as reflected in thenh / h2o and ch / h2oratios .
there was also nonsignificant increase in the nh / roh ratio and a decrease in the ch / roh ratio related to preterm births .
genomics , proteomics , mass spectroscopy , and other assays have been used for analysis of maternal and fetal biofluids with poor success rates [ 8 , 9 , 14 , 15 ] .
in contrast , the results of this pilot study showed that nir spectral waveforms differed significantly in the range 7001050 nm between term and preterm births .
varying concentrations of several functional groups contributed to the variation in these spectral analyses , confirming our hypothesis that metabolomic differences exist early in pregnancy in the amniotic fluid of second trimester pregnancies between subjects delivering preterm and those delivering at term .
although the spectral features are broad , absorbance is still directly related to the concentration of ch , nh , and oh functional groups within the matrix .
these differences seen in the preterm group corresponded to ch3 and nh ( region a ) , aliphatic alcohols and ch functional groups ( region b ) .
presence of proteins , water , and polyalcohols in af are well known and can differ in concentration from sample to sample .
our findings are intriguing in that they suggest the underlying processes ultimately leading to preterm delivery are present between 12 and 20 weeks of gestation .
prior studies have focused on identifying biomarkers present in amniotic fluid at the onset of preterm labor [ 8 , 9 , 14 , 15 ] .
likewise , limitations such as af samples taken after 22 weeks gestation , destructive analytical methods , and small populations plague many existing techniques [ 6 , 9 , 10 , 14 , 15 ] .
others have cited inability to distinguish preterm from term groups and low positive predictive values .
techniques from proteomics and genomics also have characterized few biomarkers in af , thus limiting the amount of information about bioprocesses that might be associated with the amniotic fluid matrix [ 911 ] .
the differing metabolomic profiles seen here in early pregnancy may represent an abnormal metabolic process in the fetus that ultimately predisposes the pregnancy to insults resulting in preterm labor and delivery .
in addition , many recent technologies using proteomics and genomics rely on invasive procedures , expensive equipment , and technical expertise [ 6 , 810 ] .
the use of metabolomics with nir spectroscopy is relatively inexpensive , easy to use and can characterized a metabolomic fingerprint resulting from multiple biological processes .
these results , however , should be considered preliminariy since the number of patients in the preterm delivery group is small . despite this , our data using spectral analysis were able to distinguish between preterm and term deliveries with 100% sensitivity and specificity , which supports the potential diagnostic possibilities of this technique . even though we were not able to identify specific differences in the ratios of the functional groups , probably owing to our small sample size
, the strength of our results lies in the power of the spectral analysis to extract meaningful information about the metabolic profile from the 2nd trimester af matrix that was associated with distinct metabolomic fingerprints early in pregnancy in our preterm and term deliveries .
moreover , the suggested metabolomic profiles were consistent with previous studies that show increased protein in amniotic fluid of premature infants , alterations in polyols in iugr infants and with a higher incidence of oligohydramnios .
our results raise the question of whether obtaining metabolomic profiles of amniotic fluid in early pregnancy will help obstetricians identify those pregnancies destined to deliver preterm .
additionally , it may be possible to develop a noninvasive probe to analyze the nir spectra of amniotic fluid on an ongoing basis .
if future studies using larger sample sizes confirm these findings , this information would indicate that a problematic metabolomic profile emerges very early in pregnancy and could lead to much earlier identification of prematurity and earlier decision for potential therapies . | this pilot study investigated the possibility that metabolomic differences exist in second trimester of women delivering at term ( 37 weeks ,
n = 216 ) and preterm ( 35 weeks , n = 11 ) .
for this retrospective study , biobanked af samples underwent near - infrared ( nir ) spectral analysis using wavelengths from 700 to 1050 nm .
spectral data was compressed then optimized by multilinear regression to create a calibration model .
the resultant model was able to classify term and preterm births based on differing af metabolomic profiles with a sensitivity and specificity of 100% .
when groups were classified using a prematurity index ( pi ) , there was a statistical difference ( p < 0.001 ) between the predicted preterm group ( pi 0.77 0.08 ) and the term group ( pi 1.00 0.02 ) . in conclusion ,
the 2nd trimester af samples showed distinct differences in metabolomic profiles between patients delivering preterm as compared to those at term in functional groups related to proteins , carbohydrates , fats , polyols , and water . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
Conflict of Interests |
chilli peppers ( capsicum annuum l. ) are used in a multitude of food preparations and are marketed in different regions .
the greatest genetic variety of c. annuum l. can be found in mexico , where there are chilli peppers of many different shapes , sizes , and colours , and they are among the most frequently consumed products .
the genus capsicum is classified into the family of solanaceae , which is constituted by 25 wild and 5 domesticated species ( c. annuum l. , c. frutescens l. , c. chinense jacq , c. baccatum jacq , and c. pubescens l. ) , including more than 200 varieties [ 2 , 3 ] .
chilli peppers have been object of study mainly due to containing capsaicin , which produces different pungency levels , as well as carotenoids and phenolic compounds , which are used as natural pigments and antioxidant agents .
chilli peppers may have different content and profiles of such compounds , depending on the genotype and variety , maturity of the fruit , and the environmental conditions of the cultures . in order to preserve , manage , and improve the different chilli species , the evaluation of the extent of genetic variation within species , as this has now become a fundamental tool in biology and agriculture . among the main criteria used for that purpose
the rapd ( random amplified polymorphic dna ) method is a widely used technique for molecular marking and is based on the amplification of genomic dna fragments by using primers of arbitrary nucleotide sequences ; which in turn detects polymorphisms that can be employed as genetic markers without previous genetic sequences .
in addition to being simple , fast , and low - cost , this method does not require radioactive markers and consumes minimum amounts of dna [ 911 ]
. however , one of the inconveniences reported about the rapd technique , since it was published for the first time , is the low reproducibility found mainly in low - intensity bands .
the most important factor affecting reproducibility in rapd analysis is the preparation of the dna template .
thereby , the differences in the concentration of dna template between samples are observed as the gain or loss of some bands . recently , to improve the results , phytochemical markers have been combined with molecular techniques .
silva et al . correlated the flavonoid content of medicinal plants from brazil with molecular markers obtained by rapd , whereas ercisli et al .
( 20072008 ) [ 13 , 14 ] obtained rapid results for evaluation of genotypic diversity and distances by using methyl esters of fatty acids as markers .
previously , the content and composition of fatty acids were tested as taxonomic markers in hippophae rhamnoides l. , showing that these substances , such as linoleic acid , can be used as biochemical markers for this species . on the other hand , fller et al .
analyzed the variability of morphological characteristics as well as the content of phenolic compounds and essential oils in plants from southern brazil .
described the state of preservation of a meadow by comparing the content of phenolic compounds and the rapds .
another essential technique for determining the biological nature of agriculture products is fourier transform - infrared spectroscopy ( ftir ) , which is a technique that has been widely employed for food characterization because of several important advantages , such as its nondestructive nature and its capacity to yield structural information that constitutes a molecular fingerprint of the sample . a combination of ftir and chemometric techniques like principal component analysis ( pca ) has been employed as a direct and rapid way to discriminate properties in foods , including geographic origin , adulteration , and quality control .
the aim of the present study is to contribute to management and improvement of the different chilli species by determining the relationship between molecular and biochemical markers , such as the content of carotenoids and phenolic compounds among different commercial cultivars of chilli peppers from diverse geographic origins .
the samples were collected from central and north regions of mexico ( table 1 ) .
the chilli peppers were considered fresh when the samples presented bright green colour , and smooth , firm texture .
the mature specimens were derived from the green samples , by exposure to sunlight and moisture - free storage in the laboratory for 25 to 35 days .
the samples were considered mature when the chilli peppers had a bright red , yellow , or orange colour and less firmness and texture .
length , width , and weight of the fruits were determined in accordance to the mexican official standard .
briefly , the length was taken from the base to the apex of the fruit without considering the peduncle ; the width was measured at the widest part of the chilli pepper ; for the weight , an analytical balance was used .
the extraction of carotenoids from the chilli samples was carried out with 2 g portions of pulp and peel , which were mixed with diatomaceous earth and acetone at 10c .
afterwards , the extract was filtered at vacuum through whatman grade 2-filter paper and transferred to 10 ml of petroleum ether .
the oil phase was extensively washed with distilled water to remove the residue of acetone , while the residual water was removed by shaking with anhydrous sodium sulphate .
a 1 ml aliquot was used to determine the content of carotenoids using the molar extinction coefficient for -carotene in hexane at 450 nm .
the extracts of carotenoids were reconstituted in 500 l of hplc - grade ethyl acetate and filtered through nylon membranes ( 0.45 m , pore size ) and then introduced into the c18 column ( 25 cm 4.6 mm ; 5 m ) of the hplc equipment ( perkin elmer binary lc pump 250 ; series 200 , uv / vis detector ) .
an isocratic elution system was established with acetonitrile - methanol - ethyl acetate ( 73 : 20 : 7 ) working at 0.6 ml / min for 70 min , and the absorbance was measured at 450 nm .
a total of 2 g of pulp and peel chilli peppers were mixed with 8 ml of 80% ethanol , and shaken for 10 min at 200 rpm .
the sediment was reconstituted into 8 ml of 80% ethanol , and the procedure was repeated .
thereby , three extractions were obtained , joined , and stored under a nitrogen atmosphere until analysis .
volumes of 0.75 ml of folin - ciocalteau reagent ( 1 : 10 ) were added to 100 l of the extract of free phenolics , and the mixture was left to stand for 5 min in the dark .
afterwards , 0.75 ml of nahco3 60 g / l solution was added to neutralize the reaction .
the solution was left to stand for 90 min , and the absorbance was determined at 725 nm .
aliquots of 50 l of extract were injected in the hplc equipment ( perkin elmer binary lc pump 250 ; series 200 , uv / vis detector ) using a c18 column ( 25 cm 4.6 mm ; 5 m ) .
the mobile phase used was a binary system composed of 0.1% trifluoroacetic acid ( phase a ) and 100% acetonitrile ( phase b ) .
starting with 90% phase a and 10% phase b , the mixture was eluted to 55% phase a , 45% phase b in 50 min and to 60% phase a and 40% phase b in the last 10 min .
analyses of variance ( anova ) were performed along with student - newman - keuls test for differences between means , using sigmastat 3.5 software . principal component analysis ( pca ) of the ftir extract spectra was performed by using the opus quant ( version 6.5 ) and minitab ( version 14 ) software .
the dry extracts were reconstituted in petroleum ether , for carotenoids , and in ethanol , for phenolics , in order to obtain a semisolid consistency of the sample .
afterwards , the mixtures were analyzed in a ft ( fourier transform)-infrared spectrometer ( bruker vertex 70 ) , by using the atr ( attenuated total reflectance ) sampling method previously described .
briefly , a small sample amount is placed over a znse crystal , where the infrared radiation is propagated and interacts with the sample to obtain the corresponding spectrum , which is averaged from several data acquisitions .
the method described by allers and lichten was used for dna extraction , after modifications .
all samples of chilli peppers were washed with water , disinfected with sodium hypochlorite at 10% , 30% , and 70% ethanol , exhaustively rinsed with distilled water for 20 min and stored at 70c , under sterile conditions .
portions of 100 mg per sample were mixed with 400 l of lysis buffer ( 200 mm tris - hcl ph 6.5 , 70 mm edta ph 8 , 2 mm nacl , 2% pvp 40 , 20 mm sodium metabisulphite , 1% triton x-100 , 24 mm mgcl2/mgso4 , 0.1% spermine , and 0.1% spermidine ) previously heated at 60c .
the mixture was agitated and incubated at 60c for 60 min ; then it was transferred to ice , at which time 400 l of 10% pvp was added .
the mixture was agitated by inversion of the recipient and left to stand for 60 min at 20c .
afterwards , the blend was centrifuged at 4000 rpm for 30 min , and 400 l of each supernatant was transferred into new tubes , in order to add 5 l of 10 mg / ml rnasa for 10 min at room temperature .
immediately , the supernatants were precipitated by addition of 200 l of 10 m ammonium acetate and 600 l of cold isopropanol .
the mixture was then centrifuged at 4000 rpm for 30 min at 10c , the isopropanol eliminated , and the pellet washed with 1 ml of cold 70% ethanol , followed by centrifugation for 10 min under the same operation conditions .
finally , the ethanol was removed , and the pellet was dried for 20 min at room temperature .
the pellets were reconstituted into 200 l of 10 mm tris ph 8 , and the dna was precipitated again with 200 l of peg 8000 30%-nacl 1.2 m at 4c for 60 min .
the mixture was then centrifuged for 30 min at 4000 rpm and 10c .
the supernatants from peg were discarded , and the dna pellet was washed with 1.5 ml of cold 70% ethanol , followed by centrifugation for 15 min at 4000 rpm . the ethanol was eliminated and the dna pellet left to dry , and finally reconstituted into 200 l of 10 mm tris ph 8 .
the purity of the extracted dna was determined by absorbance at 260/280 ratio , and values greater than 1.8 were accepted for future procedures .
the dna was quantified by its absorbance at 260 nm . for dna amplification ,
a thermocycler ( bioer xp cycler ) was used under the following operation settings : 1 cycle of 5 min at 94c ; 40 cycles of 1 min at 94c ; 1 min at 36c , 2 min at 72c ; a final extension step of 10 min at 72c .
pcr reactions were performed in a volume of 25 l containing 1x pcr buffer , 50 nm of mgcl2 , 200 m dntp , 1.5 units of the enzyme taq dna polymerase ( invitrogen , brazil ) , and 40100 ng/l dna samples .
four independent reactions were assayed using 0.4 m oligonucleotide ope18 ( cggcccacgt ) , or 0.2 m oligonucleotide mfg17 ( cgcgttcttg ) , 0.2 m oligonucleotide mfg18 ( cggcccacgt ) , or 0.2 m c51 ( atcaacgtacgt ) and 0.2 m c52 ( gtcgacggacgt ) oligonucleotide mixture ( invitrogen ) [ 4 , 28 ] . the products of dna amplification were analyzed by electrophoresis in 1.5% agarose gel , using tae 1x buffer .
the gels were stained with ethidium bromide ( 0.2 l / ml ) and observed with an uv - transilluminator ( stratagene eagle eye ) . in order to increase reproducibility ,
optimum conditions were established for dna extraction and amplification , without modifying mg2 + concentration , for taq polymerase enzyme and primers as well as for the dna concentration in each reaction .
the polymorphic bands and signals of the chromatograms were considered as present or
absent in a matrix of similarity . for the chromatogram , only signals above 40 milli - units of absorbance were considered
the method of average grouping upgma was used and determined by means of the analysis cluster by using the software paleontological statistics ( past ) 2.10 , taking into account the dice 's similarity coefficient and the euclidean distance .
the weight of the different varieties of chilli peppers ranged from 3 to 46 g. in general , samples with the best morphological characteristics ( color , size , texture , and weight ) were those from the northern region , except for the sample jv1 from the south - central area , the latter being the lighter weight .
the length was from 4.3 to 9.5 cm , and the width was in the range of 0.1 to 3.34 cm .
the three varieties of chilli showed differences in both weight and size , with remarkable greater similarities between the same species ( jalapeo and serrano ) as well as those grown in nearby areas .
for instance , the serrano chilli peppers grown in tx , p , and v1 from the central region were quite similar . through statistical analysis ,
significant differences were found in weight and width between varieties , but not in length between serrano and jalapeo ( i.e. , the same species ) . however , these two peppers showed significant difference with respect to habanero ( i.e. , a different species ) ( see table 2 ) .
the content of carotenoids in fresh peppers varied within the interval 0.0830.99 mg / g dry basis ( db ) , while mature peppers were in the range from 0.69 to 13.85 mg / g db .
the carotenoid content increased from the fresh to the mature state , as expected accordingly to the natural biosynthesis of pigments as the fruit matures . in accordance with the statistical analysis , of chilli peppers in the fresh stage ,
there were significant differences between jalapeo and serrano varieties , while analysis of mature peppers , showed no differences between the three cultivars ( see table 3 ) .
this is due to the ability of fruits to synthesize carotenoids during the ripening process regardless of the species . regarding the region , fresh jalapeo peppers from tx and p were found to be different than those from v and s , while fresh serrano peppers showed statistical difference in all cases except those from jc and those from si .
mature chilli peppers , from si showed differences with respect to those from t , p , v1 and v2 .
therefore , no association was found between the concentration of carotenoids and the geographical origin .
the results presented above could be affected also by the environmental conditions , such as temperature and relative humidity , as well as the type of soil , which of course are related to the proximity of the different geographical regions . among the identified carotenoids
were lutein , -cryptoxanthin , lycopene , and -carotene , with a higher concentration of lutein and lycopene .
the latter are part of the main carotenoids present in chilli [ 29 , 31 ] . during the process of maturation , lutein remained constant for jalapeo peppers , whereas it diminished for serrano ones
however , it has been reported that lutein disappears in mature peppers by the effect of the synthesis of pigments in chromoplasts .
contrarily -cryptoxanthin and the -carotene increase during the ripening process , as reported by marn et al . .
according to statistical analysis , no significant differences were found for fresh peppers in concentrations of lutein , lycopene , and -cryptoxanthin between jalapeo and serrano cultivars .
nonetheless , there were differences in -carotene content , which is the major compound in several varieties of c. annuum l. . on the other hand
, differences were found for mature peppers only between jalapeo and serrano , in this case in lutein and lycopene content ( table 4 ) .
the concentration of phenolic compounds in fresh peppers ranged from 30.2 to 128.4 mg / g , whereas in the mature peppers it ranged from 38.6 to 297.8 g/l on dry weight basis . in most of the samples
, there was a noteworthy increase in these compounds in mature chilli peppers , in agreement with the results of deepa et al .
zhang and hamauzu show that the content of phenolic compounds are significantly higher in the fresh peppers than in the mature ones . given the above
, the main factors involved in the variation of phenolic content are the maturation state , as well as the age of the plant , as reported by deepa et al . .
when doing a comparison on the total phenolic content within the chilli varieties , significant differences were found between serrano and jalapeo , for both fresh and mature stages , while between jalapeo and serrano as a group no differences were detected with respect to habanero .
these results are inconsistent with data reported by oboh et al . , where the phenolics content was significantly higher for the c. annuum l. than for the c. chinense jacq species . concerning the region ,
differences were found in most of fresh jalapeo peppers samples , except between p and v ( v1 and v2 ) and between si and jco .
for the fresh serrano peppers , there were differences between tx and p with respect to jc and v2 .
for mature peppers , the region seems to be unimportant ( see table 5 ) . from these results ,
certain patterns indicate differences between varieties and other between cultivars , in agreement with antonious et al . who reported significant differences between cultivars of c. baccatum l. and c. chinense jacq , and between two genotypes of the latter variety .
the phenolic compounds found in chilli peppers were gallic , protocatechuic , ferulic , o - coumaric , p - coumaric , sinapinic , trans - cinnamic and caffeic acids , quercetin , catechin , rutin , and vanillin .
many of these compounds belong to the pathway of phenylpropanoids which is characteristic of chilli species . among the varieties of chilli , significant differences were found between serrano and jalapeo in the gallic and p - coumaric acids content , indifferently of maturation stage .
regarding protocatechuic and caffeic acids as well as catechin and vanillin , there were differences only for fresh peppers .
for the mature stage , the only difference was found in the content of trans - cinnamic acid between the two varieties ( table 6 ) . regarding the place of origin , fresh jalapeo peppers showed differences in the content of sinapic acid between si samples and the rest , in the content of catechin of v samples compared to p and tx samples ( despite the fact that all three belong to the central region ) , and in the content of p - coumaric acid of p compared to v1 , tx , and si . the fresh serrano peppers showed differences in the concentration of gallic acid for samples from si with respect to p and v. in the content of protocatechuic acid for samples from p with respect to the other specimens , and in the content of caffeic acid and rutin for v2 with respect to the other specimens .
finally , most regions showed differences in the content of p - coumaric acid , except for v2 compared to p and s , v1 compared to jc and tx , p compared to si , and jc compared to tx , showing no association between geographical areas . among mature jalapeo peppers , t showed differences with respect to si in the content of gallic acid and with respect to v1 in protocatechuic acid .
no differences were found between the mature serrano peppers with respect to region . by taking into account the state of maturation ,
for the rest of the phenolic compounds , there were not many differences as observed by the maturation stage in the content of gallic , protocatechuic , trans - cinnamic and p - coumaric acids ; quercetin , rutin , and vanillin .
for the rest of the phenolic compounds , there were no differences that could be explained by the maturation stage . within a jalapeo variety
, differences were found between fresh and mature peppers in the content of gallic acid , while no such difference existed in the content of ferulic acid .
for mature specimens no differences were found only between the three varieties in the content of protocatechuic acid , caffeic acid , and catechin , while the fresh samples reported differences in the content of p - coumaric acid , catechin , and vanillin .
it is clear from the results that variations exist between the three variables and concentrations .
some interactions can be explained by the proximity of the geographical areas , as in the case of p , tx , v , and t. other differences may be due to the growing conditions of each geographic region or by the genotype of the chilli , as discussed earlier by silva et al . .
these authors studied the content of rutin in a medicinal fruit from brazil and found that the high variability in the concentration and the great genetic variability of the fruit may be associated not only with genetic differences , but also with environmental conditions . the typical ir spectra ( 6002000 cm ) of phenolic extracts from different geographical regions of mexico ( figure 1 )
however , by applying principal component analysis ( pca ) ( figure 2 ) to the second derivative of the spectra , four clusters were found . in the first cluster samples
, p appeared with a variation axis which is parallel to the association to a second group of tx and v , that is , with similar chemical characteristics .
the third cluster , si and t samples were able to associate and the fourth group to jc samples , but perpendicular to the previous samples , so , the latter three were related to each other , but showed differences with the former .
we can say that the associations of geographic location could be due to the environmental conditions , that is the geographical environment determines the concentration of compounds in peppers . by joining these results with the total content of phenolics , and by chromatography , the same association for p , tx , and v in concentration of trans - cinnamic , gallic , p - coumaric , protocatechuic acids , quercetin , and catechin can be found . in the present study
, it has been found that ftir spectroscopy combined with the pca method presents great potential for the verification of the geographic origin of phenolic samples .
regarding the principal components that were generated , that was too reduced to perform discrimination .
such discrimination was realized by using the second derivative of the spectra in the region ( 9001750 cm ) .
these results show that this ftir - pca analysis could be used as an alternative , quick , and low - cost method for identification of the geographic origin of mexican chilli peppers ( c. annuum l. ) through their phenolic compounds . for variables regarding type and maturation stage ,
it was not possible to find differences because the geographical variation , influenced by climatic and growing conditions , was more important than these variables . among the oligonucleotides tested , ope-18 was the most polymorphic rapd oligonucleotide generating a total 10 bands . from the rapd reactions carried out with a mixture of mfg-17 and mfg18 oligonucleotides and c51 to c52 oligonucleotides mixture
independently of the oligonucleotide used , serrano and jalapeo chilli peppers exhibit similar rapd profiles , whereas habanero chilli peppers exhibited a particular profile with only 3 or 4 common bands .
the presence / absence and similitude matrixes derived from the most informative primer ( ope-18 ) were obtained , and an euclidean distance dendogram was constructed with 10 polymorphic bands ( r = 0.94 ) ( figure 4 ) . c. chinense jacq .
( habanero chilli peppers ) samples were clearly clustered apart from the c. annuum l. ( serrano and jalapeo chilli peppers ) , confirming the clear differences ( coefficient of 2.0 ) observed among rapd profiles .
basically , all samples formed three clusters : the first group made by the habanero peppers and the second and third groups do not show a clear separation between cultivars .
a previous rapd analysis using 10 germplasm samples of c. annuum l from thailand revealed a higher diversity of rapd profiles those found in mexican samples .
moreover , other rapd diversity analysis of c. annuum variety cuneo from northwest italy distinguished five populations .
the limited profile diversity and high similitude observed in two mexican varieties studied can be explained by selective pressure derived from culture practices and commercial interests .
evidently , rapd confirmed the great morphological diversity observed among c. annuum l varieties around the world , but an extensive study is necessary for molecular intraspecific diversity and population genetics including many c. annuum l varieties collected from different parts of the world . according to the dendrogram coupled to the amplification by oligonucleotide ope-18 and the chromatographic profile of carotenoids in mature peppers ( r = 0.69 ) ( figure 5 ) , the separation between the habanero peppers with respect to jalapeo 's and serrano 's can be observed , although it was not possible to identify between varieties within the same species or to identify an association between regions .
. found that the carotenoid content and the use of molecular markers in different genotypes of bananas , provided useful information on kin selection by crossing among different genotypes in order to develop new varieties with functional properties .
our results could be influenced only by the rapd because the carotenoid dendrogram did not show differences between species ; however , this separation maintained between the species does not occur in the case of phenols .
the present study was able to establish a separation and differentiation between the chilli species c. annuum l. and c. chinense jacq , through the rapd molecular method by using different primers .
also , by chromatographic profiles of carotenoids and molecular markers in mature chilli peppers and amplification by oligonucleotide ope 18 , it was possible to distinguish between habanero and serrano / jalapeo varieties , but no differences were found between c. annuum varieties . on the other hand ,
the pca of phenolic compounds in peppers , showed four groups according to their place of origin .
hence this technique can be used to identify chilli varieties from different geographical areas , which might be used for the protection of rights in the variety , diversity and phylogenetic analysis , as well as for the confirmation of hybrids . | the genus capsicum provides antioxidant compounds , such as phenolics and carotenoids , into the diet . in mexico
, there is a wide diversity of species and varieties of chilli peppers , a fruit which has local cultural and gastronomic importance . in the present study ,
the relationship of the carotenoid and phenolic profiles with the rapd fingerprint of three different commercial cultivars of chilli peppers of seven regions of mexico was investigated . through rapd ,
the species of chilli were differentiated by means of different primers ( ope-18 , mfg-17 , mfg-18 , c51 , and c52 ) . the genetic distance found with ope 18 was in the order of 2.6 .
the observed differences were maintained when the chromatographic profile of carotenoids , and the molecular markers were analyzed , which suggest a close relationship between carotenoids and the genetic profile . while the chromatographic profile of phenols and the molecular markers were unable to differentiate between genotypes of chilli peppers .
in addition , by using infrared spectroscopy and statistical pca , differences explained by geographic origin were found .
thus , this method could be an alternative for identification of chilli species with respect to their geographic origin . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions |
alzheimer 's disease ( ad ) is a progressive neurodegenerative brain disorder that is slow in onset but leads to dementia , unusual behavior , personality changes , and ultimately death .
ad is characterized by the presence of excessive amounts of neuritic plaques containing amyloid protein and abnormal tau protein filaments in the form of neurofibrillary tangles .
loss of cholinergic cells , particularly in the basal forebrain , is accompanied by loss of the neurotransmitter acetylcholine .
a decrease in acetyl choline in the brain of patients with ad appears to be a critical element in producing dementia .
ache inhibitors from general chemical classes such as physostigmine , tacrine , galantamine , and heptylphysostigmine have been tested for the symptomatic treatment of ad
. however , nonselectivity of these drugs , their limited efficacy , poor bioavailability , adverse cholinergic side effects in the periphery , narrow therapeutic ranges , and hepatotoxicity are among the several limitations to their therapeutic success .
therefore , it is worthwhile to explore the utility of other existing medicines for the treatment of various cognitive disorders .
scopolamine , a muscarinic cholinergic receptor antagonist , has been widely adopted to study cognitive deficits in experimental animals .
injection of scopolamine , the cholinergic neurotransmission was blockaded , leading to cholinergic dysfunction and impaired cognition in rats .
recently , it has been reported that memory impairment induced by scopolamine in rats is associated with altered brain oxidative stress status .
therefore , rats with scopolamine - induced memory deficits were used as an animal model for screening antidementia drugs .
oxidative stress is also one of the affecting factors in ad , so several antioxidants have been studied for the reduction of oxidative stress occurring during alzheimer 's disease [ 10 , 11 ] .
one of the mechanisms by which the abnormal accumulation of ubiquitinated proteins may mediate neurodegeneration is by triggering an inflammatory response .
inflammation is a defense reaction against diverse insults , intended to remove damaging agents and to inhibit their detrimental effects .
those agents were found to increase neuronal levels of cyclooxygenase 2 ( cox-2 ) suggesting that the production of such inflammatory mediators can be triggered by the intracellular accumulation of abnormal proteins .
nonsteroidal anti - inflammatory drugs ( nsaids ) are the group of drugs which effectively interfere with the cyclooxygenase pathway which is involved in generation of oxidative free radicals . in rheumatoid arthritis , nsaids have showed improvement in the circulating antioxidant status on daily dosing treatment [ 14 , 15 ] . for that purpose , meloxicam ( an enolic derived nsaid ) has been taken as reference drug by basing on the possession of significant anti - inflammatory activity as well as antioxidant property .
it has preferential inhibitory activity against the inducible cyclooxygenase-2 isoform , over the constitutive isoform cyclooxygenase-1 .
therefore , meloxicam and other cox-2 selective inhibitors are promoted for their safer profile of side effects .
selegiline ( l - deprenyl ) , an irreversible inhibitor of monoamine oxidase - b ( mao - b ) , a therapeutic agent of parkinson 's disease , is known to have neuroprotective properties that may involve its regulatory effects on antioxidant enzymes .
in addition , selegiline may act as an antioxidant in neurons and protect against glutamate - receptor - mediated toxicity .
studies of selegiline on aged male laboratory animals have showed delayed cognitive impairment and behavioral deterioration when compared with control animals .
the main purpose of the present study was to investigate the synergistic action of meloxicam and selegiline in scopolamine - induced alzheimer 's disease model .
swiss mice of male sex weighing 2025 g were used in the present study .
they had free access to food and water and were maintained under standard laboratory conditions with alternating light and dark cycles of 12 h each .
all the readings were taken during the same time of the day , that is , between 10 am and 2 pm .
the institution animals ethics committee ( iaec ) had approved the experimental protocol , and care of animals was taken as per guidelines of cpcsea , department of animal welfare , and government of india .
ltd ) , selegiline ( intas pharmaceuticals ) , and donepezil ( alkem laboratories ltd . ) were purchased .
the animals ( n = 36 ) were divided into six different groups of 6 animals per each group .
scopolamine ( 1.4 mg / kg ) as a disease inducer was administered to all groups through intraperitoneal ( i.p ) route after drugs administration to all the groups except normal control group . the same procedure was carried out for 9 days ( see table 1 ) .
the maze consists of completely enclosed rectangular box with an entry and reward chamber appended at opposite ends .
the box is partitioned with wooden slats into blind passages leaving just twisting corridor leading from the entry to the reward chamber .
animals were trained prior to the experiment by familiarizing with the rectangular maze for a period of 10 min for 2 h. well - trained animals were taken for the experiment .
transfer latency ( time taken to reach the reward chamber ) was recorded . for each animal ,
four readings were taken and the average is taken as learning score ( transfer latency ) for that animal .
lower scores of assessment indicate efficient learning while higher scores indicate poor learning in animals . the time taken by the animals to reach the reward chamber from the entry chamber
was noted on day 1 , 3 , 5 , 7 , and 9 . morris water maze was used to assess learning and memory in experimental mice .
there are several advantages of morris water maze over other models of learning and memory including absence of motivational stimuli such as food and water deprivation , electrical stimulations , and buzzer sounds [ 20 , 21 ] .
briefly , it consists of a circular water tank , filled with opaque water , and one centimeter submerged platform .
trial escape latency time ( elt ) , time measure to locate the hidden platform , was noted as an index of acquisition . each animal was subjected to the four acquisition trials per day for 4 consecutive days . the time spent by the animal , searching for the missing platform in target quadrant q2 with respect to other quadrant (
q1 , q3 , and q4 ) on 5th day , was noted as an index of retrieval .
for studying the effect of drug on acquisition , the drug solution was administered before acquisition trial .
locomotor activity is influenced by most of the cns drugs in both man and animals .
the locomotor activity of drug can be studied using actophotometer which operates on photoelectric cells which are connected in circuit with a counter when the beam of light falling on photocell is cut off by the animal , then a count is recorded .
animals are placed individually in the activity cage for 10 min and the activity was monitored .
the photo cell count is noted and decrease or increase in locomotor activity is calculated . when an electrical stimulus is given to animal , it tries to escape from it and move to the near safe place .
this equipment is designed in such a way to climb the pole when stimulus is generated .
training and testing is conducted in a 25 25 40 cm chamber that is enclosed in a dimly light , sound attenuated box .
a smooth stainless steel pole , 2.5 cm in diameter , is suspended by a counter balance weight through a hole in the upper centre of the chamber .
a micro switch is activated when the pole is pulled down by 3 mm . with weight
a response is recorded when a mice jumps on the pole and activates micro switch .
after 8-day treatment , the brains of different groups were perfusion - fixed with 4% paraformaldehyde in 0.1 m phosphate buffer .
the hippocampal lesions were assessed microscopically at 40 magnification . on day 9 , after behavioral assessments
each brain was separately put on ice and rinsed with ice - cold isotonic saline .
a ( 10% w / v ) homogenate was prepared in 0.1 m phosphate buffer ( ph 7.4 ) .
the homogenate was centrifuged at 3000 rpm for 15 minutes and aliquots of supernatant were separated and used for biochemical estimation .
the cholinergic marker , acetylcholinesterase , was estimated in the whole brain according to the method of ellman method .
ellman 's reagent is 5 , 5-dithiobis(2-nitrobenzoate ) and it is also abbreviated as dtnb .
this homogenate was incubated for 5 min with 2.7 ml of phosphate buffer and 0.1 ml of dtnb . then
, 0.1 ml of freshly prepared acetylthiocholine iodide ( ph 8) was added and the absorbance was read at 412 nm [ 24 , 25 ] .
aliquots of 0.5 ml distilled water were added with1 ml of 10% trichloroacetic acid and were added with 0.5 ml of brain tissue homogenate .
this is centrifuged at 3000 rpm for 10 min . to the 0.2 ml supernatant , 0.1
total solution is placed in water bath at 80c for 40 min and cooled at room temperature .
catalase activity was assessed by the method of luck , wherein the breakdown of hydrogen peroxide is measured . in this 3 ml of h2o2 phosphate buffer
the results were expressed as micromoles of h2o2 decomposed per minute per mg protein . in this
, measurement is made from the bleaching of purple - coloured methanol solution of dpph .
to the 1000 l of diverse conc . of the sample , 4 ml of 0.004% methanolic solution of dpph was added . after 30 min incubation
inhibition of free radical by dpph in % was calculated in the following way :
( 1)%=(a
blanka
sample / a
blank)100 ,
a
blank : absorbance of control reaction .
the statistical analysis of data was done by the one way analysis of variance ( anova ) followed by the dunnett 's test . the probability level less than 0.05 was considered as significant .
the mice in all treatment groups except scopolamine - treated group showed lower transfer latency on 7th day and 9th day compared to 5th day of the same group as well as with the scopolamine group which was given in figure 1 .
donepezil ( 5 mg / kg ) treated for successive 8 days acts as positive control , possessed significant ( p < 0.05 ) decrease in transfer latency when compared to normal control and disease control ( scopolamine ) using dunnet 's test .
the activity of meloxicam and selegiline was evaluated using morris water maze . the mice treatment groups except scopolamine - treated group showed significant transfer latency on 4th day with platform and on 5th day without platform which was given in figure 2 .
donepezil ( 5 mg / kg ) treated for successive 8 days acts as positive control , possessed significant ( p < 0.05 ) decrease in transfer latency when compared to disease control ( scopolamine ) using dunnet 's test .
the mice showed significant transfer latency on 7th day compared to the 9th day in all treatment groups except scopolamine - treated group which was given in figure 3 .
this donepezil ( 5 mg / kg ) treated successive 8 days acts as positive control , possessed significant ( p < 0.05 ) decrease in number of crossings which is comparable to the other treatment groups .
the values show that there was a significant difference that has been observed on days 7 and 9 compared to the 1 , 3 , and 5 .
scopolamine - treated group took more time whereas the control and drug - treated groups showed less time to reach the pole in pole climbing apparatus .
the results showed that synergistic action of meloxicam and selegiline was significant ( p < 0.05 ) and is comparable to the standard drug ( donepezil ) .
scopolamine treatment significantly increased the brain ache level compared to control group ( figure 5 ) .
standard drug ( donepezil ) and test drugs ( meloxicam , selegiline ) treatment significantly inhibited the brain ache level compared to their corresponding scopolamine - treated groups .
scopolamine treatment significantly increased the brain mda level compared to control group ( figure 6 ) .
standard drug ( donepezil ) and test drugs ( meloxicam , selegiline ) treatment significantly ( p < 0.05 ) decreased brain mda level compared to their corresponding scopolamine treated groups .
catalase levels were decreased in scopolamine - treated groups compared to the normal control group ( figure 7 ) .
significant ( p < 0.05 ) difference has been found in drug - treated groups .
synergistic effect was observed which is comparable with the standard group than individual drug - treated groups .
antioxidant levels were decreased in scopolamine - treated group compared to the control group ( figure 8) .
drug - treated groups showed significant ( p < 0.05 ) difference compared to the disease control group . from figure 9 , it is clearly visible that in disease control group the degenerated cells are more compared to other groups .
the drug - treated groups are in between the normal control and disease control groups .
the combination group is mostly near to the control group compared to the individual drug - treated groups .
the scopolamine amnesia test is widely used as primary screening test for so - called anti - alzheimer drugs .
there recently has been an increased appreciation of the role that inflammation plays in the pathogenesis of alzheimer 's disease that has arisen principally from epidemiological studies showing a dramatic effect of long - term nsaid treatment on alzheimer 's disease risk .
however , the molecuar mechanisms by which nsaids intervene in the pathological processes that underlie cognitive decline and neuronal loss remain unclear [ 28 , 29 ] .
recently , many studies reported that memory impairment in the scopolamine - induced animal model is associated with increased oxidative stress within the brain [ 8 , 30 , 31 ] .
oxidative stress is the cytotoxic consequence of oxyradical and oxidant formation and the reaction with cellular constituents .
reactive oxidative species ( ros ) are generated continuously in nervous system during normal metabolism and neuronal activity .
the nervous system is particularly vulnerable to the deleterious effects of ros . because the brain has a high consumption of oxygen , large amount of polyunsaturated fatty acids ( pufas ) , high contents of free ions , and low levels of antioxidants defense
increased mda level as one of the ros has been shown to be an important marker for in vivo lipid peroxidation . from the behavioral test ,
that is , rectangular maze test and morris water maze test , it is clearly seen that there was a general decrease in the transfer latency in all treated groups compared to the scopolamine - treated group .
the memory loss effect of scopolamine is more prominent compared to the control group . in comparison with donepezil , the drug - treated groups had almost equal performance which indicates synergistic effect of meloxicam and selegiline against memory loss .
meanwhile locomotor activity and pole climbing avoidance tests are done which also indicate the leaning ability ( figure 4 ) .
the major antioxidant and oxidative free radical scavenging enzymes like glutathione , sod , and catalase play an important role to reduce oxidative stress in brain . in this study , from the dpph assay antioxidant levels are estimated .
these enzyme levels are decreased in the scopolamine - treated group compared to the control group .
it supports the antioxidant action of drugs . in the present study rats after scopolamine treatment showed a significant increase in the brain levels of malondialdehyde , which is the measure of lipid peroxidation and free radical generation . in the drug - treated groups ,
there is a significant decrease in the levels of malondialdehyde which is nearly equal to the standard group . from the results ,
it is clear that the anti - inflammatory activity of meloxicam decreases the disease progression .
the antioxidant activity of selegiline is clear from the biochemical tests , which includes the estimation of antioxidant enzymes .
in conclusion , the present study demonstrates that meloxicam , selegiline , and co - administration of these test drugs had potential therapeutic effects on improving the antiamnesic activity in mice through inhibiting lipid peroxidation , augmenting endogenous antioxidant enzymes , and decreasing acetylcholinesterase ( ache ) activity in brain . | alzheimer 's disease ( ad ) is a progressive neurodegenerative disorder characterized by a gradual decline in memory associated with shrinkage of brain tissue , with localized loss of neurons mainly in the hippocampus and basal forebrain , with diminished level of central cholinergic neurotransmitter - acetylcholine and also reported to be associated with accumulation of ubiquitinated proteins in neuronal inclusions and also with signs of inflammation . in these disorders , the abnormal protein aggregates
may themselves trigger the expression of inflammatory mediators , such as cyclooxygenase 2 ( cox-2 ) . in the present study , the effects of meloxicam , selegiline , and coadministration of these drugs on scopolamine - induced learning and memory impairments in mice were investigated .
rectangular maze test , morris water maze test , locomotor activity , and pole climbing test were conducted to evaluate the learning and memory parameters .
various biochemical parameters such as acetylcholinesterase(ache ) , tbars assay , catalase activity , and dpph assay were also assessed .
the present study demonstrates that meloxicam , selegiline , and co - administration of these test drugs had potential therapeutic effects on improving the antiamnesic activity in mice through inhibiting lipid peroxidation , augmenting endogenous antioxidant enzymes , and decreasing acetylcholinesterase activity in brain .
the memory enhancing capacity of the drugs was very significant when compared to disease control ( p < 0.001 ) . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
the use of acid - suppressive medication is rapidly increasing . in the western world , proton pump inhibitors ( ppis ) are second only to statins in expenditures , and antisecretory medication constitutes a substantial part of the medical budget in the denmark and other western countries [ 13 ] . most of the increased use of antisecretory medication is accounted for by long - term users [ 4 , 5 ] .
antisecretory medication is highly effective for the treatment of peptic ulcer and reflux disease , but it has only sparse effect in the treatment of patients with functional dyspepsia . in primary care , the most used strategy when investigating and treating dyspeptic patients is empirical treatment with antisecretory drugs . as no data exist regarding the optimal treatment period , and as there is a poor correlation between initial treatment response and continued effect , some patients might have an unnecessary continued use of antisecretory medication . in a swedish study ,
bjrnsson et al . found that discontinuation of antisecretory mediation was successful in 27% of long - term users of ppi .
placebo - controlled discontinuation studies of antisecretory medication with interventions carried out in primary care are however not yet published .
the aim of the present study was therefore in a placebo - controlled randomised design to investigate if patients on long - term antisecretory medication need to continue treatment to control symptoms in a primary care setting .
a total of 146 general practitioners in denmark were visited , introduced to , and educated in study procedures and signed in as coinvestigators on the required forms from the danish medicines agency ( protocol i d : 2612 - 2176 ) .
eligible patients were evaluated , enrolled , and followed by their general practitioner from january 2003 to september 2006 .
patients older than 17 years with a use of antisecretory treatment ( proton pump inhibitors or histamine-2-receptor - antagonists ) for at least 56 days during the previous six months were included .
exclusion criteria were alarm symptoms ( gastrointestinal bleeding , iron deficiency anemia , progressive unintentional weight loss , progressive dysphagia , persistent vomiting , and epigastric mass on palpation ) , drug or alcohol abuse , serious or terminal diseases , planned hospitalization during follow - up , allergy to trial medication , and pregnant or fertile females not using safe contraception .
furthermore , patients suffering from oesophagitis , having had prior complicated peptic ulcer , and patients with prior endoscopically verified ulceration and ongoing use of nonsteroidal anti - inflammatory drugs ( nsaids)/acetylic salicylic acid ( asa ) were not eligible .
long - term users of antisecretory treatment were randomised to continued antisecretory treatment or placebo .
esomeprazole ( nexium , astrazeneca ) 40 mg or identical placebo was used as the randomised trial medication with a maximum of one tablet a day mimicking the patient 's usual treatment . when trial medication was discontinued due to insufficient symptom control , reinstitution of usual antisecretory treatment was left to the gps ' discretion .
both active medication and placebo were manufactured and packed by astrazeneca , mlndal , sweden , in accordance with good manufactory practice ( gmp ) .
trial drug was delivered in identical , sealed containers holding 100 tablets . the local university hospital pharmacy ( fyns amts centralapotek , j. b. winslws vej 13 , dk-5000 odense c , denmark ) labelled and randomised the trial drug accordingly using a computer - generated random number block randomisation with a fixed block size of four and a placebo / treatment ratio of one .
the pharmacy kept the sequence of allocation concealed throughout the one - year follow - up , thus blinding patients , general practitioners , and researchers .
following seven days of discontinuation of usual antisecretory treatment with allowance of antacids as rescue treatment , helicobacter pylori status was investigated with a c - urea breath test performed in general practice . during the seven days of discontinuation symptoms , demographics and clinical baseline characteristics of participants
eradication therapy was given to any helicobacter - positive patient ( amoxicillin , clarithromycin , and esomeprazole , alternatively metronidazole if allergic to penicillin ) .
the study was monitored according to good clinical practice ( gcp ) by the gcp unit at odense university hospital , denmark ( project number 02 - 004 ) .
primary endpoint was the time to discontinuation with trial medications ( esomeprazole or placebo ) due to the participants ' need to change back to their usual antisecretory medication , that is , failure to control symptoms with the trial medication at any time point in the one - year follow - up .
the proportion of patients stopping trial medication during the one - year follow - up was estimated .
additional analysis was carried out using health - related quality - of - life ( mos short form-36 ) and generic dyspeptic quality - of - life ( gastrointestinal symptom rating scale ) questionnaires .
we assumed 1520% of long - term users to be suffering from peptic ulcer disease , 4045% to be suffering from nonerosive reflux disease , and approximately 40% to be suffering from functional dyspepsia .
based on these diagnoses , we estimated the recurrence of symptoms in 10% in the esomeprazole group and 30% in the placebo group and given a type i error of 5% and a type ii error of 20% at least 111 patients in each group were needed . during data analysis , patients remained in the two arms of the study without revealing the intervention , thus allowing a blinded data analysis of the primary endpoint .
assumptions that dropouts in both groups had insufficient control of symptoms and therefore were analysed as having stopped with trial medication were made .
a total of 171 patients found eligible by their general practitioners were accepted to participate in the study ( figure 1 ) ; 17% were helicobacter pylori - positive . during the one - year follow - up , six patients were lost to follow - up ; no adverse events were seen , except for one patient developing a black discoloration of the tongue during helicobacter pylori eradication treatment .
no differences were found between gps not including participants compared with active coinvestigators with regard to sex , single - handed or partnership practice , and number of long - term users , data not shown . at the one - year follow - up ,
the stop of the trial medication had occurred more frequently ( p < 0.0001 , log - rank ) in the placebo group compared with the group receiving esomeprazole .
a total of 18/86 ( 21% ) of participants treated with esomeprazole had stopped the trial medication compared to 62/85 ( 73% ) of participants treated with placebo ( figure 2 ) .
gastrointestinal symptom scores improved in both groups during the trial , but no statistically significant differences were found between the two groups after one year ( table 2 ) .
quality - of - life scores were unchanged , and no differences were found between groups after one year ( data not shown ) .
table 3 shows a comparison between patients continuing placebo versus patients who stopped treatment with placebo .
statistically significantly more men stopped placebo treatment during follow - up ( p = 0.003 ) . a statistically nonsignificant tendency towards a more successful continuation with placebo treatment was found for patients with less gastrointestinal symptoms ( total gsrs score , p = 0.07 ) .
the subscore regarding reflux symptoms ( heartburn and regurgitation ) did , however , show less influence on continued placebo treatment ( p = 0.29 ) .
the present study provides estimates from primary care on the need for continued antisecretory treatment among long - term users . in a randomised , placebo - controlled trial , long - term users of antisecretory treatment were randomised to either continued antisecretory treatment ( esomeprazole 40 mg ) or placebo .
we found that 27% of participants receiving placebo did not stop the trial medication during the complete follow - up . to our knowledge , this study was the first nonindustry initiated drug trial conducted in a primary care setting , which was gcp monitored by a public gcp unit .
the randomised design and blinding in the design hindered any contamination between participants in the same practice and allowed a blinded data analysis .
the primary care setting is important as 90% of antisecretory drugs are prescribed in primary care and the findings may be applicable to patients managed in primary care in similar settings .
recruitment of patients for the study tended to be slower than expected and only 38% of the coinvestigators ( gps ) recruited participants for the study .
no differences were , however , found between gps not including participants compared with active coinvestigators .
based on register data , a large group of persons met the definition of long - term use of antisecretory treatment , but only a minority of the persons was included in this particular study .
however , the register data do not provide information about indications for the prescription of antisecretory medication and some of the persons in the registers were probably meeting one or more of the exclusion criteria defined for the study .
however , we can not rule out the possibility that some of the persons , who were not included , had already tried to discontinue their antisecretory treatment . due to the absence of consensus on the optimal treatment duration , the design of a discontinuation study was chosen and the discontinuation of trial drug was the primary outcome measure .
the fluctuation of dyspepsia symptoms is well known and severity as well as symptoms may vary in the same patient over time .
this may give rise to reflections on whether disappearance of symptoms due to the natural history of symptom fluctuation would allow participants receiving placebo to continue the trial drug .
however , discontinuation of the trial drug largely took place during the first two months of follow - up and no discontinuation occurred during the last six months in the placebo group .
rebound acid secretion following discontinuation may occur [ 1720 ] . in this study , a potential rebound effect may have been an important factor and would result in an underestimation of the proportion of participants who succeeded in a continuation of placebo .
. however , the study also elucidated that if symptoms do not recur within the first 60 days of ppi withdrawal they are unlikely to recur .
no endoscopy was performed prior to inclusion , which might be a limitation , as patients with uninvestigated dyspepsia on long - term treatment with ppi could in principle suffer from erosive esophagitis and therefore should probably not have withheld treatment .
this is the first placebo - controlled study on discontinuation of an acid - suppressive drug in a group of long - term users , with intervention carried out in primary care , but the results are in line with those of bjrnsson et al . , who found that discontinuation was successful in 27% of long - term users .
their patients had symptom profiles very similar to those of our patients , but patients were allocated to taper down or constant dose before discontinuation and placebo was not introduced . a number of studies examined discontinuation of ppi but without inclusion of a placebo group .
a recent study by murie et al . included patients with a diagnosis of gastroesophageal reflux disease or functional dyspepsia for prospective intervention of patient education given at specialist nurse clinic .
ppi was successfully discontinued in 34% and more than half of the patients reduced the ppi dose . in a study by krol et al . , gps were randomized to give usual care or sending a simple information leaflet about dyspepsia to patients treated with ppi for at least 12 weeks . a higher discontinuation rate ( 24% ) was demonstrated in the intervention group , but the difference between groups did not persist after 20 weeks . it may be objected that placebo treatment is not equivalent to absence of therapy and that 5060% of patients with gastroesophageal reflux disease are satisfied with placebo on demand . on the other hand ,
the perception of placebo is changing [ 24 , 25 ] and in the present study symptoms were controlled for more than 6 months on placebo .
thus , on balance , it seems reasonable to consider stopping use of acid - suppressive drugs in a substantial number of long - term users without complicated peptic ulcer , severe oesophagitis , or need for protection against nsaids .
considerable savings could be expected if these results are applicable to the entire population of long - term users and future studies should concentrate on delineating factors predictive of successful discontinuation of proton pump inhibitors .
we recommend that symptom - based long - term antisecretory treatment in general practice should be interrupted by attempts at discontinuation . in our placebo - controlled study ,
one in five patients treated with esomeprazole had even unsatisfactory symptom control and discontinued trial medication .
deleterious effects of antisecretory drugs have been reported [ 5 , 2730 ] , and unnecessary treatment may lead to polypharmacy and unnecessary expenses for the patient and society .
randomised , controlled discontinuation trials , such as the study presented in this paper , are uncommon , but they may be useful in other areas of medicine ( e.g. , treatment with nsaids ) . |
objective . to investigate whether patients on long - term antisecretory medication need to continue treatment to control symptoms
. methods . a double - blinded randomised placebo - controlled trial in general practices in denmark .
patients aged 1890 who were treated with antisecretory drugs on a long - term basis were randomized to esomeprazole 40 mg or identical placebo .
outcome measures were time to discontinuation with trial medication due to failed symptom control analysed as survival data .
the proportion of patients stopping trial medication during the one - year follow - up was estimated .
results .
a total of 171 patients were included with a median prior duration of antisecretory treatment of four years ( range : 0.5 to 14.6 years ) .
86 patients received esomeprazole 40 mg and 85 patients received placebo . at 12 months , statistically significantly more patients in the placebo group had discontinued ( 73% ( 62/85 ) ) compared with the esomeprazole group ( 21% ( 18/86 ) ; p < 0.001 ) . conclusions .
long - term users of antisecretory drugs showed a preference for the active drug compared to placebo .
however , 27% of patients continued on placebo throughout the study and did not need to reinstitute usual treatment .
one in five patients treated with esomeprazole discontinued trial medication due to unsatisfactory symptom control .
discontinuation of antisecretory treatment should be considered in long - term users of antisecretory drugs .
this trial is registered with trial registration clinicaltrials.gov i d : nct00120315 . | 1. Introduction
2. Methods
3. Results
4. Discussion |
the development of behavioral addictions ( bas , also referred to as impulse control disorders ) in association with dopamine agonists ( das , commonly used to treat parkinson s disease ) has been reported ( dodd et al . , 2005 ; driver - dunckley , samanta , & stacy , 2003 ; szarfman , doraiswamy , tonning , & levine , 2006 ; weintraub et al . , 2010 ) .
( 2014 ) present evidence that these reported associations are evident in the us food & drug administration s ( fda ) adverse event reporting system ( faers ) , a database containing information on adverse drug event ( ade ) and medication error reports submitted to the fda
2014 ) conducted a retrospective disproportionality analysis based on the 2.7 million serious domestic and foreign ades reported to the faers from 2003 to 2012 .
a threshold consisting of a proportional reporting ratio ( prr ) 2 was used to detect signals ( drug - associated adverse events ) involving any of six das and any of 10 terms ( or ades ) in the medical dictionary for regulatory activities characterized as bas .
they identified 1,580 reports containing at least one ba , 710 of which also contained a da , and reported a prr for reports containing both a ba and a da ( bas w / das ) of 277.6 ( p < 0.001 ) .
the authors concluded that their findings confirm prior reports of a ba / da association .
however , given three factors , it is possible that their findings could have been affected by publicity .
first , vulnerability to publicity - stimulated reporting has been described as a limitation of spontaneous reporting systems like the faers ( bate & evans , 2009 ; moore et al . , 2003 ) .
second , between 2003 and 2012 , the development of bas in association with das received considerable media coverage on multiple occasions .
third , a 2014 research report , which , in its analysis of faers reporting cast a wider net than moore et al .
( 2014 ) with respect to both number of bas analyzed ( 16 versus 10 ) and type of report analyzed ( both serious and non - serious reports were included ) , found no or only very weak associations prior to publicity between das and bas that could be grouped under the general headings binge eating , compulsive shopping , and hypersexuality ( pre - publicity data on reports of gambling to the faers are not freely available ) ( gendreau & potenza , 2014 ) .
( 2014 ) briefly allude to the issue of publicity in their discussion and suggest that it can not account for their findings .
specifically , they characterize growth in ba w / da reporting ( dopamine receptor agonists in figure 1 ) between 2003 and 2012 as steady and conclude that it is therefore unlikely that a burst of publicity or specific events explain their findings .
we contend that growth in ba w / da reporting between 2003 and 2012 was not steady , but rather it fluctuated , with upticks in reporting in 2004 ( approximately 133% ) and 2011 ( approximately 49% ) , and a sustained period of growth between 2005 and 2009 ( approximately 588% ) .
given the observations listed in the previous paragraph , we hypothesized that these fluctuations may coincide with publicity .
trend over time for reports of pathological gambling , hypersexuality , and related impulse control disorders .
total domestic and foreign reports of serious injury received by the us food and drug administration containing any of 10 terms in the medical dictionary for regulatory activities that characterize the impulse control disorder under study .
( reproduced with permission from the american medical association : jama internal medicine ( moore et al .
note : in the current study , impulse control disorders are referred to as behavioral addictions , or bas
to investigate the possibility that these fluctuations may coincide with media coverage , we conducted internet searches of 16 major us and international english - language media outlets between 2003 and 2012 for the keywords parkinson s and gambling .
news stories were excluded if they only briefly mentioned a ba / da association ( 26 stories ) ; keywords were found among
comments on a story ( four stories ) ; they were in question and answer or
dear doctor format ( two stories ) ; or the webpage or article was no longer accessible ( one story ) .
both authors have conformed to the highest standards of ethical conduct in the submission of accurate data , acknowledging the work of others and divulging potential conflicts of interest . as the study involved
the use of publicly accessible , de - identified data , signed informed consent was not necessary for this study .
given the use of publicly accessible , de - identified data , the study is exempted from irb review under code of federal regulations 45 cfr 46.101(b ) .
both authors have conformed to the highest standards of ethical conduct in the submission of accurate data , acknowledging the work of others and divulging potential conflicts of interest . as the study involved
the use of publicly accessible , de - identified data , signed informed consent was not necessary for this study . given the use of publicly accessible , de - identified data , the study is exempted from irb review under code of federal regulations 45 cfr 46.101(b ) .
of the 35 included news stories , 15 were precipitated by scientific publications ( 43% ) ; 10 by lawsuits ( 29% ) ; 7 by conference presentations ( 20% ) ; 1 by a change in pharmaceutical company advertising ( 3% ) ; 1 by a patient s story ( 3% ) ; and 1 for which the catalyst was unclear ( 3% ) . results are summarized in figure 2 .
the uptick in ba w / da reporting in 2004 seen in figure 1 follows a news story in the new york times in august 2003 ( oneil , 2003 ) , precipitated by the publication in the same month of research conducted by driver - dunckley et al .
the uptick in 2011 follows four news stories in january and february of 2011 triggered by lawsuits , as well as a cbs news story in june 2010 ( edwards , 2010 ) precipitated by a scientific publication ( weintraub et al . , 2010 ) .
the approximately 588% growth in ba w / da reporting observed between 2005 and 2009 coincides with the publication of 26 news stories ( 74% of the 35 news stories included ) between july 2005 and april 2009 , most of which were prompted by scientific publications ( bostwick , hecksel , stevens , bower , & ahlskog , 2009 ; dodd et al . , 2005 ; szarfman et al . , 2006 ; voon et al . , 2007 ) , conference presentations , and lawsuits .
ba = behavioral addiction , da = dopamine agonist . note : news stories data were compiled from internet searches of 16 major us and international english - language media outlets for the terms parkinson s and gambling ( abc news , cbs news , cnbc , cnn , the globe & mail , the guardian , huffington post , msnbc , nbc news , the new york times , npr , scientific american , time , usa today , the wall street journal , and the washington post )
upticks in ba w / da reporting to the faers appear to either follow or coincide with publication of news stories .
moreover , fluctuations in ba w / da reporting are not reflected in the trajectories plotted for all faers reports or ba w / non - da reports ( figure 3 ) , which suggests the presence of some influence on the ba w / da reporting rate specifically . that fluctuations in news coverage may coincide with fluctuations in ba w / da reporting suggests the possibility that publicity may have influenced reporting .
trend over time for ba w / non - da and all faers reports , 20032012 .
faers = us food & drug administration s ( fda ) adverse event reporting system , ba = behavioral addiction , da = dopamine agonist . note : ba w / non - da reports data were estimated based on data presented in moore et al .
all faers reports data were obtained from the fda s website at http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/surveillance/adversedrugeffects/ucm083765.htm given the possibility that publicity could have influenced their findings , it would have been helpful if moore et al .
( 2014 ) had provided prrs for clinically related groupings of the 10 bas analyzed .
this would make it easier to assess the degree to which the overall prr of 277.6 may be driven by pathological gambling / gambling ( two separate ades in the faers ) , which , in 2003 , was the first ba to be reported in association with das in the mainstream media .
pathological gambling / gambling was also the ba most frequently reported to the faers between 2003 and 2012 , accounting for 42.7% of the 1,902 reports of bas identified by ( moore et al . , 2014 ) .
it would also have been interesting to see a comparison between the 2003 prrs ( pre - publicity for all but pathological gambling ) and the 2012 prrs ( post - publicity for all bas ) for clinically related groupings of the 10 bas analyzed .
it should be noted that significant complexities exist in identifying the specific factors that might cause ades and their reporting . while publicity may influence reporting , it is also possible that faers reporting may have led to increased publicity , perhaps indirectly through analysis and reporting of findings .
possible examples may include through the fda s quarterly reporting of potential drug safety issues identified via analysis of faers data ; updates to product labeling or product advertising resulting from analysis of faers reporting ; or scientific publication of analysis of faers reporting .
of the 35 news stories included in the analysis , one cites as its catalyst an update to possible adverse events listed in a product s television advertisements , a change the product manufacturer says was prompted by a request from the fda , and two stories cite as an impetus scientific publication of analysis of faers reporting .
we also note the gradual nature of the sizable uptick in ba w / da reporting between 2005 and 2009 , which occurs in the midst of several years of increased media coverage of a ba / da association .
this finding would be consistent with a possible lag , with information taking time to circulate and lead to reporting , although other possibilities also exist .
it is possible that the ba w / da data presented in figure 1 are influenced by multiple factors , some of which are indicated above .
while we do not cover an exhaustive list , the notion that reporting of bas in association with das might be influenced by publicity has important clinical and public health implications , and is thus not a notion that should be prematurely dismissed .
in summary , a ba / da association has received considerable publicity since it was first reported in 2003 .
data suggest that publicity may stimulate ade reporting , and in this case it may coincide with fluctuations in ba w / da reporting .
it is important to evaluate the potential effect of publicity on ade reporting to guard against mistaking an increased risk of an ade being reported for an increased risk of an ade occurring ( moore et al . , 2003 ) .
kg : contributed study concept and design , collected , analyzed and interpreted data , and performed initial drafting of the manuscript .
mp : obtained funding and aided in analysis and interpretation of data , manuscript review and editing .
both authors have participated sufficiently in the research endeavor in order to qualify for authorship , and take public responsibility for its content .
the corresponding author has had access to all data from the study and both authors have had complete freedom to direct its analysis , interpretation and reporting without influence from funding agencies .
potenza has consulted for and advised lundbeck , ironwood , insys , shire and rivermend health for issues relating to impulse control disorders , gambling disorder , eating disorders , substance addictions and gender - related differences ; has received research support from the national institutes of health , veteran s administration , mohegan sun casino , the national center for responsible gaming , and pfizer pharmaceuticals ; has participated in surveys , mailings or telephone consultations related to drug addiction , impulse control disorders or other health topics ; has consulted for gambling and legal entities on issues related to addictions or impulse control disorders ; has provided clinical care in the connecticut department of mental health and addiction services problem gambling services program ; has performed grant reviews for the national institutes of health and other agencies ; has guest - edited journal sections ; has given academic lectures in grand rounds , cme events and other clinical or scientific venues ; and has generated books or book chapters for publishers of mental health texts . | backgroundthe development of behavioral addictions ( bas ) in association with dopamine agonists ( das , commonly used to treat parkinson s disease ) has been reported . a recent report presented data that these associations are evident in the us food and drug administration s ( fda ) adverse event reporting system ( faers ) , a database containing information on adverse drug event and medication error reports submitted to the fda .
however , given that vulnerability to publicity - stimulated reporting is a potential limitation of spontaneous reporting systems like the faers , the potential impact of publicity on reporting in this case remains unclear.method and aimsto investigate the potential impact of publicity on faers reporting of bas in association with das ( bas w / das ) as presented by moore , glenmullen , and mattison ( 2014 ) , news stories covering a ba / da association were identified and compared with ba w / da and other reporting data in the faers.resultsfluctuations in the growth of ba w / da reporting to the faers between 2003 and 2012 appear to coincide with multiple periods of intensive media coverage of a ba / da association , a pattern that is not evident in other reporting data in the faers.discussion/conclusionspublicity may stimulate reporting of adverse events and premature dismissal of the potential influence of publicity on reporting may lead to mistaking an increased risk of an adverse event being reported for an increased risk of an adverse event occurring . | Introduction
Methods
Ethics
Results
Discussion
Conclusions
Authors contributions
Conflict of interest |
the findings suggest that it represents a genetically distinct variant of colorectal adenocarcinoma and has implications for the development of targeted therapies and their clinico - pathologic features ( 3 ) .
it is thought that the presence of mucin allows cancer cells to spread faster . as a result , it is considered more aggressive than regular adenocarcinomas ( 1 ) .
conflicting results are found in the published literature regarding the relationship between mucinous colorectal cancer and survival ( 4 ) .
the appearance of high signal t2w sequences of mucinous rectal tumours on mri help to stage disease pre and post radiotherapy period .
it is reported that mri is not useful for gauging disease activity of persistent abnormalities in mucinous tumours that often represented inactive mucin lakes(4 ) .
a 67 year old patient presented to the colorectal clinic with a history of diarrhoea and bleeding .
colonoscopy revealed a tumour 6 cm from the anal verge and biopsy confirmed intestinal poorly differentiated adenocarcinoma with mucinous component .
mri has shown a t3 n1 mucinous type rectal carcinoma 9 cm from the anal verge with negative circumferential resection margin ( crm ) .
patient had been offered neo - adjuvant chemo / radiation after mdt discussion . restaging mri revealed reduction in tumour size with areas within the mesorectum consistent with perforation / infection that lies close to anterior circumferential resection margin .
a laparoscopic low anterior resection and loop ileostomy formation was done and histology showed recto - sigmoid mucinous adenocarcinoma pt3pn1pmxr0 , duke s c1 completely excised at all margins .. no extramural venous invasion , no perforation and no growth on the mucosal site on macroscopic examination .
there were mucin deposits 14 mm beyond muscularis propria and 3 mm from the non peritonealised circumferential margin containing very occasional viable tumour cells .
several lymph nodes contained acellular mucin and a single node contained small viable tumor deposits .
a 75 year lady was referred by her community doctor with a history of rectal bleeding and palpable tumour on rectal digital examination .
mri showed bulky polypoid tumour and a lot of high signal change within the rectal lumen suggesting a mucinous nature of the polypoid mass . predicted mri staging was t2n1 with threatened resection margin due its low position and unclear separation from levator ani muscles .
final mri scans were preformed 4 weeks post chemo - radiation . though the intraluminal mass had reduced a few small high signals out - pouchings through the anterior rectal wall into thin mesorectal space .
these changes of uncertain nature could be due to perforation or mucinous lakes protruding through invaded rectal wall . subsequently the patient was offered and received a laparoscopic abdominoperineal resection .
histology revealed two polypoid lesions with residual tubulovillous adenoma with low and high grade dysplasia , with no residual invasive carcinoma identified .
mucin pools were present in the submucosa , muscularis propria and subserosa . all this was interpreted as a single large adenomatous lesion giving rise to an invasive adenocarcinoma , which had regressed with therapy .
it is the first case series of two patients with mucinous adenocarcinoma with unusual changes in post radiotherapy mri .
the post chemoradiation mri findings suggested features of perforation masking the mucin lakes .
we were unable to fully explain pathogenesis or evident reason for this type of finding after looking at the literature .
although patients post chemoradiotherapy mri reported reduction in tumour bulk , there are a few questions raised in both cases : are these mucin lakes mimicking perforation , post radiation changes or persistence of the disease ? how do mucin lakes extend beyond the muscularis propria ? there are case reports describing mucin pools extending to subserosa and submucosa , but no description of mucin lakes extending 14 mm beyond the muscularis propria .
absence of the literature evidence about it causes confusion on the pre - chemoradiotherapy mri ( reported as perforation ) .
formation is that the low - grade neoplasm produces mucin that distends the bowel lumen .
this increase in intramural pressure , where the perforating vessels traverse the muscularis propria allows neoplastic epithelium to herniate through these weak areas in the wall ( 4 ) and thereby mimic perforation . in our experience ,
patient 1 had occasional cancer cells in mucin pools extending 14 mm beyond the muscularis mucosa , but circumferential resection margin was not compromised ( 2 mm ) . in the second patient
mucin pools were acellular , histopathology showed minimal margin of < 1 mm , and again the tumour was completely excised .
one more unusual observation from comparison of these two case reports : in first case study pre - chemoradiation mri did not show obvious mucin lakes , but they became evident on post - chemoradiation mri extending beyond the muscularis propria despite tumour mass having completely regressed . does this imply that wall tissues become less resistant to cancer pool intrusion after chemoradiation ?
second patient had defunctioning colostomy after first mri and mucin pools were only at submucosa and subserosa .
does the high intramural pressure have an impact on the protruding of the cancer pools beyond the muscularis propria ?
pathogenesis and prediction of the mucin lakes behaviour prior and post chemoradiotherapy is unclear and requires future study , it might change surgical management .
mri diagnostic features are not always straight forward in the recognition of the tumour extent .
the presence of acellular mesothelial reaction and inflammation extending to the serosal surface causes considerable confusion and may result in upstaging or overstaging of mucinous adenocarcinoma . | we are reporting two cases with similar appearances on post chemo radiotherapy mri scan of the mucinous adenocarcinoma .
there was high signal on t2-weighted sequences of the mucin pools within and beyond the rectal wall .
does this imply that wall tissues become less resistant to cancer pools intrusion after chemo radiation ?
does the high intramural pressure have an impact on the protrusion of the cancer pools beyond the muscularis propria?final histology reports differ from predicted mri staging .
it highlights the difficulty of the final mri staging and outcome for mucinous adenocarcinomas .
the presence of acellular mesothelial reaction , acellular mucin pools and inflammation extending to the serosal surface causes considerable confusion and may result in over staging or under staging of mucinous adenocarcinoma.pathogenesis and prediction of the mucin lakes behaviour prior and post chemoradiotherapy is unclear and requires future study but may impact surgical management . | INTRODUCTION
CASE PRESENTATION 1
CASE PRESENTATION 2
DISCUSSION |
are currently classified as overweight or obese ( bmi 25 kg / m ) , and one - third of them are frankly obese ( bmi 30 kg / m ) ( 1 ) .
is 26.1 million in 20052010 or 12% of the population ( 2 ) . among u.s .
diabetic patients , the prevalence of overweight or obesity has increased to 80% ( 3 ) .
obesity is associated with increased risks of several cardiometabolic diseases , including hypertension ( 4 ) , diabetes ( 5 ) , coronary heart disease ( chd ) ( 6,7 ) , heart failure ( 8) , and stroke ( 9 ) .
cardiovascular diseases , especially chd , are the leading causes of death worldwide . in recent years
, several prospective studies have assessed the association between obesity and the risks of total and cvd mortality among diabetic patients , and the results are inconsistent . to date , many studies have reported positive associations ( 10,11 ) , inverse associations ( 1214 ) , u - shaped associations ( 1517 ) , or no associations ( 18 ) between bmi and mortality among patients with diabetes .
all of these studies were focused on the association between bmi and cvd mortality ; however , no studies assessed the association between bmi and the risk of incident chd among diabetic patients . in this study , we examined the association between bmi and the risk of chd among patients with type 2 diabetes in the louisiana state university hospital - based longitudinal study ( lsuhls ) .
between 1997 and 2012 , the lsu health care services division ( lsuhcsd ) operated seven public hospitals and affiliated clinics in louisiana providing quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1926 ) .
overall , lsuhcsd facilities have served 1.6 million patients ( 35% of the louisiana population ) since 1997 .
administrative , anthropometric , laboratory , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data ,
a cohort of diabetic patients was established by using the icd-9 ( code 250 ) between 1 january 1999 and 31 december 2009 . both inpatients and outpatients
confirmation of diabetes diagnoses was made by applying the american diabetes association criteria : a fasting plasma glucose level 126 mg / dl ; 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test ; and one or more classic symptoms plus a random plasma glucose level 200 mg / dl ( 27,28 ) .
the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . before diagnosis with diabetes
the agreement of diabetes diagnosis was 97% : 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes associates diabetes diagnosis criteria ( 27 ) .
the current study included 30,434 newly diagnosed patients ( 10,955 men and 19,479 women ) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables .
we only included african americans and whites because the patient numbers of hispanics , asians , and native americans are very small in the lsuhcsd hospitals .
patients were excluded if they were underweight ( bmi < 18.5 kg / m ) because of limited statistical power for this group . compared with diabetic patients excluded from the present analyses due to missing data , those included in the analyses were younger ( 51.0 vs. 57.6 years old ) , had a higher frequency of african americans ( 58.6 vs. 45.4% ) , and less males ( 37.2 vs. 47.1% ) .
the study and analysis plan was approved by the pennington biomedical research center and lsu health sciences center institutional review boards , lsu system .
we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records .
the patient s characteristics , including age at diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , glycosylated hemoglobin ( hba1c ) , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive drug , cholesterol - lowering drug , and antidiabetes drug ) within a half year before the diabetes diagnosis ( baseline ) , during follow - up after the diabetes diagnosis ( follow - up ) , and the last visit were extracted from the computerized hospitalization records . height and weight
bmi was calculated by dividing weight in kilograms by the square of height in meters .
values of bmi , blood pressure , hba1c , ldl cholesterol , and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up .
for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean values was from baseline to the year before this event occurred ( 29 ) .
follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals .
the diagnosis of chd was the primary end point of interest of the study and defined according to the following icd-9 : chd ( icd - codes 410414 ) .
follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , or the date of death , determined from linking to the louisiana office of public health vital records registry , or 31 may 2012 ( 23,26 ) . the association between bmi and the risk of chd was analyzed by using cox proportional hazards models .
bmi was evaluated in the following two ways : 1 ) as five weight categories ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) and 2 ) as a continuous variable .
the trend over different categories of bmi was tested in models with the median of each category as a continuous variable .
all analyses were adjusted for age ( continuous variable ) and race ( african american and white ) ( model 1 ) and further for smoking ( never , past , and current ) , income ( continuous variable ) , and types of insurance ( free , self - pay , medicaid , medicare , and commercial ) ( model 2 ) , and additionally for systolic blood pressure ( continuous variable ) , hba1c ( continuous variable ) , ldl cholesterol ( continuous variable ) , hdl cholesterol ( continuous variable ) , triglycerides ( continuous variable ) , egfr ( 90 , 6089 , 3059 , 1529 , and < 15 ml / min/1.73 m ) , use of antihypertensive drugs ( no use , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , and other antihypertensive drugs ) , use of diabetes medications ( no use , oral hypoglycemic agents , and insulin ) , and use of cholesterol - lowering agents ( no use , statins , and other cholesterol - lowering agents ) ( model 3 ) .
we stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd .
since the interactions between race and bmi on the risk of chd were not statistically significant , data for white and african americans were combined in some analyses .
all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss inc . ,
between 1997 and 2012 , the lsu health care services division ( lsuhcsd ) operated seven public hospitals and affiliated clinics in louisiana providing quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1926 ) .
overall , lsuhcsd facilities have served 1.6 million patients ( 35% of the louisiana population ) since 1997 .
administrative , anthropometric , laboratory , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data ,
a cohort of diabetic patients was established by using the icd-9 ( code 250 ) between 1 january 1999 and 31 december 2009 . both inpatients and outpatients
confirmation of diabetes diagnoses was made by applying the american diabetes association criteria : a fasting plasma glucose level 126 mg / dl ; 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test ; and one or more classic symptoms plus a random plasma glucose level 200 mg / dl ( 27,28 ) .
the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . before diagnosis with diabetes
the agreement of diabetes diagnosis was 97% : 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes associates diabetes diagnosis criteria ( 27 ) .
the current study included 30,434 newly diagnosed patients ( 10,955 men and 19,479 women ) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables .
we only included african americans and whites because the patient numbers of hispanics , asians , and native americans are very small in the lsuhcsd hospitals .
patients were excluded if they were underweight ( bmi < 18.5 kg / m ) because of limited statistical power for this group . compared with diabetic patients excluded from the present analyses due to missing data , those included in the analyses were younger ( 51.0 vs. 57.6 years old ) , had a higher frequency of african americans ( 58.6 vs. 45.4% ) , and less males ( 37.2 vs. 47.1% ) .
the study and analysis plan was approved by the pennington biomedical research center and lsu health sciences center institutional review boards , lsu system .
we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records .
the patient s characteristics , including age at diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , glycosylated hemoglobin ( hba1c ) , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive drug , cholesterol - lowering drug , and antidiabetes drug ) within a half year before the diabetes diagnosis ( baseline ) , during follow - up after the diabetes diagnosis ( follow - up ) , and the last visit were extracted from the computerized hospitalization records . height and weight were measured without shoes and with light clothing according to a standardized protocol .
bmi was calculated by dividing weight in kilograms by the square of height in meters .
values of bmi , blood pressure , hba1c , ldl cholesterol , and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up .
for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean values was from baseline to the year before this event occurred ( 29 ) .
follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals .
the diagnosis of chd was the primary end point of interest of the study and defined according to the following icd-9 : chd ( icd - codes 410414 ) .
follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , or the date of death , determined from linking to the louisiana office of public health vital records registry , or 31 may 2012 ( 23,26 ) .
the association between bmi and the risk of chd was analyzed by using cox proportional hazards models .
bmi was evaluated in the following two ways : 1 ) as five weight categories ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) and 2 ) as a continuous variable .
the trend over different categories of bmi was tested in models with the median of each category as a continuous variable .
all analyses were adjusted for age ( continuous variable ) and race ( african american and white ) ( model 1 ) and further for smoking ( never , past , and current ) , income ( continuous variable ) , and types of insurance ( free , self - pay , medicaid , medicare , and commercial ) ( model 2 ) , and additionally for systolic blood pressure ( continuous variable ) , hba1c ( continuous variable ) , ldl cholesterol ( continuous variable ) , hdl cholesterol ( continuous variable ) , triglycerides ( continuous variable ) , egfr ( 90 , 6089 , 3059 , 1529 , and < 15 ml / min/1.73 m ) , use of antihypertensive drugs ( no use , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , and other antihypertensive drugs ) , use of diabetes medications ( no use , oral hypoglycemic agents , and insulin ) , and use of cholesterol - lowering agents ( no use , statins , and other cholesterol - lowering agents ) ( model 3 ) . we stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd .
since the interactions between race and bmi on the risk of chd were not statistically significant , data for white and african americans were combined in some analyses .
all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss inc . , chicago , il ) .
baseline characteristics of patients with type 2 diabetes by the outcome during follow - up data represent means or percentages .
all data except age are adjusted for age and race . during a mean follow - up period of 7.3 years , 7,414 subjects ( 2,926 men and 4,488 women ) developed chd .
patients who developed chd during follow - up were older and used more glucose - lowering , lipid - lowering , and antihypertensive medication compared with those who did not develop chd .
the multivariable - adjusted ( age , race , smoking , income , and types of insurance ) ( model 2 ) hazard ratios ( hrs ) for chd at different levels of bmi at baseline ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) were 1.00 , 1.14 ( 95% ci 1.001.29 ) , 1.27 ( 1.121.45 ) , 1.54 ( 1.341.78 ) , and 1.42 ( 1.231.64 ) ( ptrend < 0.001 ) in men and 1.00 , 0.95 ( 0.851.07 ) , 0.95 ( 0.841.06 ) , 1.06 ( 0.941.20 ) , and 1.09 ( 1.001.22 ) ( ptrend < 0.001 ) in women , respectively ( table 2 ) . after further adjustment for other confounding factors ( systolic blood pressure , hba1c , ldl cholesterol , hdl cholesterol , triglycerides , egfr , use of antihypertensive drugs , use of diabetes medications , and use of cholesterol - lowering agents )
, this association remained significant among men ( ptrend < 0.001 ) and women ( ptrend = 0.006 ) .
hrs of chd according to different levels of bmi at baseline , during follow - up , and at last visit among patients with type 2 diabetes adjusted for age and race . adjusted for age , race , types of insurance , income , and smoking . adjusted for age , race , types of insurance ,
income , smoking , systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , hba1c , egfr , use of antihypertensive drugs ( none , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , other antihypertensive drugs , and any two or more of above treatments ) , glucose - lowering agents ( none , oral hypoglycemic agents , and insulin ) , and cholesterol - lowering agents ( none , statins , and other cholesterol - lowering agents ) .
when bmi was examined as a continuous variable , the multivariable - adjusted ( model 2 ) hrs of chd for each one - unit increase in bmi at baseline were 1.015 ( 95% ci 1.0111.020 ) in men and 1.004 ( 95% ci 1.0011.008 ) in women ( table 2 ) .
there was a significant interaction between sex and bmi on chd risk ( = 9.86 ; 1df , p < 0.005 ) , which indicated that this positive association was stronger in men than in women .
when we did an additional analysis by using an updated mean value of bmi , we found the same positive association between bmi and chd risk among both men ( ptrend < 0.001 ) and women ( ptrend < 0.001 ) ( table 2 ) .
when we did another additional analysis by using the last visit value of bmi , we found a positive association between bmi and chd risk among men ( ptrend < 0.001 ) and a u - shaped association between bmi and chd risk among women ( ptrend = 0.003 ) ( table 2 ) .
women who were overweight and had class i obesity ( bmi 2534.9 kg / m ) at last visit had a lower risk of chd compared with normal - weight women ( bmi < 25
kg / m ) . after excluding subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 3,207 ) , the multivariable - adjusted hrs ( model 2 ) of chd for each one - unit increase in bmi at baseline , during follow - up , and at the last visit were 1.014 ( 95% ci 1.0101.019 ) , 1.017 ( 1.0121.022 ) , and 1.015 ( 1.0091.019 ) in men and 1.005 ( 1.0011.009 ) , 1.006 ( 1.0021.010 ) , and 1.005 ( 1.0001.008 ) in women ( data not shown ) , respectively .
in stratified analyses , the multivariable - adjusted positive association between bmi and chd risk was present among men with different smoking status ( tables 3 and 4 ) . when stratified by age , race , and use of antidiabetic drugs , this positive association of bmi at baseline , during follow - up , and at the last visit with chd risk was still present among men in all subgroups and among women in some of the subgroups ( tables 3 and 4 ) .
hrs ( 95% cis ) of chd according to different levels of bmi at baseline among various subpopulations adjusted for age , race , types of insurance , income , and smoking , other than the variable for stratification .
hrs ( 95% cis ) of chd according to different levels of bmi during follow - up and at last visit among various subpopulations adjusted for age , race , types of insurance , income , and smoking , other than the variable for stratification .
our study found a positive association of bmi at baseline and during follow - up with the risk of chd among both men and women with type 2 diabetes , and this association was stronger among men than among women .
in addition , we found that a positive association between bmi and the risk of chd was present in both african americans and whites with type 2 diabetes and in nonsmokers and smokers .
the positive association did not change among men but changed to a u - shaped association among women with type 2 diabetes when we assessed bmi of the last visit with chd risk .
only a few prospective studies have evaluated the association between obesity and total and cvd mortality among diabetic patients , and the results are controversial including inverse associations ( 1214 ) , positive associations ( 10,11 ) , u - shaped associations ( 1517 ) , or no association ( 18 ) .
the current study was the first , to our knowledge , to assess the association between bmi and the risk of incident chd among diabetic patients .
the results of our study indicated a positive association between bmi and the risk of chd among patients with type 2 diabetes .
we found this positive association of chd risk by bmi at baseline and during follow - up .
in addition , this positive association was present in different race , antidiabetes medication , and smoking groups .
it is noteworthy that there was a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes in the current study .
our study found that diabetic women who were overweight and had class i obesity ( bmi 2534.9 kg / m ) at the last visit had a lower risk of chd compared with normal - weight women ( bmi < 25
it is well known that women with diabetes have a greater or equal relative risk of chd than men with diabetes ( 30,31 ) .
the current study found a significant positive association of bmi and chd risk among both men and women with type 2 diabetes , and this association is stronger among men than among women .
the finding from our study is noteworthy for us to prevent chd among patients with type 2 diabetes .
in addition , more studies are needed to confirm the different effect size of bmi with chd risk among men and women with type 2 diabetes .
it has been suggested that three potential methodological concerns should be considered when assessing the associations between obesity and health outcomes ( 32 ) .
people with a history of cvd and several other chronic diseases frequently lose weight , and thus , people with a lower weight might increase the estimated risk of death . a recent analysis pooling five longitudinal studies
has found that patients who have normal weight at the time of diabetes diagnosis have a higher mortality risk than those who are overweight or obese ( 12 ) .
they suggest that diabetic individuals with metabolically obese normal - weight may reflect underlying illness that predisposes to mortality ( 33 ) . despite having a normal bmi , these diabetic individuals have hyperinsulinemia , insulin resistance , and dyslipidemia , and
all of these factors predispose individuals to death ( 33 ) . in the current study , we excluded patients with a history of chd and stroke at time of diabetes diagnosis , which can minimize the influence of reverse causation .
moreover , we performed another sensitivity analysis by excluding the subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 3,207 ) , and the positive association of bmi at baseline and during follow - up with chd risk was still present .
the second major concern is that confounding factors may distort the association between body weight and chd .
smoking is a particularly important factor because smokers tend to weigh less and have much higher chd risk than nonsmokers . in the current study ,
smoking status was considered as a confounding factor in the multivariable model , and the positive association between bmi and the risk of chd was found in both never - smokers and smokers .
the third methodological concern in some analyses between weight and chd risk is that the physiologic effects of excess fatness , such as hypertension , diabetes , and dyslipidemia , were controlled for statistically , thus artificially removing some of the effects of being overweight .
obesity has been found as a strong risk factor for hypertension ( 4 ) , high levels of hba1c ( 5 ) , and high serum cholesterol among diabetic patients ( 34 ) and has also been the key or important component of the metabolic syndrome ( 35 ) .
all of these factors are associated with an increased risk of chd ( 3537 ) and considered as mediating factors for the physiologic effects of obesity on the chd risk . in the current study , the adjustment for systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , hba1c , egfr , and treatment attenuated the association between bmi and chd risk , but bmi as a continuous variable remained a statistically significant predictor of chd in the multivariable model .
there are several strengths in our study , including the large sample size , high proportion of african americans , and the use of administrative databases to avoid differential recall biases .
we have used baseline bmi levels , updated mean values of bmi during follow - up , and the last visit value of bmi in the analyses , which can avoid potential bias from a single baseline measurement .
in addition , participants in this study used the same public health care system that minimizes the influence of accessibility to health care , particularly in comparing men and women .
one limitation of our study is that our analysis was not performed on a representative sample of the population , which limits the generalizability of this study ; however , lsuhcsd hospitals are public hospitals and cover > 1.6 million patients , most of whom are low - income persons in louisiana
. the results of the current study will have wide applicability for the population with low income and without health insurance in the u.s .
another limitation of our study is that we did not have data on other obesity indicators , such as waist , hip , and thigh circumferences , and did not assess abdominal height , although these adiposity predictors have been shown to be associated with cvd risk ( 6,38,39 ) .
third , while body weight was measured at each clinic visit , clinically measured bmi might not be as accurate as bmi measured in carefully conducted laboratory studies ( 40 ) .
fourth , even though our analyses adjusted for an extensive set of confounding factors , residual confounding due to the measurement error in the assessment of confounding factors , unmeasured factors such as heart rates , physical activity , education , and dietary factors , can not be excluded . in summary
, we found a positive association between bmi at baseline and during follow - up with the risk of chd among men and women with type 2 diabetes , and this association was stronger among men than among women .
we also found a positive association between bmi at the last visit and the risk of chd among men with type 2 diabetes and a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes . | objectivethe association between obesity and coronary heart disease ( chd ) risk remains debatable , and no studies have assessed this association among diabetic patients . the aim of our study was to investigate the association between bmi and chd risk among patients with type 2 diabetes.research design and methodsthe sample included 30,434 diabetic patients ( 10,955 men and 19,479 women ) 3095 years of age without a history of chd or stroke in the louisiana state university hospital - based longitudinal study.resultsduring a mean follow - up period of 7.3 years , 7,414 subjects developed chd .
the multivariable - adjusted hazard ratios for chd across levels of bmi at baseline ( 18.524.9 , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m2 ) were 1.00 , 1.14 ( 95% ci 1.001.29 ) , 1.27 ( 1.121.45 ) , 1.54 ( 1.341.78 ) , and 1.42 ( 1.231.64 ) ( ptrend <
0.001 ) in men and 1.00 , 0.95 ( 0.851.07 ) , 0.95 ( 0.841.06 ) , 1.06 ( 0.941.20 ) , and 1.09 ( 1.001.22 ) ( ptrend < 0.001 ) in women , respectively . when we used an updated mean or last visit value of bmi , the positive association between bmi and chd risk did not change in men .
however , the positive association of bmi with chd changed to a u - shaped association in women when we used the last visit value of bmi.conclusionsour study suggests that there is a positive association between bmi at baseline and during follow - up with the risk of chd among patients with type 2 diabetes .
we indicate a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes . | Introduction
Research Design and Methods
Study Population
Baseline Measurements
Prospective Follow-up
Statistical Analyses
Results
Conclusions |
root tears are a subset of meniscal injuries , which have become increasingly recognized as a cause of pain and impaired mobility .
the root serves as the anchor point for the menisci . occurring on either the medial or lateral meniscus
, root tears refer to a radial tear or avulsion at the posterior horn attachment to the bone . both radial tear and posterior horn avulsion defunction the menisci as load - bearing structures , with increasing local contact pressure and premature onset of knee arthritis . because the medial and lateral menisci differ in anatomy and biomechanics , the pathogenesis of posterior root avulsions is also different .
tearing of the lateral posterior meniscal root is traumatic and always associated with anterior cruciate ligament ( acl ) injury [ 1 , 22 ] , while the medial posterior root [ 2 , 8 , 10 , 19 ] is prone to chronic degenerative meniscal disease . to date , only one case has been reported of radial tear of both roots detected two years after an acl injury .
the authors hypothesised a traumatic origin for avulsion of the lateral meniscus and a degenerative origin for the medial meniscus . here , we describe a rare case of avulsion of both posterior roots in association with an acute acl tear in a professional soccer player . a review of the video documentation of the match accident revealed the exact mechanism of injury .
a 20-year - old professional soccer player reported sustaining a forceful rotatory left knee injury during an official match of the italian second division .
the dynamics of the accident could be clearly followed on the video recording of the match .
while the player was standing with one foot fixed on the field and the contralateral limb elevated for shooting the ball , a player from the opponent team collided into the elevated limb , causing a forceful external rotation of the standing limb . at presentation two days after the injury , physical examination revealed signs of acute anterior laxity and pain was elicited over both lateral and medial joint lines .
manual knee laxity tests , including the lachman test , anterior drawer test and pivot shift test , were positive , as was the mcmurray test .
measurement using a kt-2000 arthrometer ( medmetric corporation , san diego , ca , usa ) demonstrated a 5-mm side - to - side difference .
magnetic resonance imaging ( mri ) disclosed acute rupture of the acl and tears in the roots of the posterior horns of both lateral and medial menisci ( fig . 1 ) .
arthroscopic evaluation under regional anesthesia revealed an acute tear of the acl at its midsubstance .
avulsion of the posterior root of the lateral meniscus was present in addition to acute avulsion at the posterior root of the medial meniscus with posterior displacement ( figs . 2 , 3 ) .
1a preoperative coronal tse - fat saturated mr image shows the acl tear and an area of heterogeneous intrameniscal signal intensity of both meniscal roots with a void at the posterior attachment site of both menisci ( arrow ) .
3arthroscopic view of acute lateral posterior root avulsion a preoperative coronal tse - fat saturated mr image shows the acl tear and an area of heterogeneous intrameniscal signal intensity of both meniscal roots with a void at the posterior attachment site of both menisci ( arrow ) .
the asterisk shows hyperintensity signal at femoral acl insertion arthroscopic view of acute medial posterior root avulsion arthroscopic view of acute lateral posterior root avulsion treatment consisted of arthroscopic pullout suturing of both menisci and acl reconstruction with an autologous bone - patellar tendon - bone graft .
the flat tip of an acl guide ( arthrex , naples , fl , usa ) was introduced through the anterolateral portal at the footprint of the posterior root of the previously abraded medial meniscus .
a tibial tunnel was made using a 2.9-mm guide pin from the anterolateral cortex of the proximal tibia to the footprint of the posterior root of the medial meniscus .
two nonabsorbable sutures were placed at the posterior root using a crescent - shaped suture hook .
the suture material for the lateral meniscus was pulled through the anteromedial cortex of the proximal tibia .
the lateral and medial sutures were tied over two buttons after confirming sufficient reduction and tension .
postoperatively , the knee was kept in full extension in a brace locked at 0 for four weeks .
passive motion was allowed after the first two weeks and active motion was restricted to 90 during the first four weeks .
partial weight bearing was permitted at four weeks postoperatively , followed by full weight bearing at six weeks .
after six weeks closed - chain strengthening was begun , and full flexion exercises were allowed . during the second and the third month , strengthening exercises and hydrokinetic therapy were implemented . running on a treadmill was started after two months and training on the field was permitted after four months .
six months postoperatively , the patient was able to return to play in an official match with full range of knee motion . at the last follow - up of one year , no meniscal signs and symptoms were present .
manual knee laxity tests , including the lachman test , anterior drawer test and pivot shift test , were negative .
the side - to - side difference was 0.4 mm , as measured by the kt-2000 manual maximal test .
4at mri follow - up at three months , the coronal t1-tse view ( a ) shows the tibial tunnel for acl reconstruction and the tunnels for the medial root ( white arrow ) and for the lateral root ( dashed arrow ) .
b the coronal tse - fat saturated mr image shows both medial and lateral roots healed at mri follow - up at three months , the coronal t1-tse view ( a ) shows the tibial tunnel for acl reconstruction and the tunnels for the medial root ( white arrow ) and for the lateral root ( dashed arrow ) .
b the coronal tse - fat saturated mr image shows both medial and lateral roots healed
the unusual finding in this case was avulsion of both posterior meniscal roots with a concomitant acute acl tear .
medial posterior root avulsion usually results from chronic degenerative meniscal tears [ 2 , 8 , 10 , 19 ] and is seldom associated with posterior cruciate ligament tears . according to the radiological literature , the incidence of meniscal root tear is 89.8 % [ 3 , 6 , 20 ] , whereas the orthopaedic literature reports a wider range between 6.7 % and 12.4 % [ 1 , 9 , 10 ] .
two subcategories of meniscal root tear can be distinguished : root avulsion from the tibial plateau and meniscal posterior horn tear within 1 cm from the root .
these tears are biomechanically similar because they can disrupt the circumferential fibers of the meniscus resulting in failure of the hoop strain mechanism [ 9 , 11 , 12 ] . following rupture , the ability to resist extrusion under axial loading is definitely lost [ 4 , 13 ] . in the only case described to date of a radial tear in both roots concurrent with a chronic acl tear , lee et al
. postulated that the mechanism of injury was involvement of the posterior lateral root together with an acl injury .
the medial radial tear in the posterior root was caused by forceful mechanical stress secondary to instability . in the present case , avulsion of both meniscal roots
was associated with an acute acl tear . from a review of the video recording , the mechanism of injury
was seen to be clearly due to forceful rotatory stress . as postulated by park et al .
, anterior tibial translation in an acl injury may pull the lateral meniscus forward , stripping the meniscofemoral ligament away the meniscus attachment .
the mechanism of medial meniscus root injury is more difficult to explain . due to external rotation , for stress associated with compression axial load ,
have shown that the anterior tibial force and the external tibial torque during knee loading produce relatively high posterior horn attachment forces , presumably by impinging the medial femoral condyle against the posterior meniscal rim . in our patient
the sequelae of a medial root avulsion left in situ or misdiagnosed is functionally equivalent to total meniscectomy with meniscal extrusion and rapid progression to knee arthritis [ 17 , 18 ] ; however , there is no consensus on the treatment of lateral meniscal root tear .
the fewer lateral meniscal tears in chronic versus acute acl tears have led to conservative treatment of such lesions .
there are several reasons justifying this approach : concomitant acl reconstruction creates blood clots and joint stability , increased blood supply to the posterior horn in comparison to the lateral meniscal pars intermedia , and absence of definitive clinical complaints when a lateral meniscus tear is left in situ .
so , a radial or complex ( radial and longitudinal ) tear that occurs within 1 cm of the meniscal attachment may be more likely to heal spontaneously [ 7 , 22 ] .
the lateral meniscal root has two distinct insertions : one is anterior and attached to the posterior aspect of the tibial intercondylar eminence , and the other is posterior to and confluent with the meniscofemoral ligament .
when root avulsion occurs , the latter insertion probably inhibits spontaneous healing because of continuous traction by the meniscofemoral ligament during knee movements .
recently , schillamer et al . demonstrated that posterior horn avulsion of the lateral meniscus causes peak tibiofemoral contact pressure to increase from 2.8 to 4.2 mpa , but that the peak pressure returns to normal after repair to bone via a transtibial tunnel . in this rare case of tears in the roots of the posterior horns of both menisci ,
concomitant with an acute acl tear , radiological and clinical outcome after surgery confirmed good healing . both the lateral posterior horn and the medial posterior horn need to be considered when planning acl reconstruction | a rare case of acute avulsion of both posterior meniscal roots concomitant with an acute anterior cruciate ligament ( acl ) tear in a professional soccer player is described .
while avulsion of the lateral meniscal root has been extensively reported in association with acl injuries , medial root avulsion has never been reported in association with acute acl . a review of the video documentation of the match accident revealed the exact mechanism of injury was a forceful external rotation of the standing limb . | Introduction
Case report
Discussion |
spinal meningiomas are common , comprising 25 - 46% of primary intra - spinal tumors7 ) .
they affect mostly female patients , and the average age of patients is about 50 years7,15,23,24 ) .
spinal meningiomas are located in the intra - dural and extramedullary spaces , and typically adhere to the dura matter7,15 ) . to date , few cases of non - dural - based meningiomas have been reported2,3,4,6,8,9,11,13,14,16,17,18,19,20,21,22 ) , as most are reports of clear cell meningiomas2,3,4,6,8,11,13,14,16,18,20,21,22 ) .
this case report describes the first case of a non - dural based fibrous meningioma in the cervico - thoracic region in a 49-year - old female patient .
a 49-year - old female patient was admitted complaining of persistent pain and numbness in both legs for 4 months and a recently developed gait disturbance .
a neurological examination revealed bilateral numbness of the lower extremities that was particularly right - side dominant with an unclear dermatome .
bladder and anal sphincter functions were preserved and motor and sensory functions of the upper extremities were normal .
a laboratory examination and plain radiography of the cervical and thoracic spine were unremarkable . magnetic resonance image ( mri ) demonstrated a 1.71.4-cm lobulated mass in the c7-t1 dorsal space , probably the intra - dural extramedullary space ( fig .
t2-weighted mri showed a low - signal - intensity elliptical lesion , which was enhanced peripherally on a gadolinium enhanced t1-weighted image .
the spinal cord was severely compressed and displaced anteriorly by the mass lesion . a c7-t1 cervical laminectomy and vertical incision in the dura
no dural attached or adherent point was observed around the mass lesion when the intra - dural space was explored .
the mass was covered with a white membrane , which was dissected and opened , revealing a 1.61.4-cm yellowish round mass ( fig .
immunohistochemical staining revealed vimentin - positive , epithelial membrane antigen - negative , and s-100-protein - negative tissues ( fig .
spinal meningiomas typically affect females at a female - to - male ratio of 4 - 5 : 1 and usually occur in patients > 50 years7,15,24 ) .
the most frequent site of a spinal meningioma is the thoracic spine , followed in frequency by the cervical and lumbo - sacral spinal levels7,15,24 ) .
meningothelial and psammomatous meningiomas are the most common histopathological subtypes of spinal menin - giomas15,24 ) .
a fibrous meningioma is an uncommon subtype , representing about 1% of all spinal meningiomas15,24 ) and has benign features .
spinal meningiomas typically attach to the dura , and non - dural spinal meningiomas are rare1,7,10,12 ) .
eighteen cases of non - dural based spinal meningiomas have been reported to date ( table 1)2,3,4,6,8,9,11,13,14,16,17,18,19,20,21,22 ) .
most tumors in these cases occur in the lumbo - sacral region ( 16 cases ) , including the thoraco - lumbar junction ( two cases)2,3,4,6,8,9,11,13,14,16,17,18,19,20,22 ) .
the other two cases were cervical ( c3 - 5 ) and thoracic ( t9 - 10 ) meningiomas11,21 ) .
our case is the third case that involvesd cervico - thoracic spine ( c7-t1 ) .
the average age of these cases was about 25 years , which is less than the average age of patients with general spinal meningiomas , except one case ( a 65-year - old female with a thoracic clear cell meningioma)21 ) .
most of histopathologic subtype of non - dural - based spinal meningiomas was clear cell meningioma ( 15 cases)2,3,4,6,8,11,13,14,16,18,20,21,22 ) , whereas the remaining three cases were angiomatous , transitional , and meningotheliomatous meningiomas9,17,19 ) .
in several cases of clear cell meningioma , complete total resection with postoperative radiotherapy was considered because of high recurrence rate4,13,22 ) .
therefore , in cases of non - dural based spinal mengiomas , the possibility of total resection and/or radiotherapy should be taken into consideration .
however , it is remarkable that subtype of present case was benign as fibrous subtype , and there were other three cases of benign subtypes .
mri of a spinal meningioma typically shows homogenous enhancement and the " dural tail sign " , which is dural enhancement or thickening near the tumor on enhanced t1-weighted images5 ) .
however , the meningioma in the present case showed only peripheral enhancement of the tumor on a gadolinium enhanced t1-weighted image and no " dural tail sign " .
therefore , even if no typical sign of a meningioma on mri such as the " dural tail sign " is present , the possibility of a meningioma should not be ruled out .
we report the first case of a fibrous - type spinal meningioma without dural attachment . | spinal meningiomas typically adhere to the dura matter . non
- dural based spinal meningiomas are rare and most are clear cell meningiomas .
we report here the first case of a fibrous meningioma with non - dural attachment .
the patient was a 49-year - old female , who complained of numbness in the legs and a gait disturbance .
magnetic resonance imaging revealed a 1.71.4-cm mass in the c7-t1 intra - dural extramedullary space , showing peripheral gadolinium enhancement without a " dural tail sign " .
a complete microsurgical resection was performed .
the mass was covered with a white membrane but was not adhered to the dura , and its appearance was consistent with a neurilemmoma .
the histopathological diagnosis was fibrous - type meningioma .
the recovery of the patient was uneventful .
no surgical complications and no recurrence of the tumor had occurred at the 6-month follow - up . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION |
conformer generation has been a topic of considerable interest to the modeling community for a number of years , and a large number of methods for conformer generation have been presented .
these methods have utilized a wide variety of approaches to the problem , including systematic enumeration , knowledge - based rule sets , random coordinate embedding , and energy - based or energy - biased sampling .
all these approaches attempt to generate a set of conformations designated to be different from one another by some measure , usually geometric , and thereby aim to sample some subset of the conformational space available to a molecule . the method used by omega
is based on random coordinate embedding and refinement , followed by torsion driving using a set of rules derived from experimental structures and energy profile calculations , and then subsequent sampling of the resulting conformer ensemble by geometric and energetic criteria .
publications that demonstrate the utility of conformer generators have approached the problem of validation of such tools in a number of different ways .
conformer generation is not an end in itself but a means to obtain input for other applications , most frequently shape comparison tools ( or other 3d ligand matching approaches ) and docking engines .
indeed conformer generation is often subsumed as a portion of a docking algorithm , which prevents independent evaluation of the conformer generation within such tools .
one approach to validation has , therefore , been to generate conformer ensembles for use in a downstream tool ( e.g. , pharmacophore perception).(9 ) the quality of the conformer ensembles is then judged by the quality of the output from the downstream tool . while it might be argued that the real - life use of conformer generators is well reflected in this approach , judging the quality of a conformer generator based on the performance of a separate tool is fraught with problems .
if this tool has been optimized to use conformers from a certain engine , then it would be expected to produce
better results from this engine over all others . other approaches to validating conformer generators have emphasized estimation of conformational coverage based on both pharmacophoric and geometric measures.(10 ) in studies of this kind , coverage of conformational space is held to be an absolute good and the tools under study are assessed based on their ability to cover the greatest amount of conformational space .
however , there are large areas of accessible conformational space for a flexible molecule that are populated by conformers very different in shape and geometry from any experimentally determined conformer ( be it in the solid state , in solution , or in the gas phase ) . as such
, simply maximizing coverage of conformational space may result in generation of a conformer set containing a high proportion of conformers irrelevant to any measurable experimental property of the molecule . by far , the most common , though not always
the most well - executed , method for validation of conformer generators , and the method used in this paper , is the reproduction of experimental crystal structures(11 ) ( often those retrieved from the protein databank ( pdb)(12 ) ) .
the implicit argument proffered for this approach is that coverage of conformational space near an experimental conformation of a molecule is more important than coverage in other regions of conformational space . since experimental structures , while frequently not at a local minimum for a force field,(13 ) are generally considered to exhibit low levels of strain,(14 ) the regions of conformational space near an experimental structure can be considered as basins in the molecule s energy profile .
accordingly , assessing sampling in these regions of the energy hypersurface can be a good guide to the performance of a method in generating low - energy conformations . to perform this sort of validation ,
the connection tables of these ligands are used as input for the conformer generator to provide a set of conformers for each ligand , unbiased by any structural information of the original experimental conformation .
a metric of quality is then calculated that provides a measure of the three - dimensional similarity between the experimental conformation for the ligand and the conformers in the set .
there are , therefore , two critical choices to this method of validation : selection of the appropriate metric of quality to perform the comparison and selection of suitable cocrystal structures to provide the ligands . while many metrics for comparing computed and experimental conformations are available , such as rms error in cartesian space ( rmsd ) , rms error in torsion space ( rmst ) , and relative displacement error ( rde),(15 ) choosing the metric of quality presents some subtle problems .
atom - based metrics like rmsd , while very popular , have significant problems in their interpretation and necessarily have a dependence for their significance on the experimental error / precision of the crystal structure ( a particularly difficult issue for pdb ligands ) .
in addition , rde and rmsd have no easily calculable upper bound ( unlike rmst ) , and all these measures have a size dependence that is not easily compensated for . in this paper ,
two different metrics are used to compare computed and experimental conformations of druglike molecules , one atom based ( rmsd ) and one shape based ( tanimoto combo ) .
, it is hoped to provide a more complete picture of the ability of omega ( or any other conformer generator ) to match experimental conformations . identifying suitable cocomplex structures from the pdb to provide the experimental conformations of small molecules
unfortunately , in most publications in this area , insufficient care is taken in the selection of the structures , resulting in unwarranted or questionable conclusions being drawn based on unreliable data .
some of the issues that should be considered in the selection of structures from the pdb include the following : are the deposited models of good quality at both the global and local levels ? there is little sense in trying to reproduce a deposited structure that is a poor model of the experimental density or a model that is not completely defined by the density .
when using atom - based metrics like rmsd , paying attention to the precision in the atomic coordinates is critical : an rmsd or any measure of conformer reproduction more precise than the inherent inaccuracy / experimental error in the atomic coordinates is meaningless .
significant atomic clashes , either between the ligand and the protein or between atoms of the ligand , must be artifacts of the process of fitting the ligand to the experimental electron density and not an inherent property of the structure .
the binding energy available upon proteinligand complexation is unlikely to be sufficient to drive atoms into close contact with one another .
a set of ligands with a large degree of structural redundancy does not provide as rigorous a test of conformer generators as a structurally nonredundant set does .
also , in order for the results of the validation to be useful in a predictive or prospective manner , they should be readily generalizable to any set of applicable molecules .
this can only be done with validation sets that cover chemical space in a reasonable way .
consideration of these questions has allowed the development of quantitative criteria for structural quality in the pdb .
these criteria were applied as successive filters to three previously published large data sets of cocrystal structures , pdbbind,(16 ) the sadowski set(13 ) and the kirchmair set,(17 ) to provide a high quality group of cocrystal structures to establish a gold standard for the validation of approaches to conformer sampling .
a set of small molecule structures from the cambridge structural database ( csd)(18 ) was also collected to provide complementary data to that obtained with the ligands from the pdb .
these two data sets are applied to the evaluation of the omega conformer generation application using the metrics mentioned above .
here , the omega algorithm is introduced in some detail ; the metrics for comparison of the generated conformers to the experimental structures are outlined , and the criteria for the identification of suitable proteinligand cocomplexes and small molecule structures with which to validate the algorithm are discussed . the design goal for omega is to provide a thorough , though not exhaustive , sampling of conformational space for druglike molecules at as high a speed as possible ( typically 22.5 s per molecule on a workstation running sled 10 with a 2.4 ghz processor and 4 gb of ram ) .
the process used by omega for conformer generation can be considered in several parts , two of which are generic and are precalculated and the rest of which are molecule specific and occur at run time .
the first two steps are construction of a database of fragments from which the molecule will be assembled and derivation of a torsion library ; the remaining steps generate a large ensemble of conformations and sample from this ensemble to deliver the final set of conformers .
the omega fragment database contains one or more 3d conformations for every entry ( flexible rings have multiple conformations , rigid rings and acyclic groups have one conformation ) .
the database is generated by fragmenting a very large collection of commercially available compounds into contiguous ring systems and small linear linkers .
one or more 3d conformations for each fragment are generated by the following procedure : a distance bounds matrix is generated based on the connection table of the fragment ; the distance bounds are augmented by volume constraints for chirality and planarity ; the coordinates of each atom are randomly embedded in a cartesian space and optimized to fulfill the bounds and constraints ; the fragment is further refined against a modified version of the merck molecular force field ( mmff94),(19 ) in which the electrostatic and attractive van der waals terms are removed .
the procedure is repeated hundreds of times for each fragment , with cycles being sampled more heavily than acyclics , and the lowest energy conformers that are unique in rmsd space are retained .
a hierarchy of torsions defined by smarts patterns is preorganized so that every rotatable bond in a small molecule matches at least one torsion rule .
every torsion definition is associated with a list of angles at which the torsion should be sampled . very common or structurally unique
torsions may be represented by very specific rules , while unusual torsions are only covered by generic torsion rules , often with relatively heavy sampling .
specific rules are employed when the barrier to rotation is high and the minima in the torsion profile are well - known .
the torsion angles specified by the torsion library were derived by analysis of a set of experimental crystal structures ( mostly from the pdb ) and from energy scans of certain torsions against mmff94 .
each graph is fragmented in the same manner as the fragment database and fragment conformations are drawn directly from the fragment database described in step 1 . if a molecular fragment is not found in the fragment database , the procedure described in step 1 is carried out on - the - fly to generate coordinates for the missing fragment .
the fragments are assembled into the parent molecule by overlapping fragments using geometric and chemical rules , thereby providing one or a small number of initial conformations for the molecule .
this set of conformers includes all necessary ring conformer sampling . every rotatable bond in the conformers generated in step 3
is compared to the torsion library from step 2 , and the appropriate torsion angles are noted .
a torsion buildup procedure is applied to all the torsions in the molecule to generate a large ensemble of conformations that does not contain severe internal clashes or duplicates due to common symmetries .
the design goal of omega is to produce up to a few hundred low energy , structurally diverse conformers , and as such , the much larger collection of conformers produced by step 4 must be sampled .
this process is begun by ordering the conformers using a simple scoring function that eliminates conformers with internal clashes .
while many functions are suitable for this process , omega uses a modified form of mmff94 ( vide supra ) .
next , beginning with the lowest scoring conformer , all higher scoring conformers that are less than a user - defined rmsd ( including heavy atom automorphisms ) to the lower scoring conformer are eliminated ( the default rmsd cutoff is 0.5 ) .
this process is continued with each successive conformer in the ordered list until ( a ) all conformers have a score less than 10 units higher than the lowest energy conformer produced or ( b ) 200 mutually unique conformers are identified . at no stage in this process
is minimization performed because this tends to produce highly compact ( folded ) conformations not reflective of the conformation(s ) found in solution or bound to a protein .
this is mostly due to the differences between the gas phase and the solution phase or complexed potential energy surfaces for small molecules .
extensive experimentation in - house has shown that minimization of ensembles of conformers either before or after deduplication improves neither solid - state structure reproduction nor virtual screening performance with tools like rocs or fred .
two metrics for comparison of conformer ensembles to an experimental conformation were chosen , rmsd and the tanimoto combo , calculated using the shape toolkit from openeye scientific software . the tanimoto combo ( or tc )
score represents a combination of shape matching ( shape tanimoto ) and functional group matching ( color tanimoto ) .
tc offers a complementary approach to the atom - based rmsd measure for a number of reasons treated at greater length in the discussion section .
an important difference between tc and rmsd is that tc is bounded ( by 0 and 2 ) .
it should be noted that the processes used to overlay and score conformers using rmsd or tc are very different , and therefore , the conformer giving the minimum rmsd to the experimental structure will almost always be different than the conformer giving the maximum tc to the experimental structure .
two sources of small molecule structures , the pdb and the csd , were utilized . to find small molecules from the pdb ,
first , suitable cocrystal structures were identified from larger sets using global criteria ; then , the ligands were extracted and inappropriate ligands were removed using local , ligand - centric criteria .
small molecule structures from the csd were obtained from a previously published data set,(18 ) which was subjected to some of the ligand - centric criteria used to identify suitable pdb ligands .
there are two kinds of criteria that must be considered here : global criteria for the structure as a whole and local criteria pertaining to the fit of the ligand to its local density and the properties of the ligand itself .
a number of possible criteria of quality for crystal structures were enumerated in a paper by hartshorn et al . on the selection of a set of structures suitable for validating docking programs for
were that all structures must have good nominal resolution and have deposited structure factors , while the local criteria were that the ligand must be well fit to its local density and can not be covalently bound to the protein .
this list has been extended in this work , both at the global level and the local level .
in addition to the hartshorn criteria at the global level ( good nominal resolution , deposited structure factors in the pdb ) , the model must have good overall metrics of model quality and reasonable coordinate precision . at the local level
, it is demanded that a ligand be well fit ( using different criteria to hartshorn et al .
it is further required that the ligands show no intra- or intermolecular clashes and that they be diverse at the graph level .
nominal resolution , while very commonly used as a measure of structure quality , is in fact a measure of the quantity of data gathered , not quality of the structure , and as such can only be used as a very rough guide to selecting a good structure.(21 ) in many studies in this area , the nominal resolution has been used as a surrogate for experimental accuracy , which is inappropriate ( see ref ( 22 ) and the discussion ) . however , since nominal resolution indicates the quantity of data gathered , at a resolution of 2.7 or better , the ratio of experimental data points to parameters in the model is greater than 1 .
as such , structures with a resolution of 2.7 or better offer the possibility ( but only the possibility ) of a well determined model . the quality of a deposited model can be independently checked only if structure factors ( electron density maps ) are available in the pdb , so all structures must have deposited structure factors . in order to ensure that the models used are globally well fit to the data ( and not overfit )
, we use the difference between the r - factor and the rfree , as introduced by brunger.(23 ) in this work , we demand that a structure must have a rfree within 0.05 of the r - factor.(24 ) in comparing computed and experimental poses for ligands , it is almost uniformly assumed that the experimental pose is infinitely precise and without error , which is untrue for any piece of experimental data .
the average positional errors for atoms in a crystal structure can be assessed using the diffraction - component precision index ( dpi).(25 ) we calculate dpi according to the approximation published by blow(26 ) ( using the blow rearrangement allows us to calculate an estimate for dpi based solely upon information commonly contained in the header of a pdb file ) .
the comparison of a computed conformer to the experimental conformer by rmsd obviously requires comparison of atom positions between the two conformers .
accordingly , the positional uncertainty in the atoms in the experimental structures should be less than the rmsd for the comparison to be meaningful . in this work
, we estimate positional uncertainty in atomic positions as sqrt(2 ) dpi , following goto et al.(27 ) the maximum for the average positional uncertainty in a structure was set at 0.6 , which sets the maximum for the dpi to be 0.42 . in this way ,
any rmsd result from this set greater than 0.6 is guaranteed to be a prediction greater than the error in the experimental data , and therefore , such a prediction will be significant .
whatever the magnitude of the rmsd , a correction to it will have to be made to account for positional uncertainty in the ligand atoms ( see the results section ) .
the assumption used in this paper is that the ligand atoms have average positional uncertainty .
while the real uncertainty for the ligand atoms may well be different , it is not reliably calculable at the present time ; therefore , we use dpi as our best estimate . when considering whether or not a ligand is suitable for conformer generation studies , the local quality of fit of the ligand to its density , as well as global metrics of quality , should be considered . in this study , three local metrics , the real - space correlation coefficient ( rscc),(28 ) the real - space r - value ( rsr),(29 ) and the occupancy - weighted b - factor ( owab )
were used to ensure that the ligand has been sensibly fitted to the electron density .
the use of the occupancy weighted b - factor as a criterion was based not on the idea that this number necessarily measures thermal mobility in the ligand , as is often assumed .
rather , we used the owab to indicate either structures where the ligand is genuinely disordered in the active site , and therefore , the idea of reproducing the bioactive conformation is questionable or structures where there is some pathology of the model that led , in the process of fitting the ligand to its density , to very high or very low b - factors .
these models are inherently of dubious value and should not be included in the set .
the criteria used in this work were that rscc > 0.9 , rsr < 0.2 , and 1 < owab < 50 .
these data were obtained by download from the electron density server ( eds).(30 ) when applied sequentially , these global and local filters should ensure that a ligand structure arises from a good quality overall model and has been well fit to its local density .
the filters were applied to a large set of pdb structures ( see results ) , and the cocomplexes that survived were then processed to separate the ligand and the protein .
these pairs were checked to ensure that they do not show any intermolecular clashes , and those ligands that showed no clashes were designated as well - solved structures , to be used in the final , ligand - based filters . in these final steps , we focus on identifying suitable molecules using simple physicochemical and graph - based properties . in the last filtering step , we removed ligands that are not representative of the population of molecules for which this validation is intended .
as a first step , we removed all well - known cofactors , as these were not as relevant to the goals of this study ( validation of omega s performance on druglike molecules ) .
as mentioned above , omega depends upon mmff94 for its energy calculations , so molecules that contain elements unknown to mmff94 ( such as boron ) must be removed . to ensure that we used a collection of ligands that provides as broad a set of tests for the omega algorithm as possible
, we attempted to select a structurally diverse set of ligands . at the simplest level
, the molecules can be neither too rigid ( so that the conformer sampling problem is trivial ) nor too flexible ( so that a close reproduction of the experimental conformation is more a matter of good luck than a good algorithm because of the high dimensional nature of the search space ) and neither too small nor too large .
we , therefore , applied the following criteria : 3 maximum number of rotatable bonds 16 ; 8 heavy atom count 50 .
preliminary analysis revealed that molecules designed to bind to thrombin were over - represented in the set of well - solved ligands identified by the criteria used thus far .
these ligands tended to share a high degree of substructural similarity , so a method to introduce some level of structural diversity at the graph level was sought .
we applied a simple measure of molecular similarity using the lingos method;(31 ) any pair of molecules that had a lingos tanimoto > 0.9 was flagged for removal , and the molecule with the better set of local fitting metrics for the pair ( rscc , rsr and owab ) was retained .
the use of graph - based metrics like lingos for structural diversity is a surrogate for the more relevant but more difficult to assess diversity in the torsions and flexible rings present in the molecule . to further reduce the influence of experimental error on this data set
, we demand that the ligand does not show any nonbonded intramolecular atomic clashes , as these are a mark of a poorly solved model . as further discussed in the results section , we turned to the csd for precise distributions of interatomic distances with which to identify clashing atoms .
a final visual check was performed to remove molecules that are structurally analogous but were not identified as such by the lingos comparison ( those with large common substructures and/or a large fraction of their functional groups in common ) .
the attrition rates for the different data sets and an analysis of omega s ability to reproduce these 197 surviving structures is given in the results section .
a parallel analysis was performed on high quality small molecule structures from the csd , as outlined in the following paragraphs .
an important part of the selection criteria for structures from the pdb focused on removing structures that were not reliably and accurately fit to their density , and a large majority of the candidate structures failed these criteria ( see the results section ) .
a further problem with molecules from the pdb is the high level of inherent experimental uncertainty in the atomic positions . both of these problems can be avoided entirely if small molecule structures from the cambridge structural database ( csd ) are used .
a high quality set of 492 druglike structures has been extracted from the csd as part of a previously published study,(18 ) and these were used as the basis for generating a complementary set of structures to those we obtained from the pdb .
no filtering of these structures based on model fit ( global or local ) was necessary as all atoms are accurately located in these models . applying the same physicochemical and diversity criteria as were used for the ligands from the pdb gave a set of 480 small molecule structures upon which we could perform the same validation experiments .
comparison of these results to those from the ligands from the pdb can be found in the results section .
all code was written in python ( version 2.4 ) , and statistical calculations were performed with rpy version 1.03 .
cheminformatics functions ( including rmsd and lingos calculations ) were performed using openeye s oechem ( version 1.6.1 ) , and shape calculations were performed using the openeye shape toolkit ( version 1.6 ) .
proteinligand cocrystal structures were downloaded from the pdb and filtered according to the criteria laid out in the methods section .
rscc , rsr , and owab data for each pdb ligand were obtained from the eds .
molecules from the csd were obtained from one of the authors of the original study ( personal communication , k. brameld ) .
molecules were converted into isomeric smiles format using the oechem toolkit before being used for conformer generation .
conformer databases were generated using omega version 2.3 , with an energy window for acceptable conformers of 10 kcal / mol above the ground state using a modified version of the mmff94 force field , a maximum number of conformations per molecule of 200 , and an rmsd cutoff of 0.5 ( the default settings in omega2.3 ) .
in the methods section , we presented a set of criteria for selecting suitable proteinligand cocomplexes from the pdb and then identifying suitable ligands from those complexes .
we applied these criteria to three large data sets ( two of which have previously been used in conformer validation studies ) that together consist of over 4500 cocrystal structures : 778 structures that kirchmair et al . used in their comparison of catalyst and omega,(17 ) the set used by sadowski and bostrom in their study on the omega 1.8.1 torsion library(13 ) ( 1267 structures ) and a subset of the pdbbind database(16 ) ( 2516 structures ) .
the percentages of structures remaining after each filter was applied are illustrated in figure 1 . in all three databases
, we found very large levels of loss when we applied our filtering criteria ( over 90% of the structures in every database were removed ) .
it can easily be seen from figure 1 that there are three criteria that , when applied in the given order , remove the highest percentages of the structures : nominal resolution greater than 2.7 ( criterion a ) , poor fits of the ligand to its density ( criterion e ) , and inappropriate physicochemical properties ( criterion f ) .
the filters used are ( a ) nominal resolution 2.7 ; ( b ) structure factors must be present ; ( c ) r rfree must be < 0.05 ; ( d ) dpi < 0.42 ; ( e ) rscc > 0.9 , rsr < 0.2 , owab < 50 , no intermolecular clashes ; ( f ) physicochemical properties ; ( g ) pairwise lingos similarity < 0.9 , no intramolecular clashes .
the problem of identifying atomatom clashes ( filters e and g ) based on analysis of interatomic distances in structures from the pdb is nontrivial , due to the inherent uncertainty in those atom positions .
therefore , we chose to perform an analysis of nonbonded contacts for commonly occurring atoms ( c , n , o , s , p , f , cl ) using high quality structures ( r - factor < 0.05 , no disorder ) of organic molecules from the csd , where the atoms are located with very high precision .
the cutoffs identifying clashes were set at an interatomic distance above which 95% of the distances for that ( nonbonded ) atom pair in the csd occurred .
results are shown as mean / median for each column heading ; u = the coordinate uncertainty ( sqrt(2 ) dpi ) .
the only well fit ligands that can show intermolecular clashes are those that are covalently bound to their target protein , and these are explicitly excluded from this set .
therefore , noncovalent ligands that have clashes , by these criteria , with their cognate protein are incorrectly solved .
none of the ligands that passed the previous filters ( a to e in figure 1 ) showed any intermolecular clashes , indicating that the criteria used in these filters ( especially the rsr and rscc filters ) can be useful in identifying misfitted ligands . similarly
, no ligands were removed based on the intramolecular clash filter , further reinforcing the idea that rscc and rsr can provide useful information on the quality of a ligand s fit to its density .
however , it is noteworthy that several structures from such well - known data sets as the perola and charifson set(33 ) show intramolecular clashes , as these authors selected structures using only global criteria of fit , omitting local measures . as such
, these structures should not be used in conformer generation studies , as has been done by some authors,(34 ) because the deposited coordinates for the ligand are incorrect ; atomatom clashes can not be enforced by protein binding and simply reflect an error in the solution of the structure .
after the filters had been applied , the surviving ligands from each of these three sets were combined and deduplicated and close analogues were removed as discussed in the methods section .
the mean pairwise similarity of these molecules using the lingos tanimoto measure is 0.18 , so the structures are relatively diverse from one another at a graph level .
conformers were generated for these molecules using omega with the default settings ( see the methods section ) .
the average cpu time per molecule in this set was 2.05 s. in figure 2 , we show the reproduction of the experimental conformation using the lowest rmsd and highest tanimoto combo metrics .
approximately 83% of the structures are reproduced within a 1 rmsd and 66% are reproduced with a tanimoto combo of 1.5 or better .
distribution of rmsd and tanimoto combo between the closest conformer in a set and the experimental conformation for 197 ligands from the pdb .
the relationship between best ( lowest ) rmsd and best ( highest ) tanimoto combo for this data set is shown in figure 3 , where the lowest rmsd for any conformer to the experimental conformation in a set is plotted against the highest tanimoto combo for any conformer to the same experimental conformation . as expected , the cases where the rmsd is low ( < 0.25 ) all show very high tanimoto combos .
as rmsd increases , the relationship to tanimoto combo becomes increasingly less linear , in that there are several examples where constant rmsd gives a widely varying tanimoto combo and vice versa .
quantitatively , we can assess the correlation between the two measures using either the spearman rank correlation coefficient or the kendall tau .
for this data , the spearman coefficient is 0.925 and the kendall tau is 0.775 . as such ,
the two measures of conformer reproduction are correlated ( as would be hoped for two measures attempting to assess the same thing ) but are by no means identical . best rmsd v. best 3d shape and chemical similarity ( tanimoto combo ) for reproduction of 197 ligands from the pdb .
that these two measures are assessing conformer reproduction differently is to be expected , since they are generated in rather different ways and , therefore , provide complementary means to compare conformations .
a striking difference in the two overlay methods is illustrated in figure 4 , where two different overlays of conformers of the ligand from the pdb structure 1 v2n are shown .
the experimental conformation is in green ; the best overlay based on lowest rmsd is in red , and the best overlay based on highest tanimoto combo is in blue .
the best rmsd overlay here is 0.89 , while the best tanimoto combo is only 1.06 ( tanimoto combo s range is 02 , see methods section ) .
the origin of this difference in the assessment of the quality of reproduction when using these two different metrics is most clearly seen by inspecting the alignments of the benzamidine functional groups .
comparison of the experimental conformation of the ligand 1 v2n ( green ) with the best overlay as provided by rmsd ( red ) and the best overlay as provided by tanimoto combo ( blue ) .
as can easily be seen in figure 4 , the rmsd overlay in red symmetrically , but poorly , matches both benzamidine functional groups , while the tanimoto combo overlay matches one benzamidine group very well ( the one on the left in figure 4 ) while the position of the other benzamidine group is much less well reproduced , giving the surprisingly low tanimoto combo .
this illustrates a consistent difference between the two methods of comparison ; rmsd tends to produce overlays that are equally good ( or bad ) everywhere across the molecule , while tanimoto combo tends to produce overlays that are very close in some portions but can be much more divergent in others . while all the structures in this validation set have high coordinate precision , there is still some level of inherent uncertainty in the atomic coordinates .
therefore , when comparing a computed pose to an experimental pose using atom - based metrics like rmsd , this experimental uncertainty , u , in the atom positions must be taken into account .
there are two possible approaches to this , both of which are shown in table 1 .
the first is to use as the figure of merit the maximum of the rmsd and the coordinate uncertainty , u ( max(rmsd , u ) in table 1 ) , since the prediction can not be more accurate than the error in the experimental data ( the coordinate uncertainty ) .
the second is to use the difference between the rmsd and the coordinate uncertainty ( rmsd - u in table 1 ) , which reflects the additional error in the model introduced by conformer generation .
this number is set to zero if rmsd < u. as can be seen from column 3 ( rmsd - u ) in table 1 , the average amount of noise introduced by omega in generating a conformer close to the experimental is small .
also , due to carefully choosing structures that have low levels of uncertainty , the mean and median for the maximum of the rmsd and experimental uncertainty ( column 4 ) are very close to the mean and median for the raw rmsd ( column 2 ) .
therefore , the level of accuracy in our predictions is only minimally affected by the experimental uncertainty in the structures that make up the data set .
an issue that is most often ignored in studies of this kind is the prospective utility of the performance metrics presented , or how robust are the performance metrics to changes in the composition of the data set and , therefore , how predictive are the results of future performance ?
this question is addressed here by bootstrapping : we omit a randomly selected 10% of the results ( allowing duplicates ) , recalculate the mean rmsd and repeat 10 000 times , and then find the 5% and 95% quantiles .
the 5% quantile is 0.647 , and the 95% quantile is 0.688 , thereby giving us the 90% confidence interval .
therefore , we can say that for a future experiment on a set of molecules with similar properties to those of this pdb - derived set 90% of the time the mean rmsd will lie in the range 0.6470.688 .
this small interval implies that , even though the data set is not as large as we had hoped , the mean found here is a reliable indicator of omega s average performance on as yet untested collections of ligands .
the 90% confidence interval for the mean tanimoto combo can be calculated in the same fashion and is 1.54 to 1.64 .
we may also use these confidence intervals as a method of comparing two sets of results to determine if they are significantly different from one another ( see discussion ) .
so far , we have dealt with the properties of the single conformer closest by a given metric to the experimental .
while this measure has significant utility , in another view of the problem , the proportion of conformers within a certain distance of the experimental structure is important .
this method of measuring success is very rarely used(35 ) but provides a useful complement to focusing solely on the matching of only the best single conformer from a possibly large set , as we have done to this point .
a common upper bound for successful reproduction of an experimental structure in docking is an rmsd of 2 , this number being thought of as providing a sufficiently close match of the important interaction points on the ligand ( hydrogen bond partners , etc . ) .
accordingly , a close reproduction of an experimental pose could be considered to have an rmsd of less than 2 . in this work , since we calculate each metric after aligning the conformers to the experimental structure , a smaller cutoff is more relevant , so we have selected an rmsd of 1.25 as our definition of close .
figure 5 shows the cumulative frequency of cases where conformers are close to the experimental .
distribution of percentages of conformers that are close ( rmsd < 1.25 ) to the experimental conformation of 197 structures from the pdb .
it can be seen from the figure that , for around half of the cases ( 47% ) , less than 25% of the conformers generated for a given molecule are close to the experimental conformation , while for around 8% of the cases 100% of the conformers generated are close . in total ,
19% of the conformers generated for this set are within 1.25 rmsd of the protein - bound conformation .
having a reasonable proportion of the conformers in the set close to the protein - bound structure is helpful for downstream protein - based tasks such as docking .
however , as is seen in the next section , the same molecule may have multiple solid - state structures and , therefore , have too high a fraction of the conformational ensemble , for that molecule close to only one of them would not be desirable . as mentioned previously , 492 molecules from the csd
were obtained from a previous publication , of which 480 survived the ligand filtering process ( physicochemical and graph diversity filters ) .
the mean pairwise similarity of this set using the lingos tanimoto is 0.14 , again indicating that the molecules are diverse at a graph level .
it was hoped that these molecules from the csd would represent a different set of challenges in conformer generation ; the pdb structures are obtained in an aqueous environment , and the majority of csd structures are obtained in nonpolar environments .
accordingly , using sets of high - quality structures from both sources will further challenge omega s abilities to reproduce solid - state structures . while there are large numbers of small molecule structures in the pdb and the csd ,
the two databases show only a low level of overlap ; a comparison of over 4000 druglike ligands from the pdb with over 57 000 druglike molecules in the csd finds only 224 molecules in common ( see supporting information for pdb and csd codes ) . of these 224 molecules ,
only 32 meet the quality criteria for pdb structures laid out previously ( see methods ) .
the differences in the conformations of these 32 molecules ( as measured by rmsd ) found both in the pdb and the csd sets are shown in figure 6 .
the mean rmsd for this set is 1.02 , a figure close to that found in similar analyses .
rmsd distribution for the same molecule found in both the csd and the pdb ( n = 32 ) .
clearly , the same molecule in the csd and the pdb can sometimes adopt rather different conformations ( over 7% have rmsd > 2 ) , while other protein - bound and uncomplexed conformations can be quite similar ( 16% have rmsd 0.5 ) .
it is likely that at least some of these differences in conformation are due to the crystal packing forces often found in csd structures .
these can have a profound effect on the conformation of a molecule , sometimes enforcing a conformation not found in protein - bound structures .
the larger differences in conformation for the same molecule indicate that there could be several basins in a molecule s energy hyper - surface in which solid - state structures are found , so sampling across a range of the hyper - surface could be useful .
while the molecules from the csd were selected to be druglike , they have somewhat different physicochemical properties than those from the pdb .
table 2 illustrates the differences between the csd molecules and the pdb molecules for two simple properties , heavy atom count and rotatable bond count , and figure 7 compares the distributions for these two properties .
it can easily be seen that for both properties the molecules from the csd occupy a smaller range than those from the pdb .
most importantly for this study , while the molecules from both sets are approximately equal in size ( as judged by their heavy atom counts ) , the csd - derived molecules are somewhat less flexible than the pdb derived set , having , on average , one fewer rotatable bond .
therefore , the csd derived set is expected to be an easier test of conformer sampling and reproduction than the pdb set . given as mean / median .
property distributions for molecules from the csd and the pdb . given the difference in rotatable bond count , the numbers of conformers produced by omega2 for this set and for the pdb set are rather different ; the median number of confomers per molecule was 47 for the csd set and 123 for the pdb set . in figure 8 is shown omega s performance in reproducing these structures , as judged by rmsd and tanimoto combo .
distribution of rmsds and tanimoto combos between the closest conformer in a set and the experimental conformation for 480 molecules from the csd . as can be seen in figure 8 , 441/480 ( 84.5% ) of the csd structures are reproduced within 1 rmsd , while 400/480 ( 83.3% ) are reproduced within 1.5 tanimoto combo .
note that in the case of these structures from the csd the positional uncertainty for the atoms is sufficiently small to be disregarded when calculating rmsds .
the spearman rank correlation for the two metrics in table 3 is 0.89 , and the kendall tau is 0.72 , in both cases slightly smaller than those found for the pdb set , again indicating that these two measures are assessing different aspects of omega s ability to reproduce the experimental conformation .
as with the results from the pdb ligands , these small intervals imply that our results are not strongly dependent upon the exact composition of this data set and as such are reliable indicators of future performance on data sets of similar property distributions to the one used in this study .
it can also be said with greater than 90% confidence that the csd results , either for rmsd or for tc , are better than the pdb results since the 90% confidence intervals for either metric do not overlap .
the results for close reproduction ( rmsd < 1.25 ) of the experimental conformations are much better than for the pdb ; overall , 39% of the conformations generated for the csd set are close to the experimental conformation .
this is entirely consistent with the results from the csd set relative to the pdb set for best pose reproduction ( as judged by either tanimoto combo or rmsd ) .
it is possible that the superior performance of omega on the csd set is not due to the lower average flexibility of the molecules but due to some consistent and unexpected difference in the nature of the molecules between the two sets .
therefore , a subset of the pdb ligands was derived to closely match relevant physical properties of the csd set ( heavy atom count and rotor count ) .
the aggregate performance metrics on this subset are much closer , though not identical , to those seen from the csd set , see table 4 .
reproduction of three data sets by omega , using both tanimoto combo ( tc ) and rmsd .
the 90% confidence intervals of the entire pdb set and the csd - matched subset of the pdb set do not quite overlap , though they are very close ( the difference could be entirely due to the physical property distributions of the two sets not being perfectly matched ) .
therefore , we can not with complete assurance attribute the entire change in omega s performance to differences in the rotatable bond count between the two sets .
however , we conclude that the superior performance of omega on the csd set derives to some large degree from the lower average flexibility of the csd set compared to the pdb set and not from bias in the omega knowledge base .
we used both the pdb- and csd - derived test sets to investigate another possible source of bias in the results ; the torsion library , which was assembled mostly by examination of ligand structures from the pdb .
if there is significant overlap between the molecules used to arrive at specific angles in the torsion library and the molecules in the test sets used in this study , then the results will be artificially good ( or artificially better for one test set over another ) .
we investigated possible training set bias by eliminating all training set knowledge from the torsion library . in these experiments ,
the simplest possible torsion settings were used : every rotatable bond was sampled at 30 degree increments , and new conformer sets were produced using defaults for all other settings .
interestingly , omega s ability to reproduce the solid - state structures from the pdb or the csd sets was not significantly changed ( whether measured by rmsd or tanimoto combo ) when using this naive torsion library .
results for the pdb set using rmsd are shown in table 5 , where it can be seen that the 90% confidence intervals overlap substantially . therefore , with 90% confidence , we can assert that omega s accuracy is not affected by the use of the knowledge - based torsion library . however , some other effects on performance were noted . for the pdb set , when the naive torsion library was used , the average number of conformations produced per molecule increased by almost 15% over using the default library ( from 123.3 to 139.6 conformers / molecule ) , while the total run time increased by nearly 40% .
it was also seen that the worst failures ( highest rmsd , lowest tc ) were less poor when the default , knowledge - based , torsion library was used than when the 30 degree library was used .
qualitatively similar results were obtained for the csd data set ( data not shown ) .
the object of this study was 2-fold : to arrive at reliable and challenging sets of solid - state structures to validate omega and to examine its default parameters for their effectiveness on these well chosen solid - state structures ( not to exhaustively evaluate a large number of combinations of parameters in omega for their efficacy ) .
the omega algorithm presented here combines knowledge - based and first principles approaches to conformer generation , so it can be described as systematic and rule based .
the knowledge - based part is the torsion library , while the fragment library , ensemble buildup , and sampling are all performed on a first principles basis .
we have developed a gold standard set of pdb ligand structures by paying particular attention to identifying structures that are good models for their electron density , an approach rarely taken in the literature .
many of the properties of a model that are used here are ignored or misinterpreted in other publications in this area .
for example , based on the published literature , it is commonly believed that selecting a cocrystal structure with a resolution below some cutoff value ( for example 2 ) ensures a good quality ligand structure . that this is clearly mistaken can easily be seen by inspecting the ligand models for two structures , both of 2 resolution , 1nhu and 1iy7 ( see figure 10 ) .
figure 10electron density for two ligands both solved at 2 resolution ; 1nhu on the left , 1iy7 on the right .
electron density for two ligands both solved at 2 resolution ; 1nhu on the left , 1iy7 on the right .
the 1iy7 ligand model is clearly a good model of complete density , while the 1nhu model is an interpretation of partial density that is obviously poor ( the deposited ligand coordinates show severe atomic clashes ) . in two other cases at 2 resolution or better , 1atl and 1eta
, there is no significant density for the ligand at all ( even when viewed at 1 ) making the deposited coordinates at best highly speculative educated guesses .
therefore , resolution of the parent structure alone is no guide to the quality of a ligand s conformation but should only be used as one criterion among many .
cases like 1nhu , 1atl , and 1eta also caution against using b - factors as a representation of thermal mobility in a structure .
if there is no density for a set of atoms , what physical meaning is there in the b - factors for those atoms ? models built from data collected to at least 2.7 resolution have a parameter to data point ratio of at least 1 , allowing the model to be well constructed .
the confidence in the fit of a model at the global level is increased if the difference in r and rfree is low ( < 0.05 herein ) , as a large difference in r and rfree is indicative of an overfit , though not necessarily poor , model .
a low level of difference also means that the local measures of fit , rscc and rsr , are meaningful , which is not the case for overfit models ( good values of rscc and rsr can be obtained for poor fits when r rfree is large ) . by ensuring that the structures in the set all have low experimental error in their atomic coordinates ( dpi ) ,
we can use atom - based metrics like rmsd , take appropriate account of this experimental error , and still generate meaningful measures of performance .
the rscc and rsr metrics often delineate ligands that show good fits to their local density from those that do not , and we thereby avoid many of the problems with structures from other publications . cases like 1nhu and 1atl are easily identified by these fit criteria as unsuitable for inclusion in conformation generator validation sets ( or any other kind of validation set ) , as the coordinates are not supported by the experimental data .
for example in 1nhu , the rsccs for the two versions of the ligand in the unit cell are 0.768 and 0.744 , and the rsrs are 0.27 and 0.25 ; while in 1atl , the ligand rsccs are 0.709 and 0.722 , and the rsrs are 0.35 and 0.35 , greatly exceeding the cutoffs used in this work .
sets,(33 ) and their presence in these sets only weakens the conclusions that can be drawn therefrom .
any ligand conformation from the pbd that shows intramolecular atomic clashes is an error on the part of the crystallographer , and such structures are easily avoided by the use of the rscc , rsr , and owab criteria used here .
further , by paying attention to the physicochemical properties and graph diversity of the ligands , we ensure a reasonable level of independence among the molecules in our data set . that this is a factor often ignored
is easily seen by the example of the kirchmair set,(17 ) which contains no less than 50 duplicate molecules .
such a level of duplication is likely to bias the results obtained from that data set . while individually all the criteria we deploy seem reasonable and even relatively benign , when combined , they present a significant hurdle for a structure to surmount .
even though we began to assemble our validation set using three large data sets , two of which had already been selected for validation of conformer generators , we found it impossible to assemble a substantial set of well - solved structures . by applying relatively loose criteria for the quality of the crystallographic models at a global level
overall , more than 90% of all the input structures failed our filtering criteria , a surprisingly high level .
figure 1 illustrates the attrition rates for each of the three databases used and shows that they are quite similar , which was unexpected .
given that the kirchmair and sadowski sets were selected with the explicit goal of testing conformer generators , we expected lower attrition rates for these two sets than for pdbbind , which is simply a collation of cocrystal structures for which there exists a published binding affinity .
however , the percentages of surviving structures are quite similar : 5.3% for the kirchmair set , 6.4% for pdbbind , and 8.1% for the sadowski set .
these very low levels of survival emphasize that the number of structures suitable for this sort of study in the pdb as a whole is very small , as has been seen in a study using pdb structures for docking validation.(37 ) however , given that recently the pdb has made deposition of structure factors along with coordinates a requirement , we hope that that this situation will improve quickly in the future .
the most commonly used metrics in conformer generation studies are based on comparing each conformer in the set to the experimental conformation , using some atom - based geometric measure such as rmsd .
metrics like rmsd are used almost without exception in conformer reproduction studies , probably because they are relatively easy to understand and require no specialized applications to calculate .
there are , however , a number of objections to the use of rmsd as a metric of quality ; it has no upper bound , it scales with molecular size ( so that an rmsd of 2 for a molecule of 6 heavy atoms is much different than an rmsd of 2 for a molecule with 60 heavy atoms ) , and it can give an inaccurate picture of the overall quality of a prediction.(38 ) the most serious of the problems with rmsd may be that it does not directly compare a prediction with an experimental value but rather compares a prediction or model ( the conformer set ) with another model ( the atomic positions in the crystal structure ) .
this problem has been eloquently discussed in a paper by yusuf et al.(39 ) in which the authors advocate comparison between the experimental data ( electron density ) with calculated density derived from a docking pose ( or computed conformation ) using the rsr metric ( which is bounded by 0 and 1 ) .
these objections notwithstanding the large amount of published literature using rmsd militates against not using it as one metric of quality in a validation study , but it should not be the only one . a complementary approach , pace yusuf et al . , is to use a bounded metric that is not derived directly from the atom positions in the two conformers being compared .
one recent approach in this vein has been the comparison of the overall shape of the experimental conformation to the shape of a docking pose using the shape tanimoto metric employed by warren et al.(40 ) we have extended their shape - based comparison to include an additional term ( the color tanimoto ) that compares the alignment of functional groups between the conformers .
the score representing this combination of shape matching ( shape tanimoto ) and functional group matching ( color tanimoto ) is known as the tanimoto combo ( tc ) .
since it represents the match of both shape and functional groups in space , tc allows for a greater discrimination between poses than using shape alone .
use of a metric like tc avoids some major problems with rmsd : while rmsd has no defined range , the range of tc is , by definition , 02 ; since tc is bounded by 0 and 2 comparisons using tc is independent of molecular size ; large rmsds can arise from differences in the conformations of only small parts of the molecule , while tc is not as sensitive to these divergences ; tc provides an extra weight for matching chemical functionality ( the color tanimoto term represents the matching of the chemical features only ) while rmsd weights the matching of all atoms equally ; the gaussian representations of molecular properties used in the calculation of tc are soft so that the significance of results are not as affected by experimental uncertainties in atomic positions ( though it is more difficult to quantitatively correct for their effect on tc ) .
while any use of a cutoff value for good reproduction is difficult , we find that if tc is below 1.0 the reproduction of the experimental pose is always bad and if tc is above 1.5 the reproduction is almost always satiusfactory .
another major issue with the common use of atom - based measures like rmsd , rde , etc .
is that no account is taken of experimental coordinate error in the structures being reproduced . to our knowledge , this is the first work in which reported rmsds are corrected for the atomic coordinate precision of the structure being reproduced .
we have chosen to allow for coordinate error or uncertainty by the use of , as a metric of quality , either the maximum of the rmsd and the uncertainty or the difference between them .
the second of these can be considered as an estimate of the level of computational noise introduced by the conformer generation process atop the existing experimental noise . while careful selection of our data set resulted in the corrected and uncorrected results not being significantly different
, the correction of rmsds by coordinate uncertainty as outlined herein allows the future use of interesting structures with poorer coordinate precision than used in this study .
there are clearly more sophisticated approaches that can be taken to this problem of experimental noise in pdb ligand structures , among which is to calculate a set of conformers that all fit the electron density equally well within some limit and compare these with the set computed by omega .
this more realistically reflects the fact that a crystal structure is an average over time and space , and so , a small molecule is likely to be found in a number of slightly different conformations in a solid - state structure .
the pdb - derived data set used here , while of good quality , is relatively small ( 197 structures ) , and so , a possible concern is that the results generated are not robust indicators of future performance .
we find that in both cases the 5% and 95% quantiles are close and that the standard deviations of the bootstrap means are small .
we infer that our results are quite stable to changes in the composition of the data sets used and , therefore , can be considered reliable indicators of future performance on molecule sets of similar physicochemical properties .
the usual practice in this area has been to compare an aggregate statistic such as mean or median results , from a number of different tools or parameter sets and to declare one superior , without any account of the errors in these terms . however ,
by the use of confidence intervals , we can quantitatively assign a probability that one tool or parameter set actually is superior ; for example based on data in this paper , it is over 90% likely that omega with default parameters is better at reproducing small molecule structures from the csd than from the pdb . the torsion library in omega
is based upon analysis of a number of crystal structures from the pdb , coupled with analysis of energy profiles for certain torsions in the mmff94 force field .
therefore , the problem of overtraining the torsion library arises if many of the structures used to derive the torsion library entries are also in the test sets used in this study .
this problem was addressed by the use of a naive torsion library containing no torsion specific information at all .
comparison of the results from this naive library with the default one showed that the main impact of the torsion library is not to improve omega s ability to closely reproduce experimental structures but rather to reduce the size of the conformer ensemble required for good reproduction and the run time required .
this result reduces any possible concern about the effect of over - training the torsion library so that it contains matches for many known structures . as above ,
the use of bootstrapping is key in interpreting the results ; it is over 90% certain that the use of the default torsion library produces no improvement in reproduction of the pdb structures compared to the use of a naive torsion library .
while the purpose of this study was not to extensively compare parameter sets and versions of omega , a comparison of previous versions of omega with the current version ( on the pdb ligand set ) is provided in the supporting information .
we have presented omega , an algorithm for rapid generation of conformers using a prebuilt library of fragments and a knowledge base of torsion angles and test sets of solid - state structures for its evaluation .
the test sets of structures were obtained from both the pdb and the csd . identifying a set of small molecules from the pdb suitable for validation proved difficult , with the vast majority of ligand structures used in some previous validation sets for conformer generators proving unsuitable for this purpose . however , by applying a set of sequential quality filters to over 4500 structures from the pdb , a set of 197 ligands was found that were accurate and well fit to their electron density .
most of the structures failed due to insufficient nominal resolution , poor fits of the ligand to its density , and inappropriate physicochemical properties .
it is hoped that the pdb data set used herein will be of general use to the community since it contains only highly reliable structures with well fit ligands , so conclusions based on this data set will be highly reliable .
a set of 492 druglike molecules from the csd was also subjected to a subset of these filters , and the surviving 480 molecules were tested against omega .
in contrast to most studies in this area , we have used two different metrics for success in reproducing these experimental structures , rmsd and the tanimoto combo ( which estimates overall similarity in three dimensions ) , as well as a metric of conformational coverage close to the experimental structure .
we , thereby , obtained complementary information on omega s ability to satisfactorily sample the conformational space of molecules around their solid - state structures .
omega s performance on both of the data sets was good when judged by any of the metrics used ( performance was particularly good against the csd structures , due to their lower flexibility ) .
the use of bootstrapping has allowed us to determine confidence intervals for our results and to make quantitative discrimination between the performance of omega on different data sets and with different amounts of information . in sum
, we found that omega was able to satisfactorily sample the conformational space around solid - state structures of druglike molecules , which is omega s design goal . | here , we present the algorithm and validation for omega , a systematic , knowledge - based conformer generator .
the algorithm consists of three phases : assembly of an initial 3d structure from a library of fragments ; exhaustive enumeration of all rotatable torsions using values drawn from a knowledge - based list of angles , thereby generating a large set of conformations ; and sampling of this set by geometric and energy criteria .
validation of conformer generators like omega has often been undertaken by comparing computed conformer sets to experimental molecular conformations from crystallography , usually from the protein databank ( pdb ) .
such an approach is fraught with difficulty due to the systematic problems with small molecule structures in the pdb .
methods are presented to identify a diverse set of small molecule structures from cocomplexes in the pdb that has maximal reliability .
a challenging set of 197 high quality , carefully selected ligand structures from well - solved models was obtained using these methods .
this set will provide a sound basis for comparison and validation of conformer generators in the future .
validation results from this set are compared to the results using structures of a set of druglike molecules extracted from the cambridge structural database ( csd ) .
omega is found to perform very well in reproducing the crystallographic conformations from both these data sets using two complementary metrics of success . | Introduction
Methods
Results
Discussion
Conclusion |
the genus penicillium comprises a large number of ubiquitous filamentous fungi , of which some are involved in human infections , ranging from mild to severe infections , especially in patients infected with human immunodeficiency virus ( hiv ) , patients with underlying diseases , and intravenous drug abusers ( 1 ) .
moreover , they lead to many complications , such as mycotoxicosis , allergies , and fungal sinusitis , and are frequently isolated from soil , dead plant materials , rotten wood , decaying vegetables , and foods ( 2 ) . based on phenotypic data , the genus of penicillium is regarded to be related to ascomycota , because of its sexual reproduction by means of ascospores ( 3 , 4 ) .
members of the genus are of commercial and industrial importance due to their use in production of antibiotics , anti - tumoral , anti - fungal , anti - insect , and anti - viral compounds , as well as extracellular enzymes ( 2 ) . to date , it has been revealed that various sections of penicillium encompass more than 250 species ( 5 ) .
the most common species of penicillium , p. chrysogenum , is found to be an agent of onychomycosis , keratomycosis , allergic bronchopulmonary mycosis , and asthma , which may be associated with indoor environments , deserts , dried foods , cheese , and is morphologically characterized by biverticillate , terverticillate , or quarter - verticillate conidiophores , floccose to velutinous in colony texture , and yellow exudate droplets ( 2 , 6 - 8 ) .
however , these conventional methods were noted to be tedious , time consuming and unreliable for species delineation ( 2 , 9 , 10 ) . nevertheless , molecular - based methods , such as dna sequencing , have provided powerful tools for precise identification of the penicillium species .
currently , species identification in this genus is based on polyphasic criteria consisting of morphological and biochemical traits and molecular data from internally transcribed spacer ( its ) regions of ribosomal dna ( rdna ) and the partial -tubulin ( bt2 ) sequences ( 2 , 9 , 11 ) . while the its - rdna regions have some limitations , such as less resolution in differentiation of closely related species , due to a better species - specific resolution provided by the partial bt2 sequencing , application of this locus
has substantially increased and is now an excellent marker for differentiation of penicillium spp ( 2 , 9 , 12 ) .
given that there was no comprehensive evaluation about the biodiversity of this genus in iran , this study was focused on determining the distribution profile of penicillium spp . in two provinces of iran ( mazandaran and tehran ) .
in total , 400 isolates used in this study were obtained from soil , air , cereals , and decaying vegetables from different regions of mazandaran and tehran provinces ( table 1 ) .
stock cultures were maintained on slants of 2% malt extract agar ( difco , detroit , mi , usa ) and incubated at 25c for one week .
strains were cultured on 2% malt extract agar ( difco , detroit , mi , usa ) , czapek yeast extract agar ( difco , detroit , mi , usa ) , and yeast extract agar ( himedia , india ) and incubated at 24c in the dark for one week .
microscopic observations were based on slide culture techniques using potato dextrose agar ( pda ) ( 5 ) .
mounts of four - day - old slide cultures were made in lactic acid or lactophenol cotton blue , and light micrographs were taken using a nikon eclipse 80i microscope ( nikon , tokyo , japan ) equipped with a nikon digital sight ds - fi1 camera .
moreover , strains were evaluated based on phenotypic characters , i.e. color , texture , growth rate , pigmentation , conidiophores , conidia , metulae , and phialide morphology ( 2 ) .
subsequently , a sterile blade was used to scrape off the mycelium from the surface of the plate .
genomic dna was extracted using an ultra clean microbial dna isolation kit ( mobio inc .
the bt2 gene was partially amplified and sequenced using the universal fungal primers as follows : bt2a ( 5-ggtaaccaaatcggtgctgctttc-3 ) and bt2b ( 5accctcagtgtagtgacccttggc-3 ) ( 13 , 14 ) .
pcr reactions were performed on a corbett research thermal cycler , model cg1 - 96 ( sydney , australia ) in 50 l volume containing 25 ng of template dna , 5 l reaction buffer ( 0.1 m tris - hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2 mm of each dntp , and 2.0 units taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .
amplification of bt2 was performed with one cycle of five minutes at 95c for primary denaturation , followed by 35 cycles of 95c for 45 seconds , 60c for 120 seconds , and 72c for 60 seconds , with a final extension of 72c for seven minutes .
amplicons were purified using gfx pcr dna and a gel band purification kit ( ge healthcare , buckinghamshire , uk ) .
pcr - sequencing was performed as follows : 95c for one minute , followed by 30 cycles consisting of 95c for 10 seconds , 50c for five seconds and 60c for two minutes .
reactions were purified with sephadex g-50 fine columns ( ge healthcare bio - sciences ab , uppsala , sweden ) , and pcr products were sent to bioneer company ( bioneer , daejeon , south korea ) for bidirectional sequencing with the primers mentioned previously .
sequence data obtained in this study were imported to mega software version 5 ( http://www.megasoftware.net ) and adjusted using clustalw .
ambiguous regions were excluded from the alignment , and then each trimmed sequence was exported to a blast search of the ncbi databases for species recognition .
they were compared with penicillium sequences , and query sequences with high similarity ( 99% ) were considered as the same species .
the program , raxml - vi - hpc v. 7.0.0 , as implemented on the cipres portal v. 1.10 , was used for the tree search and the bootstrap analysis ( gtrmix model of molecular evolution and 500 bootstrap replicates ) .
phylogenetic analyses were carried out based on the bt2 sequence in order to assess the phylogenetic placement of diverse penicillium species , along with their relative sequences downloaded from genbank , based on the barreto et al . design ( 9 ) .
in total , 400 isolates used in this study were obtained from soil , air , cereals , and decaying vegetables from different regions of mazandaran and tehran provinces ( table 1 ) .
stock cultures were maintained on slants of 2% malt extract agar ( difco , detroit , mi , usa ) and incubated at 25c for one week .
strains were cultured on 2% malt extract agar ( difco , detroit , mi , usa ) , czapek yeast extract agar ( difco , detroit , mi , usa ) , and yeast extract agar ( himedia , india ) and incubated at 24c in the dark for one week . identification was performed primarily based on macroscopic and microscopic morphology .
microscopic observations were based on slide culture techniques using potato dextrose agar ( pda ) ( 5 ) .
mounts of four - day - old slide cultures were made in lactic acid or lactophenol cotton blue , and light micrographs were taken using a nikon eclipse 80i microscope ( nikon , tokyo , japan ) equipped with a nikon digital sight ds - fi1 camera .
moreover , strains were evaluated based on phenotypic characters , i.e. color , texture , growth rate , pigmentation , conidiophores , conidia , metulae , and phialide morphology ( 2 ) .
subsequently , a sterile blade was used to scrape off the mycelium from the surface of the plate .
genomic dna was extracted using an ultra clean microbial dna isolation kit ( mobio inc .
dna extracts were stored at 20c prior to use . the bt2 gene was partially amplified and sequenced using the universal fungal primers as follows : bt2a ( 5-ggtaaccaaatcggtgctgctttc-3 ) and bt2b ( 5accctcagtgtagtgacccttggc-3 ) ( 13 , 14 ) .
pcr reactions were performed on a corbett research thermal cycler , model cg1 - 96 ( sydney , australia ) in 50 l volume containing 25 ng of template dna , 5 l reaction buffer ( 0.1 m tris - hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2 mm of each dntp , and 2.0 units taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .
amplification of bt2 was performed with one cycle of five minutes at 95c for primary denaturation , followed by 35 cycles of 95c for 45 seconds , 60c for 120 seconds , and 72c for 60 seconds , with a final extension of 72c for seven minutes .
amplicons were purified using gfx pcr dna and a gel band purification kit ( ge healthcare , buckinghamshire , uk ) .
pcr - sequencing was performed as follows : 95c for one minute , followed by 30 cycles consisting of 95c for 10 seconds , 50c for five seconds and 60c for two minutes .
reactions were purified with sephadex g-50 fine columns ( ge healthcare bio - sciences ab , uppsala , sweden ) , and pcr products were sent to bioneer company ( bioneer , daejeon , south korea ) for bidirectional sequencing with the primers mentioned previously .
sequence data obtained in this study were imported to mega software version 5 ( http://www.megasoftware.net ) and adjusted using clustalw .
ambiguous regions were excluded from the alignment , and then each trimmed sequence was exported to a blast search of the ncbi databases for species recognition .
they were compared with penicillium sequences , and query sequences with high similarity ( 99% ) were considered as the same species .
the program , raxml - vi - hpc v. 7.0.0 , as implemented on the cipres portal v. 1.10 , was used for the tree search and the bootstrap analysis ( gtrmix model of molecular evolution and 500 bootstrap replicates ) .
phylogenetic analyses were carried out based on the bt2 sequence in order to assess the phylogenetic placement of diverse penicillium species , along with their relative sequences downloaded from genbank , based on the barreto et al . design ( 9 ) .
all 400 penicillium isolates were screened for phenotypic characters , and 20 strains representative of 10 morphological species , including p. chrysogenum , p. polonicum , p. canescens , p. griseofulvum , p. italicum , p. raistrickii , p. expansum , p. melanoconidium , p.
palitans , and p. glabrum , were identified and selected for molecular analysis . in figure 1a - j , the colony characteristics of 20 representative isolates on cya and yes after seven days at 25c were exemplified .
some species , such as p. chrysogenum and p. italicum , due to production of typical yellow and red exudates on cya and yes , respectively , could easily be identified , while for others it was essential to apply three complementary specific media ( mea , yes , and cya ) for exact identification ( figure 1a - g ) . except for p. chrysogenum and p. expansum with floccose colony texture , others were velutinous ( table 1 and figure 1 ) . upon microscopic examination ,
nearly all identified species were one- or two - staged branched ( biverticillate , terverticillate ) , except for p. glabrum , which typically produces no branching in conidiophore ( monoverticillate ) ( table 2 ) .
definitions : monoverticillate , conidiophore without branching ; biverticillate , conidiophore one - stage branching ; terverticillate , conidiophore two - stage branch .
the species isolated in this study had globose , smooth - wall conidia , cylindrical phialide , and metula in various sizes .
interestingly , the only observed finely roughened conidium was related to p. canescens , which had been isolated from soil .
partial amplification of bt2 was successful in all isolates and yielded a single pcr band of approximately 450 base pair ( bp ) in gel electrophoresis ( figure 2 ) .
line 1 , p. chrysogenum ; line 2 , p. canescens ; line 3 , p. griseofulvum ; line 4 , p. polonicum ; line 5 , p. glabrum ; line 6 , p. expansum ; line 7 , p. italicum ; line 8 , p.raistrickii ; n , negative control ; m , dna ladder .
final identification of isolates was performed by comparing the obtained sequences with those of reference strains held in the open access validated cbs database for penicillium ( http://www.cbs.knaw.nl/penicillium/ ) .
sequences of all isolates were deposited in genbank and the accession numbers of the sequences are given in table 1 . in table 1 the results of sequence and phenotypic - based identification were compared .
figure 1 shows that phylogenetic reconstruction was successfully carried out for the bt2 region and represented the maximum parsimony phylogenetic tree reconstructed o then -tubulin gene .
the phylogenetic assessment showed that there were three major sections , consisting of viridicata , chrysogena , penicillium , and one series , glabra including p. glabrum .
the analysis showed that the environmental species including p. melanoconidium ( represented by two strains ) , p. polonicum ( represented by three strains ) , p. palitans ( represented by two strains ) , and the related standard strains ( represented by three strains ) are monophyletic and well supported ( 96.4% bootstrap , figure 3 ) .
the phylogenetic tree ( figure 3 ) shows that the penicillium section is composed of five species , p. canescens , p. italicum , p. expansum , p. griseofulvum and p. raistrickii , which are phylogenetically distinct from other species and sister to each other and standard strains in this section .
the species of the penicillium section were strongly ( 93.1% ) well supported by bootstrap . among accepted members of the chrysogena section and the glabra series , only p. chrysogenum and p. glabrum ,
the phylogenetic tree ( figure 3 ) shows that six p. chrysogenum and one standard strain ( ubocc - a 101393 ) formed a well - supported monophyletic group nested within this section of penicillium .
all 400 penicillium isolates were screened for phenotypic characters , and 20 strains representative of 10 morphological species , including p. chrysogenum , p. polonicum , p. canescens , p. griseofulvum , p. italicum , p. raistrickii , p. expansum , p. melanoconidium , p.
palitans , and p. glabrum , were identified and selected for molecular analysis . in figure 1a - j , the colony characteristics of 20 representative isolates on cya and yes after seven days at 25c were exemplified .
some species , such as p. chrysogenum and p. italicum , due to production of typical yellow and red exudates on cya and yes , respectively , could easily be identified , while for others it was essential to apply three complementary specific media ( mea , yes , and cya ) for exact identification ( figure 1a - g ) . except for p. chrysogenum and p. expansum with floccose colony texture , others were velutinous ( table 1 and figure 1 ) . upon microscopic examination ,
nearly all identified species were one- or two - staged branched ( biverticillate , terverticillate ) , except for p. glabrum , which typically produces no branching in conidiophore ( monoverticillate ) ( table 2 ) .
definitions : monoverticillate , conidiophore without branching ; biverticillate , conidiophore one - stage branching ; terverticillate , conidiophore two - stage branch .
the species isolated in this study had globose , smooth - wall conidia , cylindrical phialide , and metula in various sizes .
interestingly , the only observed finely roughened conidium was related to p. canescens , which had been isolated from soil .
partial amplification of bt2 was successful in all isolates and yielded a single pcr band of approximately 450 base pair ( bp ) in gel electrophoresis ( figure 2 ) .
line 1 , p. chrysogenum ; line 2 , p. canescens ; line 3 , p. griseofulvum ; line 4 , p. polonicum ; line 5 , p. glabrum ; line 6 , p. expansum ; line 7 , p. italicum ; line 8 , p.raistrickii ; n , negative control ; m , dna ladder .
final identification of isolates was performed by comparing the obtained sequences with those of reference strains held in the open access validated cbs database for penicillium ( http://www.cbs.knaw.nl/penicillium/ ) .
sequences of all isolates were deposited in genbank and the accession numbers of the sequences are given in table 1 . in table 1 the results of sequence and phenotypic - based identification were compared .
figure 1 shows that phylogenetic reconstruction was successfully carried out for the bt2 region and represented the maximum parsimony phylogenetic tree reconstructed o then -tubulin gene .
the phylogenetic assessment showed that there were three major sections , consisting of viridicata , chrysogena , penicillium , and one series , glabra including p. glabrum .
the analysis showed that the environmental species including p. melanoconidium ( represented by two strains ) , p. polonicum ( represented by three strains ) , p. palitans ( represented by two strains ) , and the related standard strains ( represented by three strains ) are monophyletic and well supported ( 96.4% bootstrap , figure 3 ) .
the phylogenetic tree ( figure 3 ) shows that the penicillium section is composed of five species , p. canescens , p. italicum , p. expansum , p. griseofulvum and p. raistrickii , which are phylogenetically distinct from other species and sister to each other and standard strains in this section .
the species of the penicillium section were strongly ( 93.1% ) well supported by bootstrap .
among accepted members of the chrysogena section and the glabra series , only p. chrysogenum and p. glabrum , respectively , were isolated from all the examined isolates .
the phylogenetic tree ( figure 3 ) shows that six p. chrysogenum and one standard strain ( ubocc - a 101393 ) formed a well - supported monophyletic group nested within this section of penicillium .
to evaluate biodiversity and phylogeny of the penicillium species in mazandaran and tehran provinces , the current study focused on morphology - based identification and sequencing of the -tubulin gene .
there has been no extensive attempt at this type of study , including reliable morphological and high - resolution molecular approaches for penicillium identification , until now in iran ( 15 , 16 ) . over the past decades , focusing on macro- and microscopic characteristics of the colonies has been the main strategy for classification and identification of the penicillium species ( 2 , 17 ) . in our study , which was morphologically based on three specific medium ( cya , mea , and yes ) ( 2 ) , 10 species of penicillium were identified , compared to javadi et al . who did not focus on phenotypic characteristics and whose sampling was restricted , taken only from soil ( 16 ) .
also , the study performed by sabokbar et al . showed intra - species variation of penicillium species isolated from air in iran using rapd - pcr , but did not consider identification of the penicillium species ( 15 ) .
in the present study , in descending arrangement of frequency , the isolates that were identified were : p. chrysogenum , p. polonicum , p. palitans , p. glabrum , p. melanoconidium , p. expansum , p. raistrickii , p. italicum , p. griseofulvum , and p. canescens .
contrary to its regions , the -tubulin gene is known to have more variation in penicillia and currently is considered a reliable alternative marker for differentiation of the penicillium species ( 5 ) . since identification of all penicillium species by morphology was stated to be very problematic and often impossible , relying on sequencing of the high resolution genetic marker , tubulin , allowed for accurate and rapid recognition of the isolates in our study ( 2 , 14 ) .
parsimony analysis was used in our study to determine the phylogeny of the isolates ( 9 ) .
the viridicata section is substantially divided into five series including viridicata , camemberti , corymbifera , verrucosa , and solita ( 9 ) . in the current survey , only species related to two former series , such as p. melanoconidium , p. polonicum , and p. palitans , were isolated , of which p. melanoconidium and p. polonicum are regarded to be prone to cereals such as barley , wheat , rye , oats , rice , and cause mycotoxicosis symptoms in humans ( 2 ) .
also , p. palitans may produce some significant toxins such as cyclopiazonic acid and fumigaclavine on foods , and the former is proven to be an agent of organ damage in mammals ( 2 ) . in the penicillium section , p. griseofulvum , as the main producer of griseofulvin , which is placed in ser .
urticicolae alongside p. raistrickii and p. canescens , had completely identical sequences with the standard strain ( 100% ) .
penicillium expansum and p. italicum , two members of the expansa and italica series , respectively , had 99.9% identity to the standard sequences ( psn118and ab688988.1 ) .
penicillium chrysogenum , the predominant isolate in our study , is assigned to section chrysogena , ser .
whereas p. chrysogenum , p. spinulosum , and p. oxalicum ranked as the first infrequency in kansas , p. citrinum was the main agent isolate from the taipei area ( 18 ) .
penicillium chrysogenum was the predominant agent among all assessed isolates in this study , and this finding was concordant with other reports from around the world ( 19 ) .
extra evaluation , with emphasis on polyphasic procedures , not only phenotypic methods but also molecular approaches focusing on a combination of partial -tubulin , calmodulin , and its sequence , are required to clarify the complete spectrum of the penicillium species in iran .
the current investigation found a high level of genetic variability in the -tubulin gene in various penicillium species isolated from soil , fruits , food , and grains of mazandaran and tehran provinces .
this study reconfirmed the view that bt2 pcr - sequencing is a reliable and applicable diagnostic tool for differentiating and the molecular taxonomy of closely related penicillium species .
the current investigation found a high level of genetic variability in the -tubulin gene in various penicillium species isolated from soil , fruits , food , and grains of mazandaran and tehran provinces .
this study reconfirmed the view that bt2 pcr - sequencing is a reliable and applicable diagnostic tool for differentiating and the molecular taxonomy of closely related penicillium species . | background : the genus penicillium contains a large number of ubiquitous environmental taxa , of which some species are clinically important .
identification of penicillium down to the species level is currently based on polyphasic criteria , including phenotypic features and genetic markers .
biodiversity of the genus penicillium from mazandaran and tehran provinces has not been described.objectives:the current paper focused on the environmental biodiversity of penicillium isolates within some areas of mazandaran and tehran provinces , based on morphological traits and the molecular data from partial sequence of the -tubulin ( bt2 ) gene.materials and methods : a total of 400 strains were isolated from the environment and investigated using morphological tests and sequencing of bt2 , in order to characterize the spectrum of the penicillium species.results:sequence analysis of bt2 and morphological criteria of 20 strains representative of 10 species showed that penicillium chrysogenum was the most prevalent species ( n = 6 ) , followed by p. polonicum ( n = 3 ) , p. glabrum ( n = 2 ) , p. palitans ( n = 2 ) , p. melanoconidium ( n = 2 ) , and other species , including p. expansum , p. canescense , p. griseofulvum , p. italicum , and p. raistrickii with one case each.conclusions:it was shown that partial -tubulin sequence , as a reliable genetic target , supported specific morphological criteria for identification of the penicillium species . like other assessments throughout the world , p. chrysogenum remains the most frequent environmental penicillium species in mazandaran and tehran provinces . | 1. Background
2. Objectives
3. Materials and Methods
3.1. Fungal Strains
3.2. Morphological Identification and Cultural Characterization
3.3. Molecular Characterization
3.4. Alignment and Phylogenetic Reconstruction
4. Results
4.1. Morphological Identification and Cultural Characterization
4.2. Results of BT2 Amplification, Sequencing, and Phylogeny
4.3. Viridicata Section Dataset
4.4.
4.5. Chrysogena Section and Glabra Series Dataset
5. Discussion
5.1. Conclusions |
root - end filling materials are in contact with periradicular tissues , so they should have good sealing ability and be biocompatible to promote healing . because many of these materials are not able to ensure a perfect seal , a microscopic space is likely to create at the root - end cavity / filling interface , through which micro organisms and their products can penetrate . thus , besides good sealing ability and biocompatibility , root - end filling materials should ideally have some antibacterial activity .
more over , these materials should have low solubility to avoid that components leaching from the endodontic space might exercise undesirable biologic effects on the surrounding tissues . finally , radiopacity is a very important property for all root - end filling materials because the material has to be detected radiographically , and thus distinguished from surrounding anatomic structures .
intermediate restorative material ( irm ) has addressed some drawbacks including moisture sensitivity , irritation to vital tissues and difficulty in handling .
mta was developed by parirokh and torabinejad to address shortcomings of commonly used root - end filling materials .
several studies have recognized mta as a bioactive material , that is , hard tissue conductive , hard tissue inductive , and biocompatible .
the sealing ability of mta in root - end fillings was found to be superior to that of amalgam , irm , and super - ethoxy benzoic acid ( eba ) with both dye and bacterial leakage methods . however , its handling characteristics are less than ideal as a result of long setting time and difficulty in maintaining consistency of mixture .
several new calcium silicate - based materials have recently been developed ; aiming to improve some mta drawbacks such as its difficult handling property and long setting time .
biodentine is amongst these materials and is claimed to be used as a dentine restorative material in addition to endodontic indications similar to those of mta .
the purpose of the present study is to evaluate and compare the biological ( cytotoxicity and antibacterial efficacy ) and chemical - physical ( solubility , ph , and radiopacity ) properties of four different root - end filling materials : biodentine ( septodont , saint - maur - des - fosses , france ) , mta - angelus ( angelus , londrina , pr , brazil ) , proroot mta ( dentsply , johnson city , tn , usa ) , and irm ( dentsply , johnson city , tn , usa ) .
transwell insert methodology ( sigma - aldrich , st louis , mo , usa ) was performed .
cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st .
the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively .
some wells were incubated with only tissue culture media ( negative control ) whereas others with a 10% dilution of 30% h2o2 ( positive control ) . at the end of incubation time
the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the vitality test to alamar blue reagent acts as an indicator of cell health , determining the reducing power in order to measure quantitatively the proliferative capacity .
the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c .
the degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . for a further control ,
the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) .
the mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan ( a salt blue ) in the mitochondria , giving the substance a blue / purple color .
this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) .
the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) .
the cultures were grown and maintained in a brain heart infusion ( bhi ; difco lab . , detroit , mi , usa ) . s. mutans culture medium was supplemented with 10% ( v / v ) heat - inactivated horse blood serum ( oxoid s.p.a . ,
the culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions .
this overnight culture , used as source for the experiments , was reduced at a final density of
1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number .
sterile paper discs ( diameter : 6 mm , thickness : 1 mm ) ( watman international ltd .
maidstone , uk ) were impregnated with 10 l of each root - endfilling material .
then , bhi - agar plates were incubated with 1 10 cells / ml of an overnight culture of each streptococcal strain at 37c for 20 min .
the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) .
all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone .
stainless steel ring molds with an internal diameter of 20 0.1 mm and a height of 1.5 0.1 mm were used for sample preparation .
all molds were weighed three times prior to use ( accuracy 0.0001 g ) on a mettler ae-163 balance ( mettler - toledo s.p.a .
, novate milanese , mi , italy ) , which was used throughout the experiment . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess .
all samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity .
the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places .
the specimens of each material were individually placed in tarred bottles , containing 5 ml of distilled water .
the bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water , which was then collected in the same bottle .
bottles and residues were dried in an oven at 105c , cooled down in desiccators , and weighed .
the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 .
the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) .
the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated .
biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter .
the specimens were covered with glass plates on each side to allow for the removal of excessive material .
the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) .
all materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations .
for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses .
the precision assessments were performed in routine clinical manner according to international society for clinical densitometry ( iscd ) recommendations .
college station , tx : statacorp lp . ) . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities
; the shapiro wilk test was applied ( p < 0.05 ) . the number of vital cells was analyzed with tukey 's test .
whitney test ; the inhibition zones values after 48 h were used for the statistical analysis .
whitney test . to determine whether time influenced the solubility of the pulp capping materials ,
an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) .
t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) .
tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) .
radiographic densities obtained from dual - energy x - ray absorptiometry ( dxa ) measurements were analyzed using tukey 's test .
transwell insert methodology ( sigma - aldrich , st louis , mo , usa ) was performed .
cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st .
the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively .
some wells were incubated with only tissue culture media ( negative control ) whereas others with a 10% dilution of 30% h2o2 ( positive control ) . at the end of incubation time
the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the vitality test to alamar blue reagent acts as an indicator of cell health , determining the reducing power in order to measure quantitatively the proliferative capacity .
the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c .
the degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . for a further control ,
the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) .
the mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan ( a salt blue ) in the mitochondria , giving the substance a blue / purple color .
this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) .
the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) .
the cultures were grown and maintained in a brain heart infusion ( bhi ; difco lab . , detroit , mi , usa ) . s. mutans culture medium was supplemented with 10% ( v / v ) heat - inactivated horse blood serum ( oxoid s.p.a . , rodano , mi , italy ) to improve its growth .
the culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions .
this overnight culture , used as source for the experiments , was reduced at a final density of
1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number .
sterile paper discs ( diameter : 6 mm , thickness : 1 mm ) ( watman international ltd .
maidstone , uk ) were impregnated with 10 l of each root - endfilling material .
then , bhi - agar plates were incubated with 1 10 cells / ml of an overnight culture of each streptococcal strain at 37c for 20 min .
the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) .
all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone .
stainless steel ring molds with an internal diameter of 20 0.1 mm and a height of 1.5 0.1 mm were used for sample preparation .
all molds were weighed three times prior to use ( accuracy 0.0001 g ) on a mettler ae-163 balance ( mettler - toledo s.p.a . , novate milanese , mi , italy ) , which was used throughout the experiment . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess .
all samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity .
the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places .
the specimens of each material were individually placed in tarred bottles , containing 5 ml of distilled water .
the bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water , which was then collected in the same bottle .
bottles and residues were dried in an oven at 105c , cooled down in desiccators , and weighed .
the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 .
the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) .
the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated .
biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter .
the specimens were covered with glass plates on each side to allow for the removal of excessive material .
the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) .
all materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . for the prodigy ,
encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses .
the precision assessments were performed in routine clinical manner according to international society for clinical densitometry ( iscd ) recommendations .
stata statistical software : release 12 . college station , tx : statacorp lp . ) . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro
whitney test ; the inhibition zones values after 48 h were used for the statistical analysis .
whitney test . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) .
t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) .
tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) .
radiographic densities obtained from dual - energy x - ray absorptiometry ( dxa ) measurements were analyzed using tukey 's test .
descriptive statistics analysis for biocompatibility investigation is reported in table 1 . as regards alamar blue test [ table 1 ] , after 24 h
there were no differences in the number of vital cells among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) .
irm showed a significant reduction in the number of vital cells ( p < 0.05 ) .
these results changed uniformly after 48 h and maintained no significant differences among the materials , except for irm .
after 72 h all three materials varied from the negative control , in particular proroot mta and mta - angelus gained a similar and lower number of vital cells than the control , while biodentine showed no decrease , as confirmed by the analysis for paired data .
when the mtt test [ table 1 ] was applied , no significant differences were recorded among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) , while irm maintained lower percentages of vitality ( p < 0.05 ) .
mean standard deviation of the number of vital cells for each material tested with alamar blue at different times table 2 shows the mean diameter of the inhibition zones after 48 h and the standard deviation .
except for biodentine when tested for s. mutans , the root - end filling materials caused zones of inhibition . however ; mta - angelus , proroot mta , and irm showed the highest values for the inhibition zones when tested for s. mutans ( p < 0.05 ) .
no significant differences between mta - angelus , proroot mta , and irm ( p > 0.05 ) were found when materials were tested for s. salivarius .
biodentine showed the largest inhibition zone when tested for s. sanguis ( p < 0.05 ) , while mta - angelus and proroot mta produced similar smaller inhibition zones ( p > 0.05 ) .
mean diameter and standard deviation of inhibition zones ( mm ) formed by the root - endfilling materials after 48 h by the paper disc method the results of solubility test ( after 24 h and 2 months ) are listed in table 3 .
all the materials fulfilled the requirements of the international standard 6876 , demonstrating a weight loss of less than 3% .
there was no statistical significance in solubility among the materials tested after 24 h ( p < 0.05 ) .
similar results were obtained after 2 months , except for irm for which solubility significantly raised ( p < 0.05 ) . for remnant materials the weight loss after 2 months was not significantly different from the weight loss after 24 h :
all the materials tested provided a very alkaline ph after 3 h [ table 3 ] ; proroot mta showed the highest value among the materials tested .
biodentine and irm provided similar ph values after 3 h ( p > 0.05 ) .
for all materials tested , a nonsignificant reduction in ph value was recorded after 24 h ( p > 0.05 ) . for each root - end
filling material descriptive statistics for radiographic densities and the results obtained with the analyses of multiple comparisons were calculated and reported in table 4 .
proroot mta and mta - angelus showed the highest values of density when compared with the other materials ( p < 0.05 ) .
mean percentage values of solubility and standard deviation ( sd ) for each material and for each immersion period radiographic densities : descriptive statistics for each material tested and multiple comparisons calculated with tukey test ( p = 0.05 )
when comparing the cytotoxicity of the root - end materials tested in this study , irm demonstrated the lower rates of vitality and a strong cytotoxic capability .
irm has shown the lowest mean number of cells in the colorimetric assay performed with alamar blue , with assessments at 24 , 48 , and 72 h , and in the mtt assay at 72 h. these results are in agreement with previous studies .
it has been demonstrated that cell response to irm was characterized by marked rounding of the cells and depletion of cell numbers , indicating that irm was not biocompatible .
very different results were obtained by mta - based materials . at 72 h , with alamar blue test and mtt assay ,
parirokh and torabinejad found that proroot mta was less toxic than amalgam , super eba , and irm .
biodentine , in measurements made at 24 , 48 , and 72 h ; reported percentage of vitality above the negative control .
biodentine is new bioactive cement based on calcium silicate , whose high biocompatibility has been shown in recent studies .
the agar diffusion test has been widely used to evaluate the antibacterial activity of dental materials . in our study , except for biodentine against s. mutans , all the materials demonstrate antibacterial activity , causing zones of inhibition against the three streptococcal strains tested small differences in the antibacterial effect on the different streptococcal strains were recorded .
, in which proroot mta and irm proved to be strong inhibitors . while we have little data about the antibacterial capability of biodentine
it has been shown that in aerobic conditions , mta could generate reactive oxygen species with antimicrobial activity .
parirokh and torabinejad found that mta had no antibacterial effect against any of the strictly anaerobic bacteria .
however , as showed by our results , it is possible that mta 's highly alkaline ph affords its antimicrobial activity even when in anaerobic condition .
the test was repeated after 2 months , because schafer and zandbiglari stated that the 24-h period of the specification test is not sufficient . in our study
all the materials tested showed minimal solubility , fulfilling the requirements of the iso 6876 and demonstrating a weight loss of less than 3% .
these conclusions are in accordance with our previous study about solubility of proroot mta , irm , and super - eba .
a very alkaline ph was recorded after 3 h ; with the highest value registered for proroot mta
. no significant reduction in ph value was recorded after 24 h. proroot mta showed ph values greater than biodentine .
this may be probably caused by the higher presence of metallic oxides in mta that could promote ion dissociation .
finally , as concerned for radiopacity , in our study mta - based materials demonstrated higher values of density compared to irm and biodentine .
radiopacity of biodentine has been recently evaluated . comparing the density of biodentine and irm , grech et al .
, found higher values for irm ; while the relatively lower radiopacity of biodentine , compared to mta , has been recently shown in a study by tanalp et al .
within the limitation of this in vitro study , the differences shown by the materials do not cover completely the ideal clinical requests for root - end filling .
irm could not be considered as the ideal material due to the high cytotoxicity ; while mta - based cement and biodentine proved to be biocompatible .
however , biodentine , comparing to mta , did not cause zone of inhibition for the entire microorganism tested and it showed lower values of radiopacity .
therefore , mta - angelus and proroot mta can be considered as the best material for root - end filling among those tested due to the fact that they presented at once biocompatibility , antibacterial properties , radiopacity , and low solubility . | aim : the purpose of the study is to evaluate and compare the biological and chemical - physical properties of four different root - end filling materials.materials and methods : cytotoxicity towards murine odontoblasts cells ( mdpc-23 ) was evaluated using the transwell insert methodology by alamar blue test .
streptococcus salivarius , s. sanguis , and s. mutans strains were selected to evaluate the antimicrobial activity by agar disc diffusion test .
solubility was determined after 24 h and 2 months .
ph values were measured after 3 and 24 h. to evaluate radiopacity , all materials were scanned on a ge healthcare lunar prodigy.results:excellent percentage of vitality were obtained by mineral trioxide aggregate ( mta)-based materials and biodentine .
mta - angelus , proroot mta , and intermediate restorative material ( irm ) showed the highest values for the inhibition zones when tested for s. mutans , while biodentine showed the largest inhibition zone when tested for s. sanguis .
all the materials fulfilled the requirements of the international standard 6876 , demonstrating low solubility with a weight loss of less than 3% .
no significant reduction in ph value was demonstrated after 24 h. proroot mta and mta - angelus showed the highest values of radiographic density.conclusions:the differences showed by the root - end filling materials tested do not cover completely the ideal clinical requests . | INTRODUCTION
MATERIALS AND METHODS
Cytotoxicity assay
Antibacterial test
Solubility evaluations
pH measurements
Radiopacity
DXA Measurements
Statistical analysis
RESULTS
DISCUSSION
CONCLUSIONS |
copd is a progressive disease characterized by persistent airflow limitation , chronic and progressive dyspnea , cough , and sputum production and is often complicated by exacerbations.1 copd affects approximately 24 million us adults , including 12.7 million diagnosed patients and 12 million undiagnosed , and is the third leading cause of death in the united states.2,3 the annual cost of copd in 2010 was estimated at $ 49.9 billion usd , with $ 29.5 billion attributed to direct health care costs , in which hospital care accounted for the largest share.4 treatment goals for patients with stable copd include symptom relief , prevention of disease progression , and prevention and treatment of exacerbations.1 while symptom relief is the primary target for pharmacologic therapy , prevention of exacerbations is also important because of the impact on lung function , health - related quality of life , hospitalization , and mortality .
recent copd treatment guidelines emphasize the central role of bronchodilators , including 2-agonists and anticholinergics , with drug selection depending on patient response and tolerability .
inhaled corticosteroids ( ics ) have a more limited role for patients unable to achieve symptom control with bronchodilators alone.1 roflumilast is a selective phosphodiesterase-4 inhibitor approved by the us food and drug administration in 2011 .
it is indicated as a treatment to reduce the risk of copd exacerbations in patients with severe copd associated with chronic bronchitis and a history of exacerbations.5 no studies have compared roflumilast to other copd maintenance medications , and there is very little real - world data on utilization , clinical , and economic outcomes associated with the use of roflumilast .
we hypothesized that there were no differences in the relevant clinical and economic outcomes between roflumilast and other copd maintenance medications .
this was a longitudinal , retrospective cohort study using us managed - care administrative data from the lifelink pharmetrics plus health plan claims database , which includes medical and pharmaceutical claims for over 150 million unique patients from over 90 health plans across the united states .
the prescription claims include national drug code , drug names , dose , strength , days supply , and quantity dispensed .
it also includes demographic variables , insurance information , and plan enrollment / eligibility data .
we cleaned the obtained raw data by removing the duplicates and observations with missing values for key data elements , such as age , diagnosis date , and prescription date .
the data in the claims databases are de - identified and fully compliant with the health insurance portability and accountability act privacy regulations , exempting this study from an institutional review board approval requirement .
selection of patients for the study is outlined in figure 1 . to be included in the analysis ,
patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . according to gold ( global initiative for chronic obstructive lung disease ) guidelines,1 roflumilast
is recommended as the third maintenance agent for patients with severe copd . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent
patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline .
study patients were separated into two cohorts : patients who initiated roflumilast during the cohort selection period ( roflumilast group ) and patients who initiated the third of three copd maintenance medications during the cohort selection period ( non - roflumilast group ) .
patient demographics included age at the index date , sex , insurance type , and census region .
clinical characteristics included comorbid conditions , reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period ( termed as baseline period ) ; number of copd exacerbations ( defined in the following paragraph ) ; hospitalizations ; and emergency department ( ed ) visits during the baseline period .
the primary outcome was the rate of moderate and severe copd exacerbations per patient per month ( pppm ) over the 3 months after the index date .
severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation ( icd-9-cm codes 491.21 , 491.22 , 493.22 , or 492.8 ) or a primary diagnosis of respiratory failure ( icd-9-cm codes 518.81 , 518.82 , or 518.84 ) combined with a secondary diagnosis of copd with acute exacerbation or emphysema ( 491.0 , 491.1 , 491.21 , 491.22 , 491.8 , 491.9 , 492.0 , 492.8 , 493.22 , or 496).7 moderate exacerbation was defined as a short course ( 14 days ) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis ( icd-9-cm codes 491.xx , 492.xx , and 496.xx ) ; an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema , as defined earlier ; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition ( icd-9-cm codes 136.3 , 466466.19 , 480486 , 487.0 , 490 , 491.21 , 491.22 , 493.02 , 493.12 , 493.22 , 493.92 , 494.1 , 506.0506.3 ,
507507.8 , 511.0511.1 , 512512.8 , 517.1 , 518.0 , 518.81 , 518.82 , 518.84 , 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation .
secondary outcomes included health care resource utilization ( hcru ) , including the number of inpatient hospitalization admissions , ed and office visits , and total health care costs .
costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9 bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables .
wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier .
the did model was used to control for observed baseline differences between treatment and control groups ( eg , severity of disease ) , as well as the effects of all time - varying determinants of the outcomes ( trend change in the outcomes over time ) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time .
analyses were performed using statistical analysis software version 9.2.3 ( sas institute , cary , nc ) .
for all analyses , statistical significance was defined as a p - value < 0.05 .
this was a longitudinal , retrospective cohort study using us managed - care administrative data from the lifelink pharmetrics plus health plan claims database , which includes medical and pharmaceutical claims for over 150 million unique patients from over 90 health plans across the united states .
the prescription claims include national drug code , drug names , dose , strength , days supply , and quantity dispensed .
it also includes demographic variables , insurance information , and plan enrollment / eligibility data .
we cleaned the obtained raw data by removing the duplicates and observations with missing values for key data elements , such as age , diagnosis date , and prescription date .
the data in the claims databases are de - identified and fully compliant with the health insurance portability and accountability act privacy regulations , exempting this study from an institutional review board approval requirement .
selection of patients for the study is outlined in figure 1 . to be included in the analysis ,
patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . according to gold ( global initiative for chronic obstructive lung disease ) guidelines,1 roflumilast
is recommended as the third maintenance agent for patients with severe copd . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent
patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline .
study patients were separated into two cohorts : patients who initiated roflumilast during the cohort selection period ( roflumilast group ) and patients who initiated the third of three copd maintenance medications during the cohort selection period ( non - roflumilast group ) .
patient demographics included age at the index date , sex , insurance type , and census region .
clinical characteristics included comorbid conditions , reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period ( termed as baseline period ) ; number of copd exacerbations ( defined in the following paragraph ) ; hospitalizations ; and emergency department ( ed ) visits during the baseline period .
the primary outcome was the rate of moderate and severe copd exacerbations per patient per month ( pppm ) over the 3 months after the index date .
severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation ( icd-9-cm codes 491.21 , 491.22 , 493.22 , or 492.8 ) or a primary diagnosis of respiratory failure ( icd-9-cm codes 518.81 , 518.82 , or 518.84 ) combined with a secondary diagnosis of copd with acute exacerbation or emphysema ( 491.0 , 491.1 , 491.21 , 491.22 , 491.8 , 491.9 , 492.0 , 492.8 , 493.22 , or 496).7 moderate exacerbation was defined as a short course ( 14 days ) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis ( icd-9-cm codes 491.xx , 492.xx , and 496.xx ) ; an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema , as defined earlier ; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition ( icd-9-cm codes 136.3 , 466466.19 , 480486 , 487.0 , 490 , 491.21 , 491.22 , 493.02 , 493.12 , 493.22 , 493.92 , 494.1 , 506.0506.3 ,
507507.8 , 511.0511.1 , 512512.8 , 517.1 , 518.0 , 518.81 , 518.82 , 518.84 , 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation .
secondary outcomes included health care resource utilization ( hcru ) , including the number of inpatient hospitalization admissions , ed and office visits , and total health care costs .
costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9
bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables .
wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier .
the did model was used to control for observed baseline differences between treatment and control groups ( eg , severity of disease ) , as well as the effects of all time - varying determinants of the outcomes ( trend change in the outcomes over time ) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time .
analyses were performed using statistical analysis software version 9.2.3 ( sas institute , cary , nc ) .
for all analyses , statistical significance was defined as a p - value < 0.05 .
of 818,457 patients identified as having copd using icd-9 codes , 804,246 met at least one of the criteria for exclusion ( figure 2 ) .
the major reasons for exclusion were absence of claims for copd maintenance medications during the cohort selection period ( n=501,556 ) and not receiving at least three maintenance copd medications during the cohort selection period ( n=110,320 ) .
after these exclusions , there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group .
patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period .
there were no statistically significant differences in age , sex , or charlson comorbidity score between groups . in the non - roflumilast group ,
the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group ,
33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics .
figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . during the follow - up period ,
the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group ,
however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) .
after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group .
there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) .
monthly number of office visits , ed visits , and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . during both the baseline and follow - up periods ,
patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group .
both treatment groups had increased office visits , ed visits , and hospitalizations during the follow - up period compared to the baseline period .
however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits .
after controlling for key covariates using did models , roflumilast was associated with fewer hospital admissions ( =0.003 , p=0.57 ) and office visits ( =0.46 , p=0.26 ) ; however , the differences were not statistically significant .
table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . during the baseline and follow - up periods ,
costs increased for both groups from the baseline to the follow - up period , with no significant difference in the change from baseline between groups . after controlling for the key covariates using a did model ,
the total cost increase was $ 116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast ( p=0.62 ) .
of 818,457 patients identified as having copd using icd-9 codes , 804,246 met at least one of the criteria for exclusion ( figure 2 ) .
the major reasons for exclusion were absence of claims for copd maintenance medications during the cohort selection period ( n=501,556 ) and not receiving at least three maintenance copd medications during the cohort selection period ( n=110,320 ) .
after these exclusions , there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group .
patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period .
there were no statistically significant differences in age , sex , or charlson comorbidity score between groups .
in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group ,
33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics .
figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . during the follow - up period ,
the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group ,
however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) .
after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group .
there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) .
monthly number of office visits , ed visits , and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . during both the baseline and follow - up periods ,
patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group .
both treatment groups had increased office visits , ed visits , and hospitalizations during the follow - up period compared to the baseline period .
however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits .
after controlling for key covariates using did models , roflumilast was associated with fewer hospital admissions ( =0.003 , p=0.57 ) and office visits ( =0.46 , p=0.26 ) ; however , the differences were not statistically significant .
table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . during the baseline and follow - up periods ,
costs increased for both groups from the baseline to the follow - up period , with no significant difference in the change from baseline between groups . after controlling for the key covariates using a did model ,
the total cost increase was $ 116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast ( p=0.62 ) .
this study found that patients initiating roflumilast had more severe copd in that they had a higher number of monthly exacerbations , received more copd medications , and used more hcru than patients initiating other copd maintenance medications ( based on the observation during the baseline period ) .
exacerbation rates were significantly improved for patients treated with roflumilast , with an 11% decrease in the rate of exacerbations in the roflumilast group and a 16% increase in the non - roflumilast group ( a relative 27% reduction ) . after controlling for baseline differences using a did model approach , the significant differences in exacerbation rates between groups remained . after controlling for covariates using did models , changes in hospital admissions and total costs did not differ significantly between the roflumilast and non - roflumilast groups .
the cost analysis in our study focused on the overall cost ( cost component shown in table s1 ) . in order to further understand how roflumilast impacts different cost components
, future studies should examine the copd- or exacerbation - related costs by component , especially when the roflumilast was used as an add - on treatment following the gold guidance .
randomized controlled trials evaluating the efficacy and safety of roflumilast in patients with copd found significant between - group differences in moderate or severe exacerbation rates ( a 17% reduction , p=0.0003 ) ; however , these trials were placebo - controlled and 6 months12 or 1 year13 in duration .
significant differences in the rate of moderate , but not severe , exacerbations were initially found in two pivotal 1-year trials,12,13 which may be due to a lack of statistical power given the relatively small sample sizes of clinical trial data .
similarly , in the current study , there may not have been sufficient power to detect a significant difference in the change in severe exacerbation rates between groups .
relatively , the cohort of patients who received roflumilast was large in this retrospective cohort study ; however , there may be large treatment variations from patients in real - world settings compared to the strict treatment regimens of a clinical trial .
additionally , severe exacerbations are relatively rare events , which may further reduce the power of the analysis for this outcome .
as gold recommended , roflumilast can be used an add - on treatment for patients with copd in stages 3/4 ( forced expiratory volume in 1 second ( fev1 ) < 50% predicted after bronchodilatation ) and frequent exacerbations despite a long - acting bronchodilator treatment .
it was found that the copd drugs usage before initiating roflumilast varies in our study .
it may be informative to explore the subset of patients who strictly followed the gold guidance ( ie , those receiving ics and a laba or other long - acting bronchodilators prior to the use of roflumilast ) using the real - world data .
future studies are warranted to assess the outcomes of those patients who have used ics and long - acting bronchodilator within certain window prior to initiating roflumilast using a large sample .
previous studies examined the resource utilization and health care costs associated with exacerbations among copd patients.1417 in a us managed - care population initiating copd maintenance medications , abudagga et al found that exacerbation rates continued to be high , resulting in an estimated annual cost of $ 25,747 in the overall population and $ 29,861 in the patients with history of one or more exacerbations.14 in this study , calculated total monthly costs during the 3-month follow - up period were $ 2,472 and $ 2,851 for non - roflumilast and roflumilast group , respectively , which is higher than the abudagga study14 and likely due to the selection of patients with more severe copd .
darnell et al reported an annual 3.07 clinic visits , 0.15 ed visits , and 0.41 hospitalizations per person per year , among copd patients , calculated using a large administrative database.15 the resource utilization reported here was similar or slightly higher than data in the darnell et al study15 and again , this discrepancy may be due to the selection of patients with more severe copd . of note ,
our study examined the economic burden associated with copd medications from the payer s perspective ; indirect costs , such as productivity loss , were not included , and thus , cost data may be conservative estimates .
copd exacerbations result in increases in resource uti - lization , especially for severe exacerbations.9,18,19 consistent with previous studies , the current work found that reduced exacerbation rates among copd patients were associated with reduced all - cause resource utilization and costs from a managed - care payer s perspective .
therefore , it is vital for physicians and payers to be aware that preventing copd exacerbations through appropriate treatments could minimize the need for hospitalization and other medical resource use .
in addition , previous research also suggested that reducing exacerbation frequency may improve the quality of life among patients with copd.20 strengths of this study include the use of a robust national commercial insurance database with a longitudinal data structure that allows for tracking of both inpatient and outpatient diagnoses and procedures as well as outpatient prescription records .
the availability of prescription claims provided rich information on treatment patterns and medication use as an indicator of exacerbation frequency .
the use of did models controlled for significant baseline differences in study populations , which is important as there is potential for selection bias due to the observational study design .
our study has several limitations and it is important to interpret our results in the context of these limitations .
first , some clinical details such as lung function parameters are not available in the database .
the cohort selection criteria is based on the use of copd maintenance treatments ( ie , patients who initiated roflumilast or a third copd drug ) , which may identify more severe patients by the criteria itself .
second , the definition of copd exacerbations was based entirely on claims data rather than patient symptoms .
the study by stein et al suggested that algorithms based on icd-9-cm codes may undercount the acute exacerbation for copd.21 the exacerbation rates among patients receiving roflumilast were similar to those reported in randomized trials,12,13,22 which validate our results .
third , the study sample for the roflumilast group was restricted to those with insurance covering roflumilast . because data were available only for commercially insured patients , the results may not be generalizable to older patients covered by medicare .
however , the mean age of patients in our study was only slightly lower than that of patients in randomized trials ( 63 years vs 65 years).12,13 fourth , we used a did model that mimicked an experimental study and controlled for the baseline difference between study groups .
our adjusted did regressions were based on an assumption that in the absence of treatment , the difference between control ( without roflumilast ) and treatment ( with roflumilast ) groups would be constant or
fixed over time . however , in reality , it is possible that the roflumilast group was inherently more likely to experience a reduction in exacerbation rate compared to the non - roflumilast group due to having higher exacerbation rates at baseline . in this study , we observed a decreased overall exacerbation rate in the roflumilast group contrasted with an increased exacerbation rate in the non - roflumilast group .
the magnitude of the difference suggests that the observed results can not be attributed exclusively to unobserved confounders .
fifth , it is challenging to adjust for the difference in adherence in the patients without roflumilast .
patients adherence to treatments , especially to the combination therapy , may change quickly over time because patients may discontinue some drugs after the symptoms improve .
it will be important for future studies to control for the adherence if a specific comparator drug is chosen .
sixth , our results may not be able to identify the seasonal effect as the follow - up duration covers only 3 months.23 however , both study groups have the same index period so the difference in the exacerbation outcomes between the two study groups may not be biased . finally , we did not have a large sample of the roflumilast - treated group as roflumilast is only recently approved by the us food and drug administration ( february 2011 ) .
this may affect the statistical power and our study may not be sufficient to determine the long - term effects of roflumilast on exacerbations , hcru , and costs .
patients with copd - initiating roflumilast treatment had greater reductions in exacerbation rates in the 3 months after the new therapy was added than patients initiating other copd maintenance medications .
we also found a numerically smaller increase from baseline in the monthly rates of hospital admissions , office visits , and monthly total costs between patients treated with roflumilast versus patients not treated with roflumilast .
future studies using alternative methods such as patient survey or chart review are warranted to further evaluate the impact of roflumilast on clinical outcomes , hcru , and costs within a longer follow - up period .
monthly total health care costs and cost by component ( $ ) notes : the result data represent average cost per patient per month . the total cost is more than the sum of inpatient , outpatient , er , and copd drug costs because there are other types of cost components not shown in this table .
abbreviations : baseline , 12-month preindex period ; copd , chronic obstructive pulmonary disease ; sd , standard deviation ; er , emergency room . | backgroundroflumilast is approved in the united states to reduce the risk of copd exacerbations in patients with severe copd .
exacerbation rates , health care resource utilization ( hcru ) , and costs were compared between roflumilast patients and those receiving other copd maintenance drugs.methodslifelink health plan claims database was used to identify patients diagnosed with copd who initiated roflumilast ( roflumilast group ) or 3 other copd maintenance drugs ( non - roflumilast group ) from may 1 , 2011 to december 31 , 2012 .
patients must have been enrolled for 12 months before ( baseline ) and 3 months after ( postindex ) the initiation date , 40 years old , not systemic corticosteroid dependent , and without asthma diagnosis at baseline .
difference - in - difference models compared change from baseline in exacerbations , hcru ( office , emergency visits , and hospitalizations ) , and total costs between groups , adjusting for baseline differences.resultsa total of 14,211 patients ( roflumilast , n=710 ; non - roflumilast , n=13,501 ) were included . during follow - up ,
the rate of overall exacerbations per patient per month decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( p<0.001 ) .
after controlling for baseline differences , roflumilast - treated patients experienced a greater reduction in exacerbations ( 0.0160 fewer exacerbations per month , p=0.01 ) , numerically greater reductions in hospital admissions ( 0.003 fewer per month , p=0.57 ) , office visits ( 0.46 fewer per month , p=0.26 ) , and total costs from baseline compared with non - roflumilast patients ( $ 116 less per month , p=0.62).conclusionin a real - world setting , patients initiating roflumilast experienced reductions in exacerbations versus patients treated with other copd medications . | Introduction
Methods
Study design and data source
Patient selection
Study measures
Statistical analyses
Results
Patient sample
COPD regimens exposed on the index date
Exacerbations
Health care resource utilization
Health care costs
Discussion
Conclusion
Supplementary material |
the prevalence of obesity in early childhood has shown some signs of decreasing ; however , for the hispanic population , it continues to remain high , estimated at 16.7% for 25-year - old hispanic children in the united states , double that of the general population .
there are few effective interventions that have enrolled obese , hispanic children from this age group .
there are also very few prevention studies shown to be effective in hispanic families [ 3 , 4 ] . of the intensive , clinical interventions shown to be effective ,
a major limitation is the intensity required as this limits the intervention 's potential public health scope and brings about challenges of sustainability [ 58 ] .
peer or parent mentors are an alternative to professional counseling for delivering information and assisting with behavioral change .
parent mentors have been used as an effective intervention model in coaching other parents on their child 's diabetes management and in childhood asthma .
this model may also be an attractive option for the treatment of early childhood obesity because it requires a relatively low amount of resources to initiate a parent mentoring program , and there is potential for empowerment and sustainability within the community .
head start is a government - funded program for low - income families that includes early learning and school readiness , health and development screenings as well as daily meals , and family well - being to strengthen parent - child relationships .
one of head start 's core values is the empowerment of families , and they serve a population at high risk for obesity ; therefore , the parent mentor model of intervention meshes well with their structure and operational values .
the data on obesity interventions in head start centers in other locations have yielded variable results .
the positive deviance approach was used as the premise for building the intervention as it is well suited for high - risk populations and family empowerment .
positive deviance is the idea that , even among those most at risk for an adverse outcome , there are some individuals in the community who find a way to succeed , and , by identifying how those individuals or families succeed , one can identify successful strategies and behaviors that utilize local resources and knowledge .
this approach was historically used in malnutrition though it has recently been explored in obesity [ 1417 ] .
this study was designed to evaluate the feasibility of a clinical trial to test the hypothesis that parent mentors using positive deviance - derived education could be an effective intervention for early childhood obesity in the context of a head start program serving a low - income , hispanic community .
this was a pilot , randomized clinical trial designed to evaluate the feasibility of an intervention using parent mentors trained in positive deviance behaviors to reduce adiposity among obese 25-year - old children .
all of the parent - child dyads recruited for the study were actively enrolled in a head start program , neighbors in need of services ( ninos inc . ) .
the full protocol for this trial is described in greater detail elsewhere and is also available by contacting the corresponding author .
staff identified 139 families with at least one 25-year - old obese child from the 16 centers in the geographic area of this study , cameron county , south texas .
identified families were contacted by letter and phone to explain the study and determine interest in participating .
eligibility was determined as having a body mass index ( bmi ) 95th percentile for age and gender .
exclusion criteria were intellectual disability , severe development delay , seizure disorder , diabetes , cerebral palsy , any genetic problem , and inability to communicate in either english or spanish .
sixty parents provided written consent , and they and their child were enrolled in the study .
parent - child dyads were randomized 1 : 1 to intervention or comparison in blocks of six using redcap .
the randomization allocation table was generated by a research assistant and was concealed from the principal investigator and staff . enrollment and assignment of participants to intervention and comparison arms were carried out by clinical research unit staff .
parent mentors were recruited from cameron county head start centers and trained as described previously .
briefly , these individuals were required to have a 25-year - old child at a healthy weight who was enrolled in head start at the time ; the parents themselves could be of any weight .
four parents received a one - day intensive training on the content of the parent mentor manual and on reflective listening , with three parents selected for participation based on their engagement in the training .
the parent mentor manual was developed using the american academy of pediatrics guidelines on obesity prevention and the previous study on identified positive deviance practices done in cameron county .
there were five main foci that the parent mentors were instructed on as being potentially effective strategies to share with their parent mentees : dealing with behavior problems without using food , identifying internal motivators for healthy habits , organizational strategies for feeding , accurate perceptions of weight , and effective snacking strategies .
they completed worksheets monthly for each encounter with their mentee and reported what topics were discussed .
parent - child dyads randomized to the intervention arm were assigned to one of three parent mentors .
home assessments with the parent and child were conducted by each parent mentor at baseline and three months after enrollment . a standardized approach to the visit included asking about five main areas ( positive deviance behaviors listed above ) , and
at least one phone call per month was made by each parent mentor in order to reinforce those behaviors .
meetings were conducted on a monthly basis by parent mentors , and each mentor was allowed to implement her own curriculum in accordance with the goals discussed with the participating group .
parent - child dyads randomized to the community health worker comparison arm had the opportunity to attend one of three monthly community meetings held at head start centers .
these meetings were conducted by a local promotora or community health care worker .
in contrast to the intervention arm meetings , the comparison arm meetings followed a structured setting outlined by the eatplaygrow curriculum .
topics discussed during the meetings included health benefits of fruits and vegetables , limiting unhealthy foods , physical activity , portion control , and sleep , using an interactive format via songs , exercise , story time , and snacks . during the hour - long meeting , the first ten minutes were reserved for signing in and serving fruits and vegetables , the following forty - five minutes were for content delivery , and the final five minutes were used to remind parents of the following meeting and discuss any questions .
children were allowed to attend the meetings with their parents but it was not a requirement .
in contrast to the intervention arm , parent - child dyads did not receive any home visits or follow - up phone calls throughout the study .
the only contact time between the community health worker and parents was during the hour - long community meeting once a month .
therefore , a control group was identified with no intervention other than usual care in head start which consists of providing healthy meals and messaging on healthy habits in newsletters .
the control group children were identified as obese ( 95th bmi percentile for age and gender ) , were enrolled in head start over the same period of time , attended the same local head start centers , and were matched to each study participant by sex and on their initial bmi z - score ( within 0.3 units ) .
the average baseline bmi z - score difference between participants in the trial and community control group children was 0.03 units .
all measures described below were administered in either english or spanish depending on the preference of the individual participant .
the primary outcome was bmi z - score change at the end of the six - month intervention ; bmi z - scores were calculated using centers for disease control standards . a postintervention follow - up
children were measured and weighed without shoes and in light clothes by trained research staff .
secondary outcome measures assessed at baseline , at the end of intervention ( six months ) , and six months after intervention ( 12 months from baseline ) included health - related quality of life using the pediatric quality of life inventory ( pedsql 4.0 , mapi research trust , lyon , france ) , feeding behaviors measured by the comprehensive feeding practices questionnaire ( cfpq ) , dietary intake assessed using the block kids food screener ( bkfs ) developed by nutritionquest ( berkeley , ca , usa ) , screen time , sleep , and outside play using standardized questions previously described [ 18 , 2628 ] . growth ( height ) velocity was calculated over a twelve - month period , and each individual was plotted on a sex specific growth velocity chart .
recruitment was evaluated by assessing how many parent - child dyads were screened and contacted to achieve one dyad enrolled .
participation was calculated using a point system for each potential interaction in each arm of the study with fourteen possible points in the intervention arm and six points possible for the comparison arm .
these points were categorized into high participation 65% , some participation 1% and < 65% , and no participation = 0% .
the primary clinical outcome of bmi z - score was analyzed under the intention - to - treat principle .
linear mixed models with a random intercept were used to evaluate the primary outcome using randomization group , time ( 0 , 6 , and 12 months coded as intervals rather than a continuous variable to evaluate period effects ) , and their interaction ( group time ) as main effects adjusted for baseline measurements ; an interaction term between baseline measurement and time was included if significant to account for the intercept variation .
this modeling approach was also used for the secondary outcomes of the pedsql 4.0 scales , cfpq scales , and diet and activity measures .
data were analyzed using spss ( version 23 ; ibm spss statistics , ibm corporation , chicago , il ) .
weight maintenance in this age group with continued growth in height leads to a decrease in bmi z - score among obese children of about 0.5 units over 6 months ; this approximates a moderate effect size . with an expected mean bmi z - score of 2.5 at baseline , a standard deviation of 0.5 , and a two - sided of 0.05 , 30 participants in each group provided 48% power to detect a difference of moderate effect size , 86% power to detect a large effect size ( reduction in bmi z - score of 0.8 ) , and 12% power to detect a small effect size ( reduction in bmi z - score of at least 0.2 ) .
this study was approved by the university of texas health science center at san antonio institutional review board .
the total program enrollment for the cameron county head start includes more than 2,700 students . from this cohort of students ,
139 families were assessed for eligibility to participate in the randomized clinical trial . ultimately , 79 families were excluded and 60 were enrolled in the trial . of the sixty parent - child dyads initially enrolled in the study , forty - eight completed the six - month follow - up visit ( end of intervention ) ( 80% ) and forty - one completed the twelve - month visit ( six months after intervention ) ( 68.3% ) ( figure 1 ) .
participation in both the intervention and the comparison groups overall was high , with 76% of all participants meeting the benchmark of 65% of possible interactions to qualify as having high participation .
only two of the parent - child dyads had no participation recorded while still completing all study visits for measurements , both in the comparison arm of the study . in assessing adherence
, the parent mentors followed a standardized approach of discussing five main areas ( positive deviance behaviors ) when conducting home visits and phone calls throughout the trial .
their discussion of each area by proportion of all documented interactions was perceptions of weight , 84.3% ( 95% ci : 75.9 , 92.7 ) ; snacking strategies , 95.3% ( 95% ci : 91.6 , 99.0 ) ; dealing with behavior problems and emotions , 69.2% ( 95% ci : 56.2 , 82.2 ) ; organization and taking control , 77.1% ( 95% ci : 67.8 , 86.5 ) ; and figuring out why healthy habits are important , 78.6% ( 95% ci : 69.3 , 87.7 ) . comparing the baseline demographics and anthropometrics , there were no significant differences between the randomized groups ( table 1 ) .
there was no difference in baseline demographics between completers ( n = 48 ) and noncompleters ( n = 12 ) at six months . those completing the twelve - month visit were less likely to be employed at baseline ( 44% ) compared to noncompleters ( 78% ) , p = 0.05 , and otherwise there were no differences .
for the outcome of bmi z - score , there was no difference in the mean change between the parent mentor and community health worker groups ( mean difference : 0.02 ( 95% ci : 0.26 , 0.22 ) ) .
both had a significant reduction in mean bmi z - score by time ( p < 0.001 , table 2 ) . using estimated marginal means adjusted for baseline values , the estimated change in bmi z - score from baseline to end of intervention at six months was 0.24 ( 95% ci : 0.34 , 0.15 ) , and that between the end of intervention and the twelve - month time point was 0.00 ( 95% ci : 0.10 , 0.11 ) .
when separated by baseline bmi z - score quartiles , participants starting at a higher z - score intercept had the largest overall z - score change with a mean of 0.68 ( 95% ci : 1.1 , 0.24 ) .
there was no effect on systolic blood pressure ; diastolic blood pressure percentiles decreased overall by a mean of 5.53 ( 95% ci : 9.74 , 1.31 ) at six months , with no effect by group ( p = 0.96 ) ( table 2 ) . for sleep ,
no differences by group were observed ; the mean baseline for the parent mentor group was 10.7 hours ( sd = 1.42 ) and was 10.7 ( sd = 1.40 ) for the community health worker group . in the analysis adjusting for baseline and multiple measures
, there was a significant increase from baseline to six months ( p = 0.04 ) but then there was a decrease from six months to twelve months ( p = 0.002 ) resulting in overall no difference between baseline and twelve months ( p = 0.16 ) ( table 2 ) .
weekday screen time had a trend toward significance in the interaction of randomization group time ( p = 0.08 ) with the intervention group decreasing screen time from a mean of 3.3 ( 95% ci : 2.3 , 4.2 ) at six months to 2.1 ( 95% ci : 1.5 , 2.7 ) at twelve - month follow - up .
out of the forty - one participants who completed the six - month postintervention , 12.2% ( 5 ) had a growth ( height ) velocity < 50th percentile , 29.3% ( 7 ) were between the 50th and 90th percentile , and 70.7% ( 29 ) had a growth velocity > 90th percentile .
for the nonrandomized control group comparison , their baseline bmi z - score was 2.65 ( 95% ci : 2.38 , 2.93 ) ( table 3 ) and their mean age at baseline was 45.6 months ( 95% ci : 44.0 , 47.2 ) . the difference between their baseline and six - month bmi z - score was not significant ( p = 0.08 , paired t - test ) . as a group over a twelve - month period , they did have a significant decrease in bmi z - score of 0.32 ( 95% ci : 0.53 , 0.10 ) , with a mean z - score at twelve months of 2.33 ( 95% ci : 2.02 , 2.66 ) .
using the block ffq as the primary assessment of dietary intake , there was a significant reduction in sugary beverage intake overall with a mean change of 0.14 servings ( 95% ci : 0.23 , 0.04 ) at six months but not by group ( p = 0.96 ) , with the significance occurring between baseline and end of intervention at six months ( p = 0.001 ) and sustained with no change at twelve months ( six months after intervention ) ( p = 0.83 for six versus twelve months ) ( table 4 ) .
there was also a significant decrease in sugar added to food or drink over time of 1.22 tsp ( 95% ci : 2.12 , 0.32 ) , and no changes were seen by time or group for vegetable intake ( p = 0.36 for time ) or whole grain intake ( p = 0.12 ) .
the overall caloric intake decreased between baseline and six months with a mean difference of 119.99 ( 95% ci : 252.23 , 12.25 p = 0.07 ) and was then stable between end of intervention and twelve months ( p = 0.51 ) .
the quality of life scales assessed using the pedsql 4.0 showed no significant changes from baseline to end of intervention or six months after intervention in either group , except in emotional functioning , with a significant increase occurring between baseline , 81.3 ( 95% ci : 76.9 , 85.8 ) , and end of intervention , 86.8 ( 95% ci : 83.1 , 90.6 ) ( p = 0.003 ) .
the following are mean overall scores for the intervention and comparison group throughout the trial : baseline , 84.4 ( 95% ci : 81.3 , 87.5 ) ; end of intervention , 83.6 ( 95% ci : 79.2 , 88.1 ) ; and six months after intervention , 85.7 ( 95% ci : 81.5 , 90.0 ) .
finally , all of the cfpq scales showed significance by time except the monitoring measure ( p = 0.24 ) ( table 5 ) . the encourage balance and variety in diet
scale was the only measure to show significance by group ( p = 0.009 ) with a higher score reported at the end of intervention and six months after intervention for the intervention group versus the comparison group , with mean score of 4.5 ( 95% ci : 4.2 , 4.8 ) , 4.8 ( 95% ci : 4.6 , 5.0 ) for the intervention arm and 4.3 ( 95% ci : 4.0 , 4.5 ) , 4.5 ( 95% ci : 4.2 , 4.7 ) for the comparison arm , respectively , over time .
the scales that are most important and related to the intervention are environment , emotion regulation , restriction for health , restriction for weight control , and modeling .
these scales were more heavily discussed during the parent mentor training before the beginning of the study .
this is the first clinical trial to our knowledge that uses parent mentors as an intervention mechanism for childhood obesity .
an important aspect of this trial was that it was conducted in a low - income , hispanic population who , in the united states , are at higher risk of obesity compared with white children .
the data on recruitment , participation , and retention suggest that a full - scale clinical trial would be feasible in this setting .
the high participation rates for both intervention and comparison groups in the present study indicate that our approach was successful in terms of engaging and motivating parents of obese children .
moreover , our findings provide evidence that parent mentors are effective at facilitating this type of engagement in their peers .
it has been suggested that interventions sponsored or supported by government agencies and/or big organizations ( i.e. , top - down approaches ) are more sustainable compared to bottom - up approaches like interventions driven by community - based organizations .
our parent mentor intervention was driven by an independent social network , which is different compared to standard bottom - up approaches that are dependent on formal institutional support .
this is significant because it suggests that a positive deviance approach , with peer mentors serving as agents of change , may hold the key to improving sustainability . at the same time
, the relative success of these mentors may be dependent on their own capacities , and the variation between mentors in efficacy will need further testing in full - scale trial .
both the parent - child dyads randomized to receive a parent mentor and those randomized to receive health education from a community health worker experienced a decrease in their adiposity as measured by bmi z - score .
the diet and activity changes that were measured were consistent with this decrease in adiposity , and the plateauing of those diet and activity changes between the end of the intervention at six months and the twelve - month follow - up is consistent with their weight stabilization .
a recent study comparing childhood obesity interventions with and without a true control group suggests that careful interpretation of results in those without a true control should be considered due to possible regression to the mean and biases favoring resolution . for the control group that we did employ ,
the reduction in the control group 's bmi z - scores over twelve months makes it impossible to attribute the reduction seen in both of our intervention groups to the intervention .
however , the lack of a significant effect in the first six months for the control children in contrast to the stronger reduction in the first six months of the two intervention groups suggests there is some additional benefit to receiving the intervention that should be evaluated in a larger clinical trial .
other trials demonstrating success in reducing adiposity in this age group have utilized multidisciplinary teams including a dietician , psychologist , or behavioral counselor and some physical activity coaching [ 7 , 8 ] .
one trial specifically tested a mentoring component in addition to multidisciplinary education and found a significant benefit of health coaching by a paraprofessional mentor compared with the educational intervention alone [ 6 , 32 ] . in a multidisciplinary intervention study with a usual care control , a larger effect size on bmi z - score
was seen ( mean difference 0.77 , 95% ci : 0.27 , 1.26 ) than in our study ; other studies using a degree of intensity more similar to that described in this study ( using motivational interviewing or health coaches ) have seen a similar , more modest effect .
one important aspect of this study was its conduct in partnership with a head start program .
while the head start program staff did not provide any direct education for the parents involved in the study , they did encourage their involvement .
the potential for a scalable and effective program for reducing obesity has significant potential impact given the scope of head start and the population the programs serve .
prior studies targeting obesity in head start have been mixed with one study showing no reduction in adiposity and even increases in some groups , whereas others have found decreases associated with usual head start participation .
another series of trials found no significant effect with a weight - focused intervention but then after including a parent - focused component found significant decreases from baseline in both the intervention and the control groups , suggesting a possible role for usual head start participation .
using the cfpq , we observed an increase in parental report of more child self - control of their own eating behaviors , a decrease in regulation of child 's emotional states through use of food , an increase in parent promotion of well - balanced food intake , an increase in availability of healthy foods in the home , a decrease in use of food as a reward for child behavior , an increase in encouragement of child 's own involvement in meal planning and preparation , an increase in parent demonstration of healthy eating , a decrease in parent pressure of child eating more food at meals , an increase in parent control of child 's food intake to limit less healthy foods and maintain child 's weight , and an increase in techniques to encourage consumption of healthy foods .
one challenge of using the positive deviance method to develop the intervention is that there were no specific behavioral mapping tools for the targeted behaviors .
however , given that one area of the positive deviance - based intervention was on how to address behaviors without food , the cfpq findings are encouraging that the intervention had a causal relationship with the behavior changes reported .
interestingly , the only difference between the intervention and comparison group was an increase in parent reported encouragement of a balanced and varied diet in the intervention group .
prior observational studies have shown a correlation between restrictive feeding practices and increased risk for overweight [ 3638 ] ; however , some studies have suggested a role for restriction at least in early childhood , and the role of restrictive practices in the context of an intervention for obesity where a parent is trying to change patterns is less clear and should be explored in future interventional studies .
finally , reporting bias is certainly a possible influence on the cfpq results , particularly after intervention .
the change in the health - related quality of life measure of emotional functioning may be a result of the interventions or relating to the natural maturation process . also , the initial scale ratings for quality of life are lower than previously reported means of healthy children and similar to that of another study of obese preschoolers . however , these could also be unrelated to the child 's weight status and be more related to their socioeconomic status or another confounding variable
. the limitations of this study include the secondary outcome measures of screen time , activity and sleep being by parental report , though any recall bias would probably have affected both groups equally .
the limitation of no true control that was randomized is discussed above , though this study does add to the literature on describing the potential effect of usual head start participation .
the study was underpowered to detect a difference between groups for a small effect size on adiposity , which is what we observed .
however , the focus of this study was on feasibility of a novel intervention and study design .
finally , we can not separate the effect of the content delivery versus the health coaching or support by the promotora with this study design .
a significant challenge in addressing the childhood obesity epidemic , particularly the disparities seen in minority populations , requires scalable and culturally acceptable interventions .
the data presented suggest that the method of using parent mentors with minimal training is a potentially feasible intervention with a small effect size that warrants further exploration .
positive deviance as a method of deriving solutions from a high - risk community also warrants further testing in clinical studies .
finally , the role of early education centers , particularly head start , in addressing early childhood obesity is promising from both this and other recent studies . |
objective . to assess the feasibility of a parent mentor model of intervention for early childhood obesity using positive deviance - based methods to inform the intervention
. methods . in this pilot ,
randomized clinical trial , parent - child dyads ( age : 25 ) with children whose body mass index ( bmi ) was 95th percentile were randomized to parent mentor intervention or community health worker comparison .
the child 's height and weight were measured at baseline , after the six - month intervention , and six months after the intervention .
feasibility outcomes were recruitment , participation , and retention .
the primary clinical outcome was bmi z - score change .
results .
sixty participants were enrolled , and forty - eight completed the six - month intervention . at baseline ,
the bmi z - score in the parent mentor group was 2.63 ( sd = 0.65 ) and in the community health worker group it was 2.61 ( sd = 0.89 ) . for change in bmi z - score over time , there was no difference by randomization group at the end of the intervention : 0.02 ( 95% ci : 0.26 , 0.22 ) . at the end of the intervention ,
the bmi z - score for the parent mentor group was 2.48 ( sd = 0.58 ) and for the community health worker group it was 2.45 ( sd = 0.91 ) , both reduced from baseline , p < 0.001 .
conclusion .
the model of a parent mentor clinical trial is feasible , and both randomized groups experienced small , sustained effects on adiposity in an obese , hispanic population . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion |
leptospirosis commonly presents as an acute illness occurring in a biphasic pattern of fever followed by an afebrile period and again fever with organ involvement .
neurological involvement ( neuroleptospirosis ) is rare . among the several manifestations of neural involvement ,
myeloradiculopathy , myelopathy , cerebellar dysfunction , transverse myelitis , guillain - barre syndrome , optic neuritis , peripheral neuropathyhave also been described .
the hitherto undescribed entity of bilateral abducent nerve palsy as a manifestation of neuroleptospirosis is reported herewith .
a 22-year - old male agricultural worker presented with fever , nausea , abdominal pain , yellowish discoloration of the eyes and myalgia .
he had noticed the symptoms seven days prior to admission and had been treated symptomatically at a local dispensary .
he denied a history of alcohol usage . since this was during a time when our hospital was getting numerous cases of leptospirosis , and in view of his occupation , a possible clinical diagnosis of leptospirosis
injection crystalline penicillin two million units intravenously sixth hourly was commenced while awaiting lab reports for malaria parasite and leptospira , dengue , viral hepatitis serology .
urea 28 mg / dl , serum creatinine 1.4 mg / dl and rbs 112 mg / dl .
chest x - ray was normal . on the second night of hospitalization during routine rounds at 8.30 pm ,
the patient replied in the affirmative when asked about diplopia , but did not have any pain or headache .
the diplopia was worse when he directed his gaze to the left or right ( figure 2 , diplopia chart ) .
onset was rapid since evaluation a few hours earlier had not shown any neurological deficit . limitations of eye movements were confined to abduction in both eyes ( figure 3 ) .
the size of the convergent squint or esotropia was larger on distant fixation than on near fixation .
cerebrospinal fluid ( csf ) analysis showed 16 leukocytes / mm - all lymphocytes , glucose of 59 mg / dl and protein level of 39 mg / dl .
diplopia chart depicts that diplopia was worse when gaze was directed to the left or right
. shows extra - ocular movements ; it can be seen from this chart that the limitations of eye movements were confined to abduction in both the eyes .
titer was 50 u / ml ( < 15 u / ml negative , 15 - 20 intermediate , > 20 positive ) confirming the clinical diagnosis .
he also showed complete recovery from abducent palsy over the next five days and was discharged on the eleventh day after admission .
it is an important cause of acute febrile illness in china , the indian subcontinent , southeast asia , africa , south america and central america where malaria , typhoid and dengue are also common .
human infection occurs by direct contact with urine or blood of an infected rodent or animal , or from water or soil contaminated by urine . the organisms can penetrate abraded skin or intact mucous membrane , and enter the circulation and rapidly disseminate to various tissues .
the infection is commonly seen in farmers , sewage workers , veterinarians , and animal handlers . after an incubation period of 7 - 14 days
the first phase , leptospiremic or septicemic phase , has an abrupt onset , lasts for a week and is characterized by fever , chills , headache , skin rash , myalgia and conjunctival suffusion . the second phase or the immune phase , which can last up to 30 days , is characterized by leptospiruria and the development of anti - leptospira antibodies .
. additional involvement of pulmonary , renal , cardiovascular , and neurological systems can occur . in this phase
the rarer clinical presentations described in literature are myeloradiculopathy , myelopathy , cerebellar dysfunction , transverse myelitis , cerebrovascular accident , cerebral venous thrombosis , cerebral arteritis , subarachnoid haemorrhage , optic neuritis , mononeuritis multiplex , peripheral nerve palsy , psychosis , suicidal behaviour , encephalitis , and cerebellitis .
transient paraparesis has been described in a recent report by lih - shinn et al .
although implicated in various neurological manifestations , leptospirosis is often overlooked by clinicians in the evaluation of febrile illnesses with neurological manifestations . in the case
reported by kavitha and shastry , their patient had fever , jaundice and lower limb weakness , urinary retention and decreased sensory perceptions below t4 level . as in our case , the confirmation of the diagnosis was by positive anti - leptospira igm serology . in the report by costa et al . , facial palsy was observed following an illness consistent with leptospirosis and in their case , microscopic agglutination test ( mat)- the confirmatory test was not done , but seroconversion was observed in the macroagglutination test . in the report by mumford et al . , flaccid paraplegia was observed .
in contrast to our patient , this case ended with fatality ascribable to renal and hepatic involvement .
the sixth cranial nerve ( abducens nerve ) innervates the lateral rectus muscle in the ipsilateral eye .
paralyses can be due to direct damage of the sixth cranial nerve , encephalon nuclei or less frequently , diffuse axonal damage .
bilateral sixth nerve palsy is a rare clinical condition . unlike unilateral palsy , which is often due to local causes such as trauma , increased intracranial pressure , brain stem glioblastoma etc .
, the bilateral form is due to systemic causes such as vasculopathy secondary to atherosclerosis and immune mediated vasculitis .
patients with bilateral sixth nerve palsies complain about binocular horizontal diplopia being worse in the gaze direction of the paretic lateral rectus muscle .
currently , there is paucity of knowledge regarding the pathogenetic pathways involved in the above neurological manifestations
. the molecular mechanisms by which spirochetes interact with cellular barriers and the chain of events involved in leptospira meningitis and other leptospirosis - related neurological phenomena remain unknown . after invading the bloodstream
, leptospira plays a role in the activation of innate immunity cells , such as macrophages , via interaction among the transmembrane toll - like receptor 2 ( tlr2 ) , cd14 , and leptospiral lipopolysaccharide .
the organisms migrate from the bloodstream to the csf by crossing the blood brain barrier .
the ensuing neurological disease is dependent on the virulence of strains and on the development of an inflammatory response . yet , interestingly , most of the current animal models for the spirochetoses do not recreate the manifestations of the neurological spectrum .
experimental approaches to the human neurological manifestations of the spirochetoses have shown inability and difficulty in reproducing aspects of the human disease in animal models .
though reports of other cranial nerve palsies in leptospirosis are made in literature , abducent paralysis has not been mentioned hitherto . in our patient , typical clinical presentation of leptospirosis
the other causes of abducent palsy were excluded by medical history and by other investigations .
it is also to be noted that the patient developed abducent palsy approximately on the ninth day of symptomatic disease , when the clinical manifestations attributed to leptospirosis had subsided .
this is consistent with the concept that cranial palsies , including the present bilateral abducent palsy in patients with leptospirosis is mediated by immunological mechanisms .
the novelty of this case is that whereas numerous case reports exist of other cranial nerve palsies occurring in leptospirosis , the present case is probably the first one that exemplifies abducent nerve palsy developing as a result of antecedent leptospira infection .
the present case report has highlighted the hitherto unreported manifestation of bilateral abducent nerve palsy .
neuroleptospirosis should be considered in the differential diagnosis of all neuroinfections especially in endemic areas .
practicing physicians in developing countries , where advanced diagnostic facilities are not available , should be aware of the different patterns of clinical presentation and the various rarer manifestations of leptospirosis to be able to make a clinical diagnosis even when confronted with the unusual associations of leptospirosis . | leptospirosis , a disease of great significance in tropical countries , presents commonly as a biphasic illness with acute febrile episode in the first phase followed by a brief afebrile period and then by the second phase of fever with or without jaundice and renal failure .
however , it has varied manifestations and unusual clinical features ascribed to immunological phenomena can occur due to the additional involvement of pulmonary , cardiovascular , and neurological systems . among the various neurological features ,
aseptic meningitis is the most common myeloradiculopathy , myelopathy , cerebellar dysfunction , transverse myelitis , guillain - barre syndrome , optic neuritis , peripheral neuropathy hare also described .
cranial neuropathy involving facial nerve is a rare , but known neurological manifestation .
sixth nerve palsy in neuroleptospirosis has so far not been reported .
we hereby present the occurrence of bilateral abducent nerve palsy in a patient with leptospirosis . | Introduction
Case Report
Discussion
Conclusion |
any variation in root canal morphology should be recognized before or during root canal treatment .
an untreated canal , which a clinician fails to detect , may be a cause of failure .
various methods to assess the internal anatomy of root canal systems are : ( i ) conventional radiographs ( ii ) canal staining and tooth clearing ( iii ) plastic resin injection ( iv ) digital and contrast medium enhanced radiographic techniques ( v ) sectioning ( vi ) in vitro microscopic examination ( vii ) computed tomography ( ct ) techniques ( vii ) micro - ct and ( viii ) cone beam ct ( cbct ) .
cbct was developed in the late 1990s by italian and japanese groups to produce undistorted three - dimensional images of the teeth and their surrounding tissues .
it helps in visualizing the number of root canals and their convergence or divergence from each other .
the principle goal of endodontic therapy is to retain every functional component of the dental arch , including third molars .
when reviewing the literature regarding root and canal morphology using the pub med database ( national library of medicine ) , there were no studies on the root canal morphology utilizing cbct images of permanent maxillary third molar in indian population .
the aim of this study was to investigate the root canal morphology of maxillary third molars and to evaluate the number of roots and canals and classify them according to vertucci .
9000c 3d , 70 kvp , 10 ma , 36 s scan time , 10.8 s exposure time , 5 cm diameter5 cm height scan volume , france ) images of 325 patients who presented at a private clinic for various dental problems in bhopal between june 2011 and march 2015 was done .
all cbct image was observed and evaluated for the presence and absence of maxillary third molars .
the scans were produced by a technician according to the manufacturer 's recommended protocol using a minimum field of view of 40 mm 40 mm or 60 mm 60 mm and lowest dose radiation .
the cbct images were analyzed with the inbuilt cs3d imaging software ( nnt ) in a hp workstation ( hp compaq le 1911 ) with a 19 inch hp led screen with a resolution of 1280 1024 pixels on a dual monitor .
the images were visually enhanced by adjusting contrast , brightness and grey scale using the image processing tool in the software to ensure better visualization .
three endodontists and a trained technician concurrently evaluated all the images to reach an agreement in the interpretation of the radiographic findings .
type of canal configuration , presence of extra root and numbers of canals were evaluated using the nnt toolbar by carefully rolling downward through the images from the pulp chamber to the root apex at the axial tomographic slices .
the images were rotated in the entire axis , and slices were taken in all the three planes , coronal , sagittal , and axial planes to confirm the findings .
( ii ) third molars with no periapical lesions ; ( ii ) no endodontic treatment performed ( ii ) no root canals with open apices , resorption or calcification ; and ( iii ) the cbct images of good quality .
the teeth with root canal fillings , restorations , posts , or crowns were excluded from the study .
the final sample group included data from 116 patients ( 66 males and 50 females ) .
following information from the individual images was recorded ( i ) the number of roots and their morphology ; ( ii ) the number of canals per tooth ; ( iii ) the canal configuration in each root using vertucci 's classification ; and ( iv ) the frequency of additional roots and the frequency of c - shaped canals in the third molars . frequency and percentage of variables were calculated using statistical package for social sciences ( spss v.21.0 , ibm , armonk , new york ) version 21.0 and data was further analyzed by pearson chi - square test and fisher 's exact test .
56.9% of the teeth belong to males , and 43.1% were of females , 43.1% were of right side , and 56.9% were of the left side .
maximum maxillary third molars ( n = 116 ) had three root ( 55.2% ) followed by one root ( 31.0% ) and two roots ( 13.0% ) .
analysis of number of canals revealed that maximum number of maxillary third molars had 3 canals ( 37.9% ) followed by 04 canals ( 24.1% ) and 1 canal ( 19% ) , 3.4% of teeth had 5 canals .
in single rooted maxillary third molar , vertucci type i ( 47.1% ) was predominant .
most of the double - rooted teeth have vertucci type i ( 42.9% ) and type iv ( 42.9% ) in buccal root and type i in the palatal root ( 62.5% ) .
mesiobuccal root of three - rooted maxillary third molars had type i ( 43.8% ) and type iv ( 40.6% ) , distobuccal root and palatal root had type i ( 87.5% and 100% ) , respectively [ table 1 ] .
vertucci classification of canals in maxillary third molars comparison of vertucci classification revealed that type i was prevalent in both males and females in single rooted maxillary third molars , and the difference was statistically significant .
the percentages of other vertucci types in males were type iv ( 14.3% ) , v ( 28.6% ) , and vi ( 14.3% ) and in females were type ii ( 30.0% ) and type iii ( 20.0% ) . in double - rooted teeth ,
buccal root had type i ( 75% ) predominant in males as compared to females where type iv ( 66.7% ) was predominant .
in distobuccal root , vertucci type i was predominant in both males ( 85.7% ) and females ( 90.9% ) [ table 2 ] .
vertucci classification of canals in maxillary third molars according to gender when vertucci classification of canals in maxillary third molars was compared between the right and left side of the oral cavity , it showed that in single rooted teeth type i was predominant in both the sides .
type v classification was observed in 28.6% of right side single rooted teeth whereas in left side 20% were type iii , 10% were and type iv .
chi - square test showed a significant difference between the right and left side of the oral cavity . in double - rooted maxillary third molar , vertucci type i and
type iv was predominant in left side whereas on the right side type i , type ii , and type iv were equally present , and the difference was not statistically significant . in three - rooted maxillary third molars in mesiobuccal root ,
vertucci type iv ( 46.7% ) was predominant on right side as compared to left side whereas type i ( 58.8% ) were predominant , and the result was statistically significant ( p = 0.013 ) . in distobuccal root on both sides ,
these variations in the root canal morphology are thought to be racially and genetically determined .
therefore , it is necessary to investigate variations in tooth anatomy and their characteristic features in different racial groups .
the multiplanar cbct scans obtained from axial sections of 116 maxillary third molars revealed that frequency of three - rooted maxillary third molars was maximum ( 55.2% ) followed by one root ( 31.0% ) [ figure 1 ] and two roots ( 13.8% ) .
similar results were observed in burmese population by clearing technique where 51% of teeth had three separate roots , and 49% had fused roots . a study conducted by ng et al . in the same population using canal staining and tooth clearing technique showed 25% had three roots , and 31% had fused roots . cone beam computed tomography image showing two canals in single rooted third molar in the present study , maximum maxillary third molars showed 3 canals ( 37.9% ) followed by 4 canals ( 24.1% ) [ figures 2 and 3 ] and 1 canal ( 19% ) .
the majority of palatal ( 100% ) and distobuccal ( 96% ) roots possessed one canal ( type i ) . according to literature ,
vertucci type i was more common in distobuccal root ( 98.1% ) and palatal root ( 100% ) .
cone beam computed tomography image of maxillary third molar showing vertucci v configuration cone beam computed tomography image showing maxillary third molar with mb2 mesiobuccal root shows considerable morphological variations in vertucci classification . a conventional radiograph can detect only 55% of mb2 due to the superimposition of anatomic structures in a buccolingual plane which fail to detect small structural density changes .
stropko found 20.0% of third molars had mb2 , whereas it was 37% in a study done by green .
the prevalence of mesiobuccal roots with two canals molars was 39% with vertucci type ii and type iv configurations .
study conducted on maxillary molars in south asian indian population using clearing technique showed that mesiobuccal roots of maxillary third molars exhibited 57.4% had type i , 32% had type ii , 2.1% had type iii , 8.5% had type iv , and 1% had type v canal anatomy . in this study , single rooted maxillary third molars , the palatal root of double - rooted maxillary third molars , and all three roots of a triple rooted maxillary third molars
in buccal root of double - rooted maxillary teeth , the most common type was vertucci type i and type iv .
a similar retrospective study in mandibular molars in a turkish population using cbct showed that majority of mandibular molars were two rooted with three canals .
mesial roots had more complex canal systems with more than one canal , whereas most distal roots had a type i configurations .
gender predilection for the prevalence of vertucci type showed that vertucci type i was predominant in single rooted teeth in both males and females .
topological predilection of vertucci classification revealed that three rooted maxillary third molars showed mesiobuccal root vertucci type iv ( 46.7% ) predominant on right side in mesiobuccal root as compared to left side whereas type i ( 58.8% ) were predominant and the result was statistically significant ( p = 0.013 ) .
prevalence of c - shaped canals in maxillary third molars was 3.4% in this study [ figures 4 and 5 ] . according to sidow et al .
, 4.67% were identified in maxillary third molars ( all of them were double - rooted ) .
cone beam computed tomography image showing c - shaped canal in maxillary third molar cone beam computed tomography image of sagital view of c - shaped canal cbct offer high - resolution isotropic images which helps in identification of canals which is not possible in conventional radiograph if canal cortication is lost .
it offers three - dimensional geometric accuracy compared with conventional radiographs without superimposition of anatomical structures where data can be reoriented in their true spatial relationships .
however , consideration to use cbct should only be done when the conventional periapical radiographs fail to provide adequate information about the area of interest , as cbct views may also show some misleading findings .
there was a high prevalence of three - rooted maxillary molars with three canals in central india population .
vertucci type i classification was most common in single rooted maxillary molars , distobuccal roots and palatal roots of three - rooted maxillary third molars . | objective : the aim of this study was to investigate the root and canal morphology of maxillary third molars in central india population using cone - beam computed tomography ( cbct ) analysis.materials and methods : cbct images of 116 maxillary third molars were observed , and data regarding the number of roots , the number of canals , and vertucci 's classification in each root was statistically evaluated.results:majority of maxillary third molars had three roots ( 55.2% ) and three canals ( 37.9% ) . most mb root ( 43.8% ) , db root ( 87.5% ) , and palatal root ( 100% ) of maxillary third molars had vertucci type i. mesiobuccal root of three - rooted maxillary third molars had vertucci type i ( 43.8% ) and type iv ( 40.6% ) configuration
. overall prevalence of c - shaped canals in maxillary third molars was 3.4%.conclusion : there was a high prevalence of three - rooted maxillary molars with three canals . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest |
the first line therapeutic modality for non - surgical patients with large and multifocal hepatocellular carcinoma ( hcc ) who do not have vascular invasion or extrahepatic spread is transcatheter arterial chemoembolization ( tace ) .
however , transcatheter arterial radioembolization ( tare ) with yttrium-90 ( y)-labeled microspheres has an emerging role in treatment of unresectable hcc patients with or without portal vein thrombosis , or as a bridge to transplantation.1,2 radioembolization refers to injection of embolic beads loaded with radioisotopes using transarterial technique .
microsphere radioembolization allows the delivery of radioactive beads directly to the site of malignancy , thereby minimizing toxicity to adjacent organs .
despite direct delivery to tumor site , complications still occur when the microembolic effect or radiation injury occurs in organs other than liver .
reported side effects are predominantly constitutional symptoms such as fever while others are gastrointestinal ulceration or bleeding , cholecystitis , pancreatitis , radiation pneumonitis and hepatic decompensation.3 herein , we report a case of severe gastric ulceration with bleeding that eventually required surgery due to aberrant deposition of microspheres .
a 67-year - old male , inactive hepatitis b virus ( hbv ) carrier ( hbeag negative with undetectable serum hbv dna level ) , was admitted for further evaluation and treatment of hepatic masses .
laboratory tests showed serum albumin level of 3.7 g / dl , bilirubin level of 1.5 mg / dl , and prothrombin time international normalized ratio of 1.40 .
neither ascites nor hepatic encephalopathy was found , which was compatible with child - pugh class a. serologic marker for hepatitis c was negative .
dynamic enhanced computed tomography ( ct ) revealed underlying liver cirrhosis with a hepatic mass measuring 2.7 cm in segment 4 and another lesion measuring 0.6 cm in segment 7 with typical patterns of arterial enhancement and delayed washout .
gadoxetic acid - enhanced liver magnetic resonance imaging showed slightly high signal intensity in the t2 and diffusion - weighted image with a defect in 20-minute delayed image for these two lesions ( fig .
1 ) . these findings were compatible with hcc ( t3n0m0 ) , stage iii .
radiofrequency ablation ( rfa ) or tace can be considered as locoregional treatments for these unresectable hcc nodules in this cirrhotic patient who had no living donor for liver transplantation .
however the lesion in segment 4 located adjacent to small bowel and gallbladder , the interventional radiologist concerned about the risk of ablation - related complications and recommended tare .
angiographic studies to detect the variation of arterial vessels that distribute to the liver and other vessels that give access to extrahepatic organs as well as the review of the portal vein patency were done .
after angiographic study , technetium ( tc)-99 m macroaggregated albumin ( maa ) was injected to determine the degree of shunt to the lung as well as gastrointestinal tract . the calculated extrahepatic shunt was 10.71% . in the pre - evaluation study ,
prior to the embolization , empirical coil embolization for the gastroduodenal artery was done to prevent complication of non - target lesion .
a dose of 0.85 gbq of y microspheres was delivered via left hepatic artery and right posterior segmental artery , through 2.7f microcatheter ( fig .
immediately after the procedure , patient complained severe abdominal pain but post - tare scan without injection of tc-99 m maa demonstrated tumoral uptake of y but no gastrointestinal uptake was observed .
pain was controlled with tramadol and the patient was discharged one week after tare . however , the patient was readmitted due to severe abdominal pain four days after discharge .
upper endoscopy revealed geographic active ulcer at the lesser curvature of gastric antrum and mid - body .
histopathologic findings of endoscopic biopsy showed several deposited microspheres without evidence of helicobacter pylori infection .
gastric ulcer was treated with full ulcer medications including an intravenous bolus followed by continuous - infusion of proton pump inhibitor .
follow - up enhanced computed tomography which was performed one month after tare showed diffuse gastric wall thickening of antrum and lower body while hcc nodules in segment 4 and 7 were completely treated without recurrence .
as abdominal pain failed to resolve with continuous medications , follow - up upper endoscopy was done two weeks later .
worsening of ulcerative mucosal lesions with exudative bases accompanied by diffuse mucosal friability and easy - touch bleeding were noted .
three additional endoscopic examinations were done to assess the lesions , but the aforementioned ulcers as well as the territory of the lesion were not grossly changed ( fig .
surgical intervention was recommended due to persisting excruciating abdominal pain and refractory endoscopic findings , and finally , subtotal gastrectomy was performed three months after tare .
gross pathologic specimen revealed diffuse ulceration with multiple scattered microspheres deposited in the whole gastric layers , vessels and perigastric lymph nodes ( fig .
the patient 's symptom resolved after surgery and he was discharged two weeks later without any complication .
patients treated with chemoembolization for the treatment of hcc are reported to show similar survival time to those treated with y labeled microspheres radioembolization .
one of the advantages of tare is decreased embolic effect compared to tace allowing treatment for those with main portal vein thrombosis .
furthermore , tare resulted in longer time - to - progression and less toxicity than tace.4,5 although complication rate may be less than tace , gastrointestinal ulceration following tare can lead to fatal bleeding , small bowel obstruction and perforation.6 these complications occur due to non - target spheres distributed to aberrant vessels supplying gastrointestinal tract as well as changes in flow dynamics during infusion .
early studies showed high incidence ( 25% ) of gastrointestinal ulceration , but the incidence dropped to less than 5% after more extensive angiographic search to assess the hepatic vasculature and its collateral vessels that supply gastrointestinal tract as well as lung shunting.6,7,8 one of the studies reported that median ulceration rate was 8% , ranging from 0 to 20% , with 6% ( 0.4% of the entire cohort ) requiring surgery.6 prior to treatment , angiographic study is performed to minimize any complication . according to study by song et al .
based on 250 patients,9 the most common aberrant artery was the right gastric artery ( rga ) 78.4% and left gastric artery 17.2% while hepatic falciform artery , posterior superior pancreaticoduodenal artery and left inferior phrenic artery comprising the rest of the aberrancy . in our case ,
celiac and superior mesenteric arteries were evaluated , especially the right , and left gastric arteries which supply gastrointestinal tract with high aberrancy rate but no aberrant vessel was observed .
microcoil embolization is the most widely adopted method for preventing extraheptic arterial embolization , while balloon occlusion technique is another choice to occlude hepatopetal flow in the common hepatic artery which is less commonly used.10,11,12 finally , tc-99 m maa is injected into hepatic artery followed by single photon emission computed tomography to determine the degree of pulmonary and extrahepatic shunting.7,8 all these planning studies were performed in this patient and coiling was done for gastroduodenal artery but not for rga before tare . despite aggressive pre - evaluation , 3 - 5% incidence of gastrointestinal complication occurs due to failure in detecting smaller vessels or collaterals developing in several weeks after empirical coil embolization.13 another possibility of gastrointestinal injury is due to dynamic changes which may occur from embolic effect of microspheres due to slowing of antegrade flow that increases the risk of particle reflux.14 besides this , catheter stimulus may cause vessel spasm , infrequently . in this case
, complication could have been prevented if the rga was coil embolized prior to radioembolization considering the high incidence of gastric ulceration due to reflux of particles into rga .
small collateral vessels and hypertrophy of other vessels might develop after coil embolizations which were unnoticed during the procedure .
our case emphasizes the importance of finding collateral vessels meticulously even after coil embolization to ensure that there is no other vessels that might lead to reflux of particle into vessels feeding gastrointestinal tract .
time to diagnosis varies from less than 1 month to over 9 months and mean time to diagnosis is reported to be 3.2 months.8 therefore gastric ulceration due to tare should be suspected if severe abdominal pain persists and clinicians must not hesitate to perform endoscopy .
antral and pyloric channel ulcerations with extension to duodenum are common.13 endoscopic findings including erosions , erythema , exudation , friability and bleeding are not specific findings , so biopsy is required to definitely distinguish radioembolization - induced ulceration from other causes of ulcer .
the characteristic finding that allows diagnosis of radioembolization - induced ulceration is microsphere particles lodged in vessels of hematoxylin and eosin stained specimen .
other supporting findings of radioembolization - induced ulceration are apoptosis , epithelial flattening , and glandular cystic dilatation to nuclear atypia , capillary ectasia , and prominent endothelial cells.15 since radioembolization associated ulceration is diagnosed pathologically , repeated endoscopic examinations and multiple biopsies are needed if initial biopsy reveals negative result .
treatment of radioembolization - induced gastric ulcer is challenging because there is no consensus for management .
currently , the treatment is no different from medications that are prescribed for peptic ulcer disease , namely acid suppression .
ulceration result from ischemic injury due to occlusion of arteries supplying gastrointestinal tract , direct toxic effect of radiation or both .
radiation destroys parietal cells leading to decreased gastric secretion , thus unlike peptic ulcer disease that originates from acid - induced mucosa , injury , acid suppression treatment may not be effective.16 additionally radioembolization induced ulcers occur at the serosal surface not mucosal surface causing lack of response of acid suppression therapy which acts primarily at the mucosa.3 radiation enteritis is known to be caused by excessive stimulation of transforming growth factor ( tgf)-1 which leads to fibrosis and organ failure .
interferon- inhibits the effect of tgf-1.17 therefore , pentoxyfylline , a phosphodiesterase inhibitor that reduces inflammation mediated by cytokines including tgf- can be effective in radioembolization associated gastric ulcer.7 besides this , the effectiveness of anti - inflammatory agent such as sulfasalazine and antioxidant that reduces radiation induced free radical formation such as tocopherol can be considered as treatment options.8 since there is no one validated effective treatment , outcome can be fatal if appropriate time for surgical approach is missed .
early surgical intervention should be considered when abdominal pain persists and mucosal ulceration aggravates in endoscopic findings . in summary
, radioembolization - induced ulcer can be irreversible and can be life - threatening and therefore , aggressive pre - evaluation angiography and preventive coiling of aberrant vessels should be done before performing the procedure .
this is the first reported , preceptive case of radioembolization - induced severe gastric ulcer in korea which emphasizes the operator perception of the possibility of reflux into gastrointestinal artery and the need of surgery when gastric ulcer is refractory to medical treatment . | transcatheter arterial radioembolization ( tare ) with yttrium-90 ( 90y)-labeled microspheres has an emerging role in treatment of patients with unresectable hepatocellular carcinoma .
although complication of tare can be minimized by aggressive pre - evaluation angiography and preventive coiling of aberrant vessels , radioembolization - induced gastroduodenal ulcer can be irreversible and can be life - threatening .
treatment of radioembolization - induced gastric ulcer is challenging because there is a few reported cases and no consensus for management .
we report a case of severe gastric ulceration with bleeding that eventually required surgery due to aberrant deposition of microspheres after tare . | INTRODUCTION
CASE
DISCUSSION |
the theme of infanticide
has received increasing attention from historians in recent years . reasons for this growing interest include curiosity into the daily lives of poor women , and also the sharp contrast between a strong ideology of motherhood and a crime that seems to completely contradict the image of maternity .
scholars have studied the socio - economic backgrounds of these women , their trials and punishments ,
as well as the influence of enlightenment humanitarianism , including sympathy for the plight of child murderers , who came to be seen as victims rather than criminals .
dutch historians have mostly focused on the socio - economic backgrounds of child murderers in the eighteenth , and to a lesser extent , nineteenth century .
these studies all indicate that most of the women accused of newborn child murder were unmarried servants who tried to hide their pregnancy and birth out of shame .
fearing the loss of their jobs and livelihood as unmarried mothers , as well as their honour , they resorted to murdering their babies .
similar to research in other countries , dutch historians have assumed only a limited number of cases of child murder were actually tried before the courts , pointing to an unknown dark number.
besides the socio - economic background and the presumed motivations of these murdering mothers , historians have studied the dutch laws on infanticide , including the sentences in practice , which i will elaborate on below . here , it is relevant to note that dutch scholars have paid scant attention to the role of forensic medicine and psychiatry in court cases of infanticide , even though doctors were indispensable in cases of infanticide . from the early modern period physicians
were called in to examine a dead baby s body , to establish whether it was stillborn or born alive and possibly murdered .
sometimes the bodies of women who were accused of infanticide were searched for signs of recent pregnancy .
physicians vehemently debated the crime of child murder and the weight of their medical findings in these cases , both amongst themselves and with legal scholars .
this article explores the influence of forensic doctors and psychiatrists in dutch court cases of infanticide in the period 18111911 . in 1811
the french code pnal was introduced in the netherlands and in 1911 the concept of puerperal insanity ( insanity during or directly after birth ) was used for the first time in court , as part of the question whether jannetje j. , a servant girl , was accountable for her crime .
in contrast to england , where reference to puerperal insanity was used as a line of defence since 1822 , in the netherlands this concept did not play a role in nineteenth and early twentieth century court cases of child murder . in order to account for this absence ,
first i suggest more attention is paid to the connections between body and mind in contemporary formulations of affect , which provided room to discuss forms of insanity without the need to resort to the new science of psychiatry .
second , i propose a new approach to the history of forensic medicine , emphasising
travelling knowledge. i have adapted the concept of travelling knowledge from insights presented by scholars from different disciplines .
historians and sociologists of knowledge have already stressed how knowledge is socially constructed and negotiated .
in particular , science and technology studies have demonstrated how knowledge is produced in laboratories and how factual information comes to be seen as such .
the idea of travelling knowledge has mostly been applied to the appropriation of scientific knowledge from other continents in the early modern period .
historians have traced how scientific knowledge has been passed on between people , countries and continents , showing that the process of travel [ ] involves much more than simply moving something from one place to another so that , in the end , it remains the same even if found elsewhere .
there is no displacement , whether it be of people , instruments , objects , or data , without some sort of work , material as well as social , which enables their extraction from a given place and context , and insertion in a new place and new set of relations .
the process of travel [ ] involves much more than simply moving something from one place to another so that , in the end , it remains the same even if found elsewhere .
there is no displacement , whether it be of people , instruments , objects , or data , without some sort of work , material as well as social , which enables their extraction from a given place and context , and insertion in a new place and new set of relations .
not only in the field of history of science has the mobility of knowledge been emphasised .
cultural theorist mieke bal , as well , has introduced the notion of travelling concepts in the humanities , showing how certain concepts travel between academic disciplines , in the meantime changing these disciplines , while also changing themselves .
in a similar vein , literary critic and theorist stephen greenblatt recently presented a mobility studies manifesto , urging scholars to take seriously the concept of mobility in cultural history .
greenblatt advocates studying mobility in a literal sense , pointing to the physical , infrastructural , and institutional conditions of movement , but he also encourages us to pay attention to hidden as well as conspicuous movements of peoples , objects , images , texts , and ideas .
importantly , greenblatt underscores that mobility studies should identify and analyse the contact zones
where cultural goods are exchanged.
applying these ideas on cultural mobility to medical history , i propose to trace the way medical knowledge
travelled to the courtroom and , on its way , was adapted to the judicial context . from this perspective
, the courtroom might be regarded as a contact zone with its own rules of behaviour , shaping the insights from the medical field as doctors and psychiatrists were called in as expert witnesses .
these rules might change when the law is reformulated , when medical expertise changes , or when other cultural images or expectations influence the judicial process . in this article ,
i aim to show how medical knowledge of mania puerperalis , or puerperal insanity , travelled from england and germany to the netherlands , but , more importantly , which barriers subsequently prevented this knowledge from being applied in court cases of infanticide .
the courtroom might be seen as a contact zone between the medical and judicial fields , both fields stimulating the exchange of knowledge , but also dismissing the appropriation of new ideas .
the concept of travelling knowledge might help the formulation of theoretical approaches to the history of forensic medicine .
although the history of forensic medicine has recently received more attention , the field remains undertheorised .
often , it is presented as the unfolding of better technological knowledge , like the introduction of chemical tests , culminating in the application of dna , that ultimate instrument of truth , in court cases in the 1980s .
however , progressivist histories of medicine have been under scrutiny for some decades , for instance by cultural historians , who underline the social construction of science , medicine and disease .
social - constructionist approaches have , however , hardly been applied to the history of forensic medicine
some important works excepted .
for instance , in his study of nineteenth - century norwegian practices of forensic psychiatry , sklevg distinguishes between two approaches . the first he designates as the historical sociology of professions , exploring the growing power of medicine and the law as professions , their competition and their pursuit for greater public esteem .
the second perspective revolves around a foucauldian discourse analysis and regards medicine and the law as cooperative , not as competing branches of the medico - legal apparatus in the nineteenth century . in the latter approach , an impersonal scientific discourse , disciplining criminals ,
is seen to have taken precedence over a specific body of lawyers , judges or doctors .
sklevg eventually regards both approaches as complementary , but found little evidence for prestige as a strategic professional goal , thereby concluding that the model of professional interest does not carry sufficient analytical potential . rather than describing the history of forensic psychiatry as a
medicalization of law , sklevg shows how , in norway , psychiatrists in the nineteenth century also cooperated with the law , and sometimes opposed general medicine . by focusing on broader contemporary cultural conceptions of mankind
, he concludes that a separation between body and mind in the course of the nineteenth century opened up a space for psychiatrists to claim expertise on the mind .
in addition to his emphasis on the body mind dichotomy and his focus on shifting alliances between medicine , psychiatry and law , i would like to propose another perspective , that of travelling knowledge , to show how knowledge from the field of medicine was transferred to the courts , or , in the case of puerperal insanity , was barred from the legal arena . in order to apply this concept to dutch court cases of infanticide ,
i have studied the legal records of all ( fifty - seven ) cases of infanticide tried before the courts of north - holland and amsterdam .
in addition to these records , my sources include textbooks of forensic medicine as well as legal and medical texts on the role of physicians and psychiatrists in cases of child murder .
before i analyse these sources , however , i will sketch the characteristics of dutch laws on infanticide in the nineteenth century .
in 1774 the enlightened humanitarian dutch doctor petrus camper advocated leniency for women accused of child murder .
camper pointed to the stringent social laws on marriage which made unmarried mothers into victims , rather than perpetrators .
however , he was also criticised for his views .
in other countries , as well , enlightened ideas on crime and punishment influenced debates on infanticide .
thomas laqueur has shown how humanitarian narrative in its attention to the body not only as the locus of pain but also as the common bond between those who suffer and those who would help , fostered compassion and empathy .
sheena sommers , applying laqueur s concept of the humanitarian narrative to eighteenth - century court cases of infanticide , argues that medical expertise of physicians , having replaced the traditional knowledge of midwives , left room for empathy towards the murdering mothers , regardless of or even because of its uncertainty .
mark jackson critically discusses the contemporary , progressivist cultural discourse on humanitarianism in relation to crime and punishment . according to jackson ,
a new medical discourse and the increasing influence of scientific testimonies in court were more influential in the changing view on crime and punishment .
moreover , the role of forensic medicine and psychiatry in promoting legal reform was small , especially in comparison with england .
in the eighteenth century the numbers of women prosecuted for infanticide in the netherlands were relatively low and the verdicts lenient : judges were unwilling to sentence the perpetrators to death .
although punishment became less severe from the end of the eighteenth century , resulting from increasing sympathy for the plight of unmarried mothers , the death penalty for child murder was not abolished in the netherlands until 1854 . in 1811
the dutch adopted the french code pnal , which penalised infanticide with the death penalty , leading to a high number of acquittals since the materialist french law hardly allowed for extenuating circumstances and the death penalty was seen as too severe by many people .
between 1811 and 1870 about fifty death sentences were proclaimed for child murder in the netherlands , but most of the convicts received pardon from the king and were subsequently given prison sentences of fifteen to twenty years .
generally , acquittal on the basis of insanity was possible , but partial non - responsibility did not exist , probably explaining why , until the late nineteenth century , few medical doctors were called on to establish the sanity of the accused in dutch courts of law .
the code pnal , used in the netherlands until 1886 , was based on the enlightenment principle of sentencing criminals proportionately to the crime committed , dismissing the personality of the criminal to avoid arbitrariness .
this focus on material evidence is also thought to have contributed to the small role of psychiatrists in court cases .
liberal and social reformers succeeded in reforming infanticide law only in 1854 , when the death penalty was abolished for unmarried women who committed infanticide for the first time and was replaced by a prison sentence of five to ten years . in 1870
the death penalty was abolished for all other cases of child murder . in 1886 ,
when a new dutch criminal law was introduced , the death penalty as such was abolished and more attention was paid to the strong fear experienced by the murdering mother .
the law from 1854 explicitly mentioned the unmarried mother s fear of discovery of her pregnancy , and her resulting abnormal psychic and physical condition before and during delivery , as a reason to kill her child . in 1886
importantly , this law stated that , unlike in other crimes , a plea for extenuating circumstances was impossible , since these circumstances had already been assumed in the specific description of the crime in the law : the section on child murder mentioned the execution of a decision made under the influence of fear of the discovery of her upcoming delivery. this probably contributed to a dismissal of the overall mental condition of the mother .
since the law already assumed these women s state of bewilderment during delivery , further inquiry as to the cause would be unnecessary .
the new dutch penal code from 1886 furthermore introduced a distinction between murder and manslaughter of children , the difference depending on the moment ( before or during the delivery ) when the mother had decided to kill her baby .
in contrast to england , where infanticide was mostly prosecuted as murder or manslaughter until 1922 , child murder was a separate section in dutch law and a law punishing the hiding of a pregnancy in and of itself did not exist .
the age of the newborn child was , moreover , neither specified in the french code pnal nor in the new dutch laws introduced in the second half of the nineteenth century .
these new laws took into account that child murder was a last resort for mostly poor , unmarried mothers and therefore showed awareness of the difficult plight of these women . this new cultural awareness dovetailed with sympathy for prostitutes , who were increasingly regarded as victims of male lust as well .
in the netherlands , christian politicians and feminists advocated the abolition of the regulation of prostitution , especially in the second half of the nineteenth century and the first decades of the twentieth .
nevertheless , prostitution and adultery were much more important themes in this political struggle over female sexuality than infanticide . in the netherlands
so far only one case of infanticide has been found during which psychiatrists were called in as expert witnesses to establish whether the accused had been suffering from insanity , a condition that could potentially exonerate her .
in general , few dutch doctors were invited into the courtroom to assess the mental condition of criminals .
in france , for instance , psychiatrists became involved in court cases to establish the accountability of suspects in the 1820s , coining the concept of monomania .
nevertheless , despite many french medical studies on puerperal insanity , it seems that the latter notion was rarely applied in cases of child murder before 1901 .
in germany as well , physicians expertise was part of legal proceedings from the eighteenth century .
in cases of infanticide
, eighteenth - century german doctors sometimes described the mental condition of the mother , but did not use the concept of mania puerperalis ,
although german physicians had published on that topic since the seventeenth century .
subsequently , psychiatrists seem to have played a minor role in dutch , german and french cases of infanticide .
their lack of involvement stands in sharp contrast to their colleagues in england , however , probably relating to the different legal systems .
the adversarial nature of legal proceedings in england , as contrasted with the inquisitorial system in continental law , could explain this difference .
local physicians and professors in medicine were only called in randomly as expert witnesses in court .
in contrast to france , there were no court - appointed physicians in the netherlands .
a law from 1838 determined that expert statements were not to be considered as evidence by and of themselves .
donker and faber have suggested that the late application of the psychiatric concept of mania puerperalis in the netherlands was due to the specific formulations of the dutch laws on infanticide , in combination with the emphasis on material evidence like physical proof and witness statements in the dutch legal system .
however , they have neither done extensive research to back up this suggestion , nor examined the much broader and important role of forensic medicine as opposed to forensic psychiatry in cases of infanticide .
specifically , their isolated focus on the mind dismisses in advance more complicated and entangled body mind constellations .
this article , therefore , further explores the cause of the extremely late reference to potential insanity in cases of infanticide , compared to britain , by analysing textbooks of forensic medicine , medical and legal journals , in addition to court cases .
i will describe the relationship between body and mind in court cases of infanticide and argue that the emphasis on material evidence and the particular formulation of dutch laws on infanticide were indeed grounds for a late application of the concept of mania puerperalis , as donker and faber have suggested .
in addition i propose to take into account the contemporary cultural and medical discourses on the connections between body and mind , which left enough space to articulate mental confusion without referring to academic psychiatric concepts and thus made the acceptance of new psychiatric knowledge less urgent , thus explaining why this knowledge did not travel to the courtroom .
therefore in the following paragraphs i explore legal records , medical textbooks and articles in medical and legal journals to see whether the possible causes of the scant dutch attention to mental aspects in cases of infanticide ( a general focus on material evidence in the code pnal ; no options for partial accountability ; and the impossibility of pleas for extenuating circumstances from 1854 ) , as suggested in the secondary literature , can be corroborated .
i will argue that all of these factors played a role , but do not suffice to explain the minor significance of states of mind . equally important are the presence of a vocabulary on emotion , both medical and vernacular , which accorded room to mental aspects . in this particular discourse , body and mind
in order to account for the late entrance of psychiatric expertise to dutch cases of infanticide , the first question we need to address is whether the concept of mania puerperalis was known in the netherlands .
whereas french , german and english physicians and psychiatrists developed this notion , sometimes in relation to infanticide , this knowledge did not complete its journey to the netherlands , as this section will show .
interestingly , the notion was introduced by a dutch doctor who had read foreign texts describing the concept , but for several reasons it was not regarded as important enough to be used on a regular basis by dutch physicians or legal scholars .
although medical theories on madness caused by pregnancy and childbirth had circulated since antiquity mostly seen as caused by the sedimentation of the mother s milk in the brain the french psychiatrist jean - tienne dominique esquirol ( 17721840 ) dismissed the paradigm of humours and began the empirical observation of women s psychoses directly after childbirth .
he located the origins of mental illness in the passions of the soul , rather than in organic causes .
several german doctors had written on post - partum mania and melancholia from the seventeenth century , viewing them until the beginning of the nineteenth century as intellectual disturbances and regarding the affective components of the psychoses merely as epiphenomena . until the middle of the nineteenth century ,
german psychiatrists generally included all forms of post - partum psychosis under the heading of mania puerperarum , including those with melancholic symptoms , not yet distinguishing between insanity and mood disorder .
nancy theriot has described how the term puerperal insanity was constructed by nineteenth - century american doctors and patients .
most physicians divided the disease into phases of the reproductive process ( pregnancy , lactation , parturition ) or into maniacal , melancholic and depressive puerperal insanity .
insanity of parturition or puerperal mania , characterised by maniacal symptoms like an abnormal state of excitement and a hostility to husband or child , was regarded as the most common type of puerperal insanity .
theriot argues the disease was not only a product of male physicians notions of proper femininity , but also reflected contemporary ideas on the connections between mind and body in mental illness .
puerperal insanity can be interpreted as a socially constructed disease , reflecting both the gender constraints of the nineteenth century and the professional battles accompanying medical specialization.
in these french , german and american medical discussions on the aetiology of puerperal insanity , the crime of infanticide hardly played a role .
another french psychiatrist , however , louis - victor marc , elaborated on the connection between puerperal madness and child murder .
marc regarded women s reproductive functions as vital causes of mental illness , but also left room for hereditary predispositions and emotional triggers . focusing mostly on physical causes of the mental instabilities associated with pregnancy and the postpartum period ,
marc suggested that , although more research had to be done into the possible exoneration of the mother accused of infanticide based on her mental conditions , this was certainly an option .
in england ,
the london obstetrician dr robert gooch produced the first detailed account in english of puerperal insanity , described by hilary marland as very much a disorder of the nineteenth century
and from 1822
puerperal insanity was used in defence pleas , mediating between the wrath provoked by high levels of child murder and the sympathetic approach towards mothers who committed the crime.
as daniel grey has recently shown , the diagnosis remained a frequent feature in englishwomen s infanticide trials until the first two decades of the twentieth century .
in ireland ,
as well , the concept was often put forward in cases of newborn child murder , and the link was made in the medical literature from the second half of the nineteenth century .
however , in america considerable resistance was shown toward the application of this psychiatric notion to late nineteenth- and early twentieth - century infanticide cases .
janet galley has suggested three reasons why american doctors , lawyers and the general public rejected the connection between infanticide and female insanity .
first , the american system of state - based criminal law implied that there was neither a uniform legal response to infanticide or insanity , nor national studies into the phenomenon .
second , at the end of the nineteenth century , the number of infanticide cases in america decreased due to the creation of foundling homes and homes for unwed mothers , as well as the declining cost of rubber condoms .
the lower number of cases led to fewer demands on state legal communities to change the existing laws in relation to the crime of infanticide .
third , many americans regarded the insanity defence in general as a way for criminals to escape their punishment .
american beliefs in free will and the republican ideal of individual responsibility worked against the notion of unaccountability on the basis of mental illness .
these american arguments against the use of the insanity defence in cases of infanticide differed completely from the dutch ones .
in the netherlands , mania puerperalis was described in dutch for the first time by dr a. moll in 1822 in a medical journal , summarising the research by gooch , based on an article in a german journal .
moll , who wrote the first dutch textbook of forensic medicine , published in 1825 , briefly mentioned mania puerperalis in his chapter on insanity and accountability , classifying it under
melancholia , but did not refer to the psychological condition of parturient women in the chapter on infanticide , which was limited to the material and physical traces on the bodies of baby and mother .
therefore ,
contrary to what other dutch historians have assumed ,
the notion of mania puerperalis or puerperal insanity was introduced in the netherlands in 1822 .
nevertheless , in the medical literature few connections were made with insanity pleas in cases of infanticide .
it would be incorrect , however , to state that forensic doctors did not display any interest in the mental state of the mother in these cases .
although most of the authors of textbooks of forensic medicine did not explicitly mention mania puerperalis , several devoted some lines to the psychic condition of the mother and its relationship to legal accountability . already at the end of the eighteenth century ,
the aforementioned enlightened doctor petrus camper referred to the emotions experienced by women during pregnancy and delivery . whereas pregnancy makes even married women melancholic , camper noted , unmarried , solitary women , who are despised by the community , experience fear , pain and desperation during delivery , leading in that terrible moment of deep emotion to the killing of their child .
camper s reference to emotions as causing women s disturbance during childbirth was very common in medical and alienist literature and quite easily overlapped with early psychiatric notions . in 1824 , for instance , medical doctor j.h . de he s agreed with camper s plea for a more humane treatment of
he claimed that , in the netherlands , the advice given by camper fifty years ago , to give heed to the psychic condition of the mother , seems to have disappeared.
de he s located the cause for this disappearance in the decline of dutch forensic medicine , especially in comparison with germany , thus siding with many other doctors who complained about the sorry state of dutch forensic science during the nineteenth century .
camper was regarded by de he s as far ahead of his time , applying the teachings of psychic diseases to the practice of law in a period in which this was still very imperfect .
referring to moll and van eldik s article from 1822 ,
de he s underlined the existence of melancholia and mania before , during and after childbirth .
he located the cause of puerperal insanity in the genitals , triggered by emotional upheaval .
he left open the possibility of these women being prone to the disease by a certain condition of the brain .
referring to wigand , de he s saw the sudden surge of blood to the head , increasing the passions , as prompting madness .
it was the task of the forensic physician to find out if violence committed to a child had been performed in a state of melancholia or mania .
de he s , then , was one of the few forensic doctors who explicitly advocated attention to the mind of the mother .
he regarded this focus , however , as in line with older notions of emotional upheaval , indicating how the
new psychiatric discourse fused with an already existing discourse on emotions . thus the notion of puerperal mania travelled to the netherlands from france , germany and england .
although it was taken up by moll and de he s , several factors might have contributed to halting this notion from further travel to the courtroom .
second , it overlapped to an important extent with an older vocabulary of emotion , thus making its application less urgent , as i will explain below .
third , a contemporary , omnipresent notion of the inadequacy of dutch forensic medicine might also account for the lack of interest in the application of new research to forensic medicine .
moreover , another explanatory factor might have been the disagreement over the mania women possibly suffered in regard to childbirth .
legal scholar de feyfer in his dissertation treatise on child murder ( 1866 ) pointed to the lack of agreement between scholars in regard to the temporary insanity of child murderesses during and after birth .
still , he admitted that unmarried women , who often had a very quick delivery because of their vehement emotions , could easily attain a state of mental confusion .
thus far , forensic doctors had not paid enough attention to the psychic condition of the mother , according to de feyfer , having focused too much on the material facts , whereas there was every reason to regard them as not fully accountable for their actions . heeding the tight connection between soul and body in human beings ,
these women s physical sufferings caused a limitation of the rational mind .
in de feyfer s text , the connection between body and mind was central , emotions being inextricably connected to the psyche
moreover , he confirmed the neglect by dutch forensic doctors of the mental condition of criminal mothers , pointing to the material aspects of dutch law .
this claim was underlined by dr w. koster , who also concluded in his textbook of forensic medicine from 1877 that , in most cases , the forensic doctor only had to unveil the facts relevant to finding whether the baby was violently killed , without mentioning the psychic state of the mother .
whereas during the first half of the nineteenth century , doctors were mostly concerned with classifying puerperal insanity and discussed its existence in general , during the second half of the nineteenth century they debated whether post - partum madness could serve to claim diminished legal responsibility .
as i have shown above , the french psychiatrist marc was the first to suggest that puerperal insanity could actually function as such .
however , the famous french forensic doctor tardieu disagreed with him in a text written in 1868 .
both tardieu and , in 1897 , pathologist and forensic scholar paul brouardel doubted the existence of acute insanity during and directly after childbirth .
in their textbook of forensic obstetrics ( 1908 )
, the dutch doctor hector treub and the legal scholar a. tak refuted the opinions of these french scholars .
treub and tak , referring to austrian - german psychiatrist richard von krafft - ebing s studies on consciousness and mania , regarded attacks of mania transitoria in parturient and recently delivered women and transitory deliria on an epileptic and hysterical basis as potential grounds for non - accountability .
simon thomas had argued in the dutch journal for criminal law that the emphasis on fear in the 1886 dutch law on infanticide , and the description of special affects in the accompanying explanatory memorandum , did not imply a reference to a psychic condition that made women unaccountable for their actions .
thus medical and legal opinions on the existence of puerperal insanity , as well as its relationship to unaccountability , continued to differ at the beginning of the twentieth century . to summarise
, we have seen that knowledge of the concept of mania puerperalis existed in the netherlands , introduced by moll .
fleeting references in dutch medical journals ,
as well as a limited number of allusions in other texts on forensic medicine , indicate an awareness of the condition . however , several barriers served to keep this knowledge from being generally accepted and used in court cases .
some regarded the mentioning of fear in the law itself as a reason not to look for further evidence of psychic illness .
last , it seems that the discussion of emotions in relation to infanticide left considerable room to include psychic components .
for example , in an article in a dutch medical journal , the anonymous authors vaguely referred to new research into anthropology and psychology to argue that the term
this adjective was intended to leave space for a lenient sentence for the mother , since the unmarried mother , directly after birth , and after the recently suffered labour pains , has not yet felt full love for the new - born baby , and the birth itself so powerfully impacts on the body and mind of the woman , to be able to assume , that the murder committed immediately after birth was committed in a sickly and psychically excited condition . since , according to the authors , this phase of physical and psychic confusion could not be pinned down to an exact time frame , the term
importantly , the authors use the term abnormal somatic condition , referring to the interconnection of body and mind .
i shall explore this relationship between body , mind and affect further by looking at the role of forensic doctors in dutch court cases of infanticide in order to account for the barriers that prevented psychiatric expertise from travelling to the courtroom , in contrast to medical expertise on the body of the child , which did find its way to court . the unmarried mother ,
directly after birth , and after the recently suffered labour pains , has not yet felt full love for the new - born baby , and the birth itself so powerfully impacts on the body and mind of the woman , to be able to assume , that the murder committed immediately after birth was committed in a sickly and psychically excited condition .
physicians played an important role in cases of infanticide . in nearly every instance , doctors were called in to inspect the body of the dead baby , to establish whether it had been born alive .
they assessed whether it was full term , and they searched for signs of violence .
an autopsy was also performed to show the condition of the inner organs , especially of the lungs , and included the findings of the infamous lung test .
this test , invented at the end of the seventeenth century , consisted of placing the lungs in a bucket of water in order to see whether they sank or floated .
floating lungs were thought to indicate the presence of oxygen , hence pointing to a live birth .
it was considered to be faulty , especially since the lungs could contain gas that was often present in corpses , which could also make them float . throughout the eighteenth and nineteenth centuries , physicians studied more accurate lung tests and other signs to use in addition to the test .
nevertheless , it continued to be used , although only as one part of increasingly extensive post - mortem reports .
in all the cases consulted for which sufficient documentation has been preserved , the lung test was executed and , in all of these except for one where the results were inconclusive it was concluded that the baby had been born alive .
it then depended on the confession of the accused , witness statements and other material evidence whether the judge would convict the mother .
it is , however , difficult to reconstruct the exact weight of the forensic evidence . especially in cases dating from the first half of the nineteenth century
, judges and lawyers often approached forensic evidence critically . in a case from 1839 , for instance , a judge criticised the results of a lung test , indicating that the child , having respired , had been born alive and strangled by the umbilical cord .
he argued that a child could have breathed before and during birth , yet still be born dead . since neither these signs , nor the other evidence , provided enough certainty , the accused was acquitted .
similarly , in a case from 1852 the judge deliberated that the lung test was considered an
one explanation for the qualms expressed by judges in regard to the lung test might be that they were reluctant to sentence women accused of infanticide to death , a dilemma they no longer faced in the second half of the nineteenth century . in addition , as the visum repertum ( autopsy report ) became more extensive , the lung test decreased in importance , as other bodily evidence was increasingly taken into account .
still , until the beginning of the twentieth century lawyers for the defence would invite professors of medicine to court to underscore the uncertainty of the lung test .
the different values attached to the lung test by people in different locations with differing interests points to the shape that knowledge might take while travelling to other places .
whereas in medicine , as evidenced by textbooks of forensic medicine , the lung test could be promoted wholeheartedly or with some reserve , in the courtroom different criteria applied . for example , in a case from 1845 the defence called in a professor of medicine and a medical doctor as additional witnesses , who claimed the lung test only presumed that breathing took place , and that there were examples of breathing and life before birth .
similarly , in a case from 1857 the defence lawyer included professor of medicine van geuns , who was critical of the premises of the medical report . that the child had been mature and breathing at birth
could be concluded from the report with some measure of probability , but not as strong evidence. the judge admitted that the medical report had not provided
undisputed certainty , but a high degree of probability was enough to give reinou vette , a thirty - four - year - old female worker , the death penalty .
thus forensic doctors appointed by the authorities often claimed certainty in regard to the results of the lung test , but when this knowledge travelled to the courtroom the judge or the medical experts hired by the defence transformed this into probability or a lack of certainty .
disagreement amongst the several experts involved in the investigation and court case could cause severe problems . in a case from 1822 the defence s request for more experts was rejected by the judge , since that would give the impression that the report had been subjected to the judgement of other experts , which would be little respectful to the experts appointed by the judges , and which will make it impossible to find experts in future , which is already difficult now . to question further experts , professors against professors cannot be advantageous for the interests of justice .
the report had been subjected to the judgement of other experts , which would be little respectful to the experts appointed by the judges , and which will make it impossible to find experts in future , which is already difficult now . to question further experts , professors against professors
equally , in texts on forensic medicine authors were concerned with the image of forensic medicine if several doctors disagreed with each other in public court cases .
ten houte de lange complained that forensic medicine in holland was a disgrace for science , a
plaything for the cleverness of jurists.
in a case dating from 1884 , the accused was acquitted because the three experts interrogated at the trial contradicted the findings of the post - mortem performed by two other doctors , even though she had made a confession : she had put her fingers in the mouth and throat of the baby , but the experts disagreed over whether that had actually caused the death or whether the child had died because of problems experienced during birth .
the court eventually ruled that , because of the great differences in expert opinion , the guilt of the accused had not been sufficiently proved .
in a similar case ,
the doctors disagreed over the cause of death ( bleeding or suffocation ) and therefore it could not be proved that the mother had murdered her baby by pulling a string attached to the baby s neck .
paradoxically , although medical expertise was considered a vital element of the evidence sought in cases of infanticide , it was not accepted uncritically by judges , especially in the first half of the nineteenth century , possibly to avoid sentencing the accused women to death .
generally , however , in the second half of the nineteenth century , the court agreed with expert opinion when it came to bodily and material evidence .
the important role of the forensic doctor in regard to the examination of bodies contrasts with the lack of expertise sought in the realm of the mind .
the absence of forensic psychiatrists and their scientific vocabulary , however , does not imply reference to the mind was completely absent . to express the state of mind of the accused during or directly after delivery , as well as her accountability , the vocabulary of emotion was appropriated .
confusion or a baffled emotional state . however , the language of affect could probe more deeply into the mind , as evidenced by expressions like bewilderment of affect ( verbijsterde gemoedsaandoening ) ,
fears in movements of the soul ( angsten in zielsontroering ) ,
utterly moved , desperate , and sufficiently devoid of senses ( geheel ontroerd , wanhoopend en genoegzaam zinnenloos ) .
when , for instance , in 1882 twenty - one - year - old servant elisabeth kelder told the court that , during the delivery , her heart had felt like stone and she herself had felt
mad like a crazy person , the records do not evidence any response by the judge and a medical expert was not called in .
similarly , in 1885 twenty - two - year - old servant jantje van tuil described her condition during her delivery as nearly bordering on madness , preventing her from thinking .
they underlined her rational behaviour , leading the court to hold the defendant accountable for her crime .
these could refer to a state of consciousness , and hence to potential responsibility and accountability . in the following case , dating from 1888 ,
this allusion to affect comes clearly to the fore : twenty - four - year - old servant helena van krenk at first claimed not to remember whether she had pinched her baby in the neck before or after its death . neither
could she recall if the umbilical cord was torn before or after the child had suffocated .
dr luchtmans stated that women in the circumstances experienced by the accused were not generally in a normal condition .
however , in his opinion this also depended on the individual , since one person gets into affect
more easily than others. since he had neither observed the suspect before , nor after the birth , he had never seen her in affect. he had the impression she had a calm character .
the term in affect was not a conventional medical term and here equated emotion , consciousness and memory .
the lower court first exonerated her from child murder , since premeditation had not been proved .
she was convicted of manslaughter and sentenced to a prison sentence of one year and five months .
the court of appeal , however , ruled that the killing of her child had been premeditated and convicted her of infanticide , with a prison sentence of two years and six months .
thus eventually the medical evidence on the accused s mental condition did not contribute to her acquittal .
expert opinion could , however , endorse exoneration in regard to the speed of birth , corroborating women s claims .
many women testified in court that their babies had escaped them during birth and had fallen on the floor or in the lavatory , causing their deaths .
interestingly , forensic doctors connected body and mind in their explanation of fast deliveries , as in the case of maria van dam , a thirty - three - year - old servant accused of infanticide : the inconsistency in her answers [ during interrogation wr ] testifies to the desperation of the prisoner both before , during and after the delivery , causing her to be unable to notice the events and to give a full testimony of them [ ] the emotions of a girl surprised by labour can be deregulated by fear so badly that she is hardly conscious of all that is happening , and can not clearly represent them ; also , these affects influence these parts used during labour this fear especially works on the functioning of the womb which might cause convulsions of the womb , possibly triggering a faster delivery .
the inconsistency in her answers [ during interrogation wr ] testifies to the desperation of the prisoner both before , during and after the delivery , causing her to be unable to notice the events and to give a full testimony of them [ ] the emotions of a girl surprised by labour can be deregulated by fear so badly that she is hardly conscious of all that is happening , and can not clearly represent them ; also , these affects influence these parts used during labour this fear especially works on the functioning of the womb which might cause convulsions of the womb , possibly triggering a faster delivery .
importantly , this reference to the mind is very much connected to the body and used to explain the fast and sudden delivery , rather than the motive of the accused and her possible unsound mind and lack of responsibility for her actions .
the sheer importance of the discourse on affect is shown in the terms used during the trial of jannetje jurgens , the first case in the netherlands where psychiatrists asserted that the female perpetrator probably suffered from a mental condition while committing child murder .
dr herman schoo , who was one of the two physicians who conducted the autopsy , but was also asked for his opinion on the mental state of the suspect , stated that in his opinion the suspect had committed the crime in a
fear psychosis. the other expert , dr jacob voorhoeve , suggested that the accused might not have known what she was doing , that is , did not commit the crime in full consciousness. physician dr frederik meijers wrote in his report on the mental state of jannetje that she was suffering from acute nervous oedema , indicated by her thick , swollen tongue. meijers described how the fear of her pregnancy being discovered led to a state of sickly excitement of the nerves , which decreases her accountability to a minimum , but can not be thought to be completely absent. dr schoo agreed with him during the appeal and added that , although he had spoken of
fear psychosis during the first trial , he had meant a sickly excitement of the nerves , the wording used by meijers in his report .
both experts furthermore stated that the fact that jannetje had hardly felt any pain during the delivery pointed to her pathological excitement of the nerves , the lack of feeling being one of the symptoms of this nervous condition .
clearly , the physical experience of pain during delivery was directly connected to the woman s mental condition .
this should not surprise us , considering that from the early days of psychiatry for instance , in the description of monomania the status of the passions or affections had been unclear and the difference between normal and pathological passions was not specified .
thus a considerable overlap between the language of psychiatry and the more common language of affect remained .
this language could , but did not always , explain the particular mental and physical condition of the female suspect and exonerate her .
the strong focus of the dutch law on material evidence , however , meant that the state of mind of the accused was hardly ever the sole focus of the interrogation and therefore rarely served as the only line of defence .
this article aimed to provide new source material to study the absence of psychiatric expertise in dutch cases of infanticide , and to explore the use of the concept of travelling knowledge as an approach to the history of forensic medicine .
travelling knowledge might refer literally to medical knowledge and its potential application in the field of law , being received and dispersed in a different country . in the case of mania puerperalis ,
the dutch doctor moll introduced it into the netherlands by referring to the work of the english doctor gooch , having read about it in a german journal .
more interestingly , however , travelling knowledge can point to the fields of medicine and law as contact zones inviting , promoting or barring the transmission of knowledge . in the netherlands ,
the concept of mania puerperalis was not applied to actual court cases of infanticide for several reasons .
first , physicians and legal scholars continued to doubt the existence of this form of mania .
also , the specific formulation of the law strongly determined what medical evidence was needed in court cases .
not only did the code pnal generally emphasise material evidence , the laws on infanticide specifically mentioned fear and therefore additional reference to a mental condition of the accused was not needed .
most importantly , i argue , the existing vocabulary on emotion , both vernacular and medical , allowed for an analysis of psychic components , connecting body and mind in the description of the mental state of the mother during and after birth , as well as of the rapidity of her delivery . to conclude , specific national formulations of the law might have been the biggest barrier to the application of psychiatric knowledge , while the presence of an older , useful vocabulary of affect might have been a more hidden obstacle to the introduction of new mental concepts . | this article aims to explain why the notion of mania puerperalis , or puerperal insanity , was not used in the netherlands to exonerate women accused of infanticide , in contrast to other countries .
it applies the concept of travelling knowledge as an approach to the history of forensic medicine , pointing to the fields of medicine and law as contact zones inviting , promoting or barring the transmission of knowledge .
although the notion of mania puerperalis was known in the netherlands from 1822 on , psychiatric expertise was not requested in actual court cases of infanticide for several reasons .
first , physicians and legal scholars continued to doubt the existence of this form of mania .
moreover , it was not always directly connected to infanticide .
also , the specific formulation of the law strongly determined what medical evidence was needed in court cases .
not only did the code pnal generally emphasise material evidence , the laws on infanticide specifically mentioned fear and therefore an additional reference to the mental condition of the accused was not needed .
most importantly , the article argues that the existing vocabulary on emotion , both vernacular and medical , already allowed for an analysis of psychic components . | Introduction
Dutch Laws on Infanticide
Puerperal Insanity and Travelling Knowledge
Court Cases of Infanticide
Expert Opinion and Affect
Conclusion |
osteosarcoma ( os ) is the most frequently diagnosed malignant bone tumor in children and adolescents , and about half of the cases have lesions localized in the distal femur and proximal tibia .
the incidence of os is estimated to be about three to five cases / million / year , accounting for about 5% of childhood malignancies and about 9% of malignancy - related deaths in children , .
the long - term survival rate of os patients was less than 20% after surgical resection alone prior to the availability of neoadjuvant and adjuvant chemotherapy in the 1980s .
the 5-year survival rate improved to 6070% after the development and use of multi - agent chemotherapy regimens .
however , the improvement of survival has not changed significantly for the past 30 years since chemotherapy was developed , and chemoresistance has become a troublesome obstacle during management of os .
there is an urgent need to elucidate the molecular mechanisms of chemotherapy resistance and find reliable biomarkers of its development .
this would greatly help identify more effective biological - based therapies and optimize the treatment strategies .
post - transcriptional regulation by micrornas ( mirnas ) has been identified as an important mechanism underlying oncogenesis , invasiveness , proliferation , and migration of malignant tumors , .
growing evidence indicates that various mirnas ( including mir-92a , mir-99b , mir-132 , mir-193a-5p , mir-422a , and mir-125b ) are involved in the development of resistance to chemotherapy , , .
it is thought that mir-125b can act as both an oncogene and a tumor suppressor , depending on the cellular context , , .
a previous study indicated that mir-125b was significantly reduced in os tissues , and that it suppresses proliferation and migration of os cells through down - regulation of stat3 .
another study showed that mir-125b increases the sensitivity of os cell lines to cisplatin by targeting bcl-2 .
therefore , this study was designed to explore the possible use of mir-125b levels in patients to predict the response of os to cisplatin - based chemotherapy .
the study protocol was approved by the medical ethics committee of the second clinical hospital of lanzhou university .
informed consent was obtained from all adult participants prior to the start of the study . for children under 18 years of age
patients with os who presented to our department between january 2010 and july 2015 were screened for enrollment .
exclusion criteria were previous malignant tumors in another organ or system ; hematological disorders ; end - stage patients not qualified for chemotherapy ; patients with no pathological data ; and any patients unwilling to participate .
patients were treated according to national comprehensive cancer network practice guidelines for os , and any treatment decisions were not affected by participation in the study .
patients with resectable os received cisplatin - based neoadjuvant and adjuvant chemotherapy and surgery , while cisplatin - based aggressive chemotherapy was given to os patients with unresectable lesions .
the demographic and clinical characteristics of the study participants were obtained from their medical records on admission , and follow - up of patients receiving surgery was performed by the combination of outpatient visits , letters , and telephone calls .
the patients had follow - up visits with physical examinations and radiography every 3 months , as well as computed tomography scans or magnetic resonance imaging when necessary .
disease - free survival was calculated from the date of surgery until an event for each patient . for patients with unresectable os tumors , the response to chemotherapy
was evaluated by the response evaluation criteria in solid tumors ( recist ) . in this study ,
chemotherapy sensitivity was defined as complete remission or partial remission , whereas stable disease and disease progression were taken as signs of chemotherapy resistance .
the patients were also followed for overall survival after therapy , which was calculated from the date of the beginning of chemotherapy in our department , until the date of death . in each participant , a 10 ml peripheral venous blood was collected in edta anticoagulation tubes before any therapy was begun .
the blood samples were centrifuged at 3,000 rpm for 10 min within 20 min after collection . in order to completely remove the cellular debris ,
the supernatant was separated and further processed by 15 min of high - speed centrifugation at 12,000 x g. the final plasma was then stored in rnase - free tubes ( axygen , union , ca ) at 80 c for further analysis .
total rna was extracted from specimens using a mirvana paris kit ( applied biosystems , usa ) according to manufacturer 's protocol . a nanodrop 1000 spectrophotometer ( nanodrop technologies inc .
, wilmington , de , usa ) was used to determine the concentration of extracted rnas . taqman microrna reverse transcription kits ( applied biosystems , foster city , ca , usa )
a 5 l reaction system comprising 0.5 l of different primers , 0.063 l of 20 units/l rnase inhibitor , 0.33 l of 50 units/l multiscribe reverse transcriptase , 0.05 l of 100 mm dntps , 0.5 l of 10 reverse transcription buffer , the rna sample , and rnase - free water was incubated at 30 c for 10 min , followed by 30 min of incubation at 50 c , at 95 c for 5 min , and then held at 4 c . a bio - rad iq5 ( bio - rad laboratories inc . )
a 10 l qpcr reaction solution with 5 l of taqman 2 perfect master mix , 2 l of cdna solution , 0.25 l of specific primers , and 2.75 l of rnase - free water was used .
the qpcr primers were mir-125b : sense , 5-gcucccugagacccuaac-3 , and antisense , 5-cagtgcagggtccgaggt-3 ; u6 : sense , 5-ctcgcttcggcagcacatatact-3 and antisense , 5-acgcttcacgaatttgcgtgtc-3. the pcr amplification was performed as follows : an initial denaturation at 95
c was carried out for 2 min , followed by 40 cycles of 95 c for 15 s and 60 c for 1 min . the reaction was terminated by incubation at 95 c for 15 s , and the products held at 4 c .
the expression level of mir-125b was calculated using the ct method , and each reaction was repeated in triplicate to avoid bias .
medcalc for windows , version 13.0 ( medcalc software , ostend , belgium ) and spss version 16.0 ( spss , chicago , il , usa ) were used for statistical analyses .
the kolmogorov - smirnov test was applied to test the normality of mir-125b expression in both groups , and one - way analysis of variance testing , student 's t - test and spearman correlation analysis were flexibly used as appropriate .
receiver operating characteristic ( roc ) curve analysis was conducted to evaluate the efficacy of mir-125b in distinguishing chemotherapy resistance in the os group with unresectable lesions .
the association between mir-125b expression and survival was assessed by the log - rank test and cox proportional hazard regression analysis , and age , gender , tumor location , tumor stage , histologic grade , metastasis status , and mir-125b expression level were entered into the multivariate analysis .
the mean value of mir-125b expression level was set as the cut - off point to differentiate patients with high or low mir-125b expression in the resectable and unresectable groups .
the study protocol was approved by the medical ethics committee of the second clinical hospital of lanzhou university .
informed consent was obtained from all adult participants prior to the start of the study . for children under 18 years of age
patients with os who presented to our department between january 2010 and july 2015 were screened for enrollment .
exclusion criteria were previous malignant tumors in another organ or system ; hematological disorders ; end - stage patients not qualified for chemotherapy ; patients with no pathological data ; and any patients unwilling to participate .
patients were treated according to national comprehensive cancer network practice guidelines for os , and any treatment decisions were not affected by participation in the study .
patients with resectable os received cisplatin - based neoadjuvant and adjuvant chemotherapy and surgery , while cisplatin - based aggressive chemotherapy was given to os patients with unresectable lesions .
the demographic and clinical characteristics of the study participants were obtained from their medical records on admission , and follow - up of patients receiving surgery was performed by the combination of outpatient visits , letters , and telephone calls .
the patients had follow - up visits with physical examinations and radiography every 3 months , as well as computed tomography scans or magnetic resonance imaging when necessary .
disease - free survival was calculated from the date of surgery until an event for each patient . for patients with unresectable os tumors , the response to chemotherapy
was evaluated by the response evaluation criteria in solid tumors ( recist ) . in this study ,
chemotherapy sensitivity was defined as complete remission or partial remission , whereas stable disease and disease progression were taken as signs of chemotherapy resistance .
the patients were also followed for overall survival after therapy , which was calculated from the date of the beginning of chemotherapy in our department , until the date of death .
in each participant , a 10 ml peripheral venous blood was collected in edta anticoagulation tubes before any therapy was begun .
the blood samples were centrifuged at 3,000 rpm for 10 min within 20 min after collection . in order to completely remove the cellular debris ,
the supernatant was separated and further processed by 15 min of high - speed centrifugation at 12,000 x g. the final plasma was then stored in rnase - free tubes ( axygen , union , ca ) at 80 c for further analysis .
total rna was extracted from specimens using a mirvana paris kit ( applied biosystems , usa ) according to manufacturer 's protocol . a nanodrop 1000 spectrophotometer ( nanodrop technologies inc . , wilmington , de , usa )
was used to determine the concentration of extracted rnas . taqman microrna reverse transcription kits ( applied biosystems , foster city , ca , usa )
a 5 l reaction system comprising 0.5 l of different primers , 0.063 l of 20 units/l rnase inhibitor , 0.33 l of 50 units/l multiscribe reverse transcriptase , 0.05 l of 100 mm dntps , 0.5 l of 10 reverse transcription buffer , the rna sample , and rnase - free water was incubated at 30 c for 10 min , followed by 30 min of incubation at 50 c , at 95 c for 5 min , and then held at 4 c . a bio - rad iq5 ( bio - rad laboratories inc . )
thermocycler was applied for the qpcr reaction . a 10 l qpcr reaction solution with 5 l of taqman 2 perfect master mix , 2 l of cdna solution ,
0.25 l of specific primers , and 2.75 l of rnase - free water was used .
the qpcr primers were mir-125b : sense , 5-gcucccugagacccuaac-3 , and antisense , 5-cagtgcagggtccgaggt-3 ; u6 : sense , 5-ctcgcttcggcagcacatatact-3 and antisense , 5-acgcttcacgaatttgcgtgtc-3. the pcr amplification was performed as follows : an initial denaturation at 95 c was carried out for 2 min , followed by 40 cycles of 95 c for 15 s and 60 c for 1 min . the reaction was terminated by incubation at 95 c for 15 s , and the products held at 4 c .
the expression level of mir-125b was calculated using the ct method , and each reaction was repeated in triplicate to avoid bias .
medcalc for windows , version 13.0 ( medcalc software , ostend , belgium ) and spss version 16.0 ( spss , chicago , il , usa ) were used for statistical analyses .
the kolmogorov - smirnov test was applied to test the normality of mir-125b expression in both groups , and one - way analysis of variance testing , student 's t - test and spearman correlation analysis were flexibly used as appropriate .
receiver operating characteristic ( roc ) curve analysis was conducted to evaluate the efficacy of mir-125b in distinguishing chemotherapy resistance in the os group with unresectable lesions .
the association between mir-125b expression and survival was assessed by the log - rank test and cox proportional hazard regression analysis , and age , gender , tumor location , tumor stage , histologic grade , metastasis status , and mir-125b expression level were entered into the multivariate analysis .
the mean value of mir-125b expression level was set as the cut - off point to differentiate patients with high or low mir-125b expression in the resectable and unresectable groups .
from january 2010 to july 2015 , 176 patients who presented with os to our orthopedic department were screened . of these , 138 ( 78% ) were enrolled as participants in the study , 82 with resectable os and 56 with unresectable os .
the median follow - up time in the resectable os group is 23.110.9 months , while it was 13.96.6 months in the unresectable os group .
the mir-125b expression level in the resectable os group was normally distributed with a mean of 0.970.55 ( p>0.05 , table 2 ) , and no significant correlation was found between mir-125b expression and patients age , gender , tumor location , tumor grade , histologic grade , number of metastases , or location ( table 2 ) .
the samples with mir-125b expression less than 0.97 were assigned to the low - expression group ( n=28 ) , while samples with expression more than the mean value were taken as high mir-125b expression ( n=54 ) .
kaplan - meier analysis indicated that patients with low mir-125b expression suffered shorter disease - free survival ( p<0.01 , fig .
1(a ) ) , which was verified by the univariate and multivariate cox proportional hazard regression analysis ( p<0.001 , table 3 ) .
the multivariate cox proportional hazard regression analysis also showed that patients with low tumor stage ( t1 ) experienced longer disease - free survival than those with high tumor stage ( t2+t3 ) ( p=0.036 , table 3 ) , which was not found by univariate cox proportional hazard regression analysis ( p=0.447 , table 3 ) .
the mir-125b expression level in the unresectable os group was also normally distributed ( p>0.05 ) , and averaged 0.500.25 .
no significant correlation was found between mir-125b expression and patients age , gender , tumor location , histologic grade , number of metastasis , or metastatic location ( table 2 ) .
low mir-125b expression was found to be associated with advanced tumor stages ( p=0.006 ) .
no complete remission was observed , but there were 13 patients with partial remission , 21 with stable disease , and 22 with disease progression in the study .
there was a significant difference of mir-125b expression with different tumor responses to chemotherapy ( p<0.001 ) .
spearman correlation analysis showed a negative correlation between mir-125b expression and tumor response to chemotherapy ( p<0.001 , r=0.606 ) .
furthermore , roc analysis indicated that mir-125b at the cut - off point of 0.61 yielded an area under the roc curve of 0.793 ( p<0.001 , 95% confidence interval ( ci ) : 0.6640.890 ) in distinguishing chemotherapy - resistant os from chemotherapy - sensitive os , the sensitivity and specificity of which were 76.9% and 79.1% , respectively ( fig .
kaplan - meier analysis showed that the study participants with low mir-125b expression ( mir-125b<0.50 ) had shorter overall survival ( p=0.014 , fig .
1(b ) ) , which was consistent with the univariate and multivariate cox proportional hazard regression analysis ( p=0.024 and p=0.049 , respectively , table 4 ) .
from january 2010 to july 2015 , 176 patients who presented with os to our orthopedic department were screened . of these , 138 ( 78% ) were enrolled as participants in the study , 82 with resectable os and 56 with unresectable os .
the median follow - up time in the resectable os group is 23.110.9 months , while it was 13.96.6 months in the unresectable os group .
the mir-125b expression level in the resectable os group was normally distributed with a mean of 0.970.55 ( p>0.05 , table 2 ) , and no significant correlation was found between mir-125b expression and patients age , gender , tumor location , tumor grade , histologic grade , number of metastases , or location ( table 2 ) .
the samples with mir-125b expression less than 0.97 were assigned to the low - expression group ( n=28 ) , while samples with expression more than the mean value were taken as high mir-125b expression ( n=54 ) .
kaplan - meier analysis indicated that patients with low mir-125b expression suffered shorter disease - free survival ( p<0.01 , fig .
1(a ) ) , which was verified by the univariate and multivariate cox proportional hazard regression analysis ( p<0.001 , table 3 ) .
the multivariate cox proportional hazard regression analysis also showed that patients with low tumor stage ( t1 ) experienced longer disease - free survival than those with high tumor stage ( t2+t3 ) ( p=0.036 , table 3 ) , which was not found by univariate cox proportional hazard regression analysis ( p=0.447 , table 3 ) .
the mir-125b expression level in the unresectable os group was also normally distributed ( p>0.05 ) , and averaged 0.500.25 .
no significant correlation was found between mir-125b expression and patients age , gender , tumor location , histologic grade , number of metastasis , or metastatic location ( table 2 ) .
low mir-125b expression was found to be associated with advanced tumor stages ( p=0.006 ) .
no complete remission was observed , but there were 13 patients with partial remission , 21 with stable disease , and 22 with disease progression in the study .
there was a significant difference of mir-125b expression with different tumor responses to chemotherapy ( p<0.001 ) .
spearman correlation analysis showed a negative correlation between mir-125b expression and tumor response to chemotherapy ( p<0.001 , r=0.606 ) .
furthermore , roc analysis indicated that mir-125b at the cut - off point of 0.61 yielded an area under the roc curve of 0.793 ( p<0.001 , 95% confidence interval ( ci ) : 0.6640.890 ) in distinguishing chemotherapy - resistant os from chemotherapy - sensitive os , the sensitivity and specificity of which were 76.9% and 79.1% , respectively ( fig .
kaplan - meier analysis showed that the study participants with low mir-125b expression ( mir-125b<0.50 ) had shorter overall survival ( p=0.014 , fig .
1(b ) ) , which was consistent with the univariate and multivariate cox proportional hazard regression analysis ( p=0.024 and p=0.049 , respectively , table 4 ) .
accumulating knowledge of the molecular pathogenesis of malignant tumors has provided new insights for disease characterization and therapy ; these insights may be applied to selected patients for more rational treatment decisions and better clinical outcomes . in recent years , mirnas , classified as oncogenes or tumor suppressor genes , have been widely studied and have the potential to serve as classification criteria . in the present study
, we found that os patients with low mir-125b expression had shorter disease - free survival in the resectable os group and shorter overall survival in the unresectable os group .
additionally , we found a negative correlation between mir-125b expression and tumor response to chemotherapy .
mir-125b is a highly conserved mirna among different species , which includes the mir-99/mir100 and the let7 family members .
it has been reported that mir-125b is involved in control of cell proliferation , cell cycling , and inflammation , , .
the role of mir-125b in malignant tumors is controversial : it has been reported to be overexpressed in prostate cancer and hematological malignances and to act as oncogene , , and is also reported to be down - regulated in breast , ovarian , and thyroid carcinomas , as well as hepatocellular cancer , and is assumed to be a tumor suppressor . , , . in os , mir-125b was down - regulated in human osteosarcoma tissues compared with the adjacent tissues , .
down - regulation of mir-125b was reported to be associated with advanced tnm stage , metastasis , and higher tumor size , and it was said to be acting as a tumor suppressor . in the present study , a significant correlation was found only between mir-125b expression and tumor stage in the unresectable os group .
various factors may contribute to this difference , such as race , sample category , sample size , detection techniques , and experimental reagents .
one important factor may be that the prior study analyzed mir-125b expression in tissue , while we analyzed mir-125b in plasma .
the key issue in this study is the role of mir-125b in predicting os patients response to cisplatin - based chemotherapy . for the resectable os group ,
therefore , we applied disease - free survival to indirectly assess this , and found that os patients with low mir-125b expression suffered shorter disease - free survival .
we also found that patients with low mir-125b expression experienced shorter overall survival in the unresectable os group .
a previous study analyzing resected os tissue found that the overall survival time was significantly shorter in patients with low mir-125b expression compared with those with high mir-125b expression .
in the present study , recist was utilized to assess patients response to chemotherapy in the unresectable os group , and a negative correlation was found between mir-125b expression and the tumor response to cisplatin - based chemotherapy . in vitro , similar results were reported previously by wang et al . , who found that stable overexpression of mir-125b in os cell lines u2os and mg-63 inhibited cell proliferation , migration , and invasion , and mir-125b increased the sensitivity of os cell lines to cisplatin .
the mechanisms for development of chemotherapy resistance are complicated , including decreased intracellular drug accumulation , drug inactivation , enhanced dna repair , perturbations in signal transduction pathways , apoptosis , cell cycle - related gene expression turbulence , autophagy - related chemoresistance , and mirna dysregulation , .
furthermore , mirnas levels have been shown to be associated with the development of chemoresistance in os ; specifically , mir-34a , mir-92a , mir-99b , mir-132 , mir-193a-5p , mir-422a , mir-140 , mir-215 , mir-15a , and mir-161 have been identified , , , , .
wang et al . showed that bcl-2 was inversely associated with mir-125b in os tissues , and that mir-125b could bind to the 3 untranslated region of bcl-2 and therapy reduced its expression .
the combined treatment of mir-125b and cisplatin significantly induced cell apoptosis , while overexpressed bcl-2 partially reduced the effect induced by mir-125b plus cisplatin treatment .
additionally , they found that the activity of caspase 3 , a key factor in cell apoptosis , was significantly improved with treatment by mir-125b plus cisplatin compared with mir-125b or cisplatin treatment alone .
in contrast , up - regulation of bcl-2 attenuated the activation of caspase-3 induced by mir-125b plus cisplatin treatment .
these results showed that mir-125b improved os cells sensitivity to cisplatin treatment by targeting bcl-2 in os cells .
mir-125b could also promote apoptosis by diminishing the expression of mcl-1 , bcl - w , and il-6r .
in contrast , mir-125b could play an anti - apoptotic role through suppressing bcl-2 antagonist killer 1 ( bak1 ) .
moreover , it is reported that mir-125b could suppress the proliferation and migration of os cells through down - regulation of stat3 .
determining the exact mechanism involved in mir-125b regulation of os response to cisplatin - based chemotherapy will require further investigation .
although the results of the present study are encouraging , several limitations need to be acknowledged . first , the sample size of this study was relatively small , and confirming data from larger groups of patients are needed .
second , all participants were drawn from a limited geographic area from a single center , and so whether the results apply to the whole population is open to question .
fourth , the conclusions of the study are derived from the clinical data ; evidence from laboratory studies and the mechanism by which mir-125b affects os response to cisplatin - based chemotherapy are lacking .
this study presents evidence that mir-125b levels have the potential to be a valid biomarker to predict the effect of cisplatin - based chemotherapy and prognosis in os .
the limitation of the study is that it was derived from a retrospective analysis , and the positive and negative predictive values of mir-125b testing need to be evaluated prospectively with predefined cut - off levels and in a variety of clinical settings and centers before we can be certain of its efficacy . | backgroundit is known that mirnas play various roles in malignant tumors .
this study is designed to investigate whether mir-125b levels can be used to predict the clinical response of patients with osteosarcoma ( os ) to cisplatin - based chemotherapy.methodsfrom january 2010 to july 2015 , 82 patients with resectable os and 56 patients with unresectable os were enrolled .
blood samples were collected and quantitative real - time pcr was applied to determine mir-125b expression .
clinical data was collected through medical records , and patients were treated according to national comprehensive cancer network guidelines on os.resultsour study found that patients with low mir-125b expression had shorter disease - free survival ( p<0.001 ) in the os group , which was verified by kaplan - meier analysis and univariate and multivariate cox analyses ( p<0.001 ) . for patients with unresectable os ,
low mir-125b expression was found to be associated with advanced tumor stages ( p=0.006 ) .
no complete remission was observed , and there were 13 patients with partial remission , 21 with stable disease , and 22 with disease progression .
negative correlation was found between mir-125b expression and response to chemotherapy ( p<0.001 , r=0.606 ) .
furthermore , roc analysis indicated that mir-125b at the cut point of 0.61 yielded an area under the roc curve of 0.793 ( p<0.001 , 95% ci : 0.6640.890 ) in distinguishing chemotherapy - resistant os from chemotherapy - sensitive os , with sensitivity and specificity at 76.9% and 79.1% , respectively .
kaplan - meier analysis and univariate and multivariate cox analyses showed that patients with low mir-125b expression suffered shorter overall survival ( p=0.014 , p=0.024 , and p=0.049 , respectively).conclusiondown - regulation of circulating mir-125b might have the potential to predict cisplatin - based chemotherapy resistance and poor prognosis in os . | Introduction
Patients and methods
Ethical considerations
Patients and samples
Sample collection, RNA isolation and quantitative real-time PCR (qRT-PCR) analysis
Statistical analysis
Results
Patients characteristics
miR-125b expression profile in the resectable OS group
miR-125b expression profile in the unresectable OS group
Discussion
Conclusion |
a 36-year - old , previously healthy , asian woman ( gravida 2 , para 1 ) at 19 weeks gestation with a low - lying placenta traveled in late february from the united states to hong kong with her husband and child . before departing from the united states ,
the patient had been complaining of a mild , intermittent cough without fever for approximately 10 days .
the cough , similar to one she had during her previous pregnancy , did not impair her ability to function . while in hong kong , between february 19 and march 2 , 2003 , she stayed at the same hotel and on the same floor as a physician from southern china , who is believed to have been the source of infection for patients who were the index case - patients in subsequent outbreaks of sars in hong kong , singapore , hanoi , and toronto , canada ( 11 ) . on february 24 , fever , headache , weakness , anorexia , increasing cough , and shortness of breath developed in the patient . the next morning , she sought medical attention and was prescribed chlorpheniramine and acetaminophen .
her symptoms worsened , prompting her to see another physician 2 days later . a fetal ultrasound performed at this time was reportedly normal .
cephalexin was added to her regimen , but her condition did not improve ; that night , she noted blood - tinged sputum . on march 2 , the patient returned to the united states where , acutely short of breath , she was hospitalized with pneumonia .
although chest auscultation was normal , chest radiography showed diffuse bilateral lower lobe infiltrates ( figure , part a ) .
admission arterial blood gas analysis showed ph 7.47 , paco2 31 mm hg , and pao2 75 mm hg on room air .
other pertinent laboratory findings included a leukocyte count of 3,300/mm ( normal range 4,50011,500/mm ) with a differential of 83% polymorphonuclear cells , 12% lymphocytes , and 5% monocytes ; platelets of 103,000/mm ( normal range 150,000450,000/mm ) ; and alanine aminotransferase of 42 u / l ( normal range 1040 u / l ) .
she was given supplemental oxygen for hypoxia and intravenous azithromycin and ampicillin to treat typical and atypical respiratory pathogens associated with community - acquired pneumonia .
a fetal ultrasound performed on march 3 demonstrated a live intrauterine fetus of approximately 21 weeks gestational age and complete placenta previa . despite antibiotic therapy , over the next 3 days ,
the patient became increasingly dyspneic ; rales and decreased breath sounds developed , and she had radiographic evidence of progressive pulmonary infiltrates ( figure , part b ) . during this time , ticarcillin - clavulanate was added to her antimicrobial regimen , and rifampin was initiated as adjuvant therapy for possible legionellosis . because the patient s diagnosis remained elusive ,
tuberculosis was considered , and she was placed in airborne isolation . arterial blood gas analysis on march 5 showed : ph 7.48 , paco2 31 mm hg , and pao2 57 mm hg on a 100% nonrebreather mask .
the patient was subsequently placed on a mechanical ventilator . when avian ( h5n1 ) influenza was considered in the differential diagnosis
chest auscultation demonstrated few bibasilar rales , and a chest radiograph showed interval improvement ( figure , part c ) .
, she had a fetal ultrasound that showed fetal growth consistent with dates and complete placenta previa . on march 17
sputum , blood , and urine cultures ; smears for acid - fast bacilli ; and tests for legionella urinary antigen , influenza nasopharyngeal antigen , and cold agglutinins were negative .
serum specimens collected 12 and 29 days after illness onset were tested at the centers for disease control and prevention ( cdc ) and found to be positive for sars - cov antibody .
chest radiographs of case - patient with severe acute respiratory syndrome ( sars ) while pregnant .
a , day 6 of illness ; b , day 10 ; c , day 13 .
a follow - up ultrasound examination on april 29 during routine prenatal care showed fetal growth consistent with dates and persistent complete previa . on may 2 ( approximately 30 weeks gestation ) , the patient was diagnosed with gestational diabetes after an abnormal oral glucose tolerance test .
because serial ultrasounds performed on may 28 and june 24 demonstrated complete placenta previa , she underwent a cesarean section at 38 weeks gestation .
after informed consent was obtained , the following specimens ( collected approximately 130 days after illness onset ) were submitted to cdc for coronavirus testing : serum , whole blood , nasopharyngeal and rectal swab specimens from the mother , postdelivery placenta , cord blood , amniotic fluid , and breast milk .
antibodies to sars - cov were detected in maternal serum , cord blood , and breast milk by enzyme immunoassay and indirect immunofluorescence assay ( table ) .
sars , severe acute respiratory syndrome ; rt - pcr , reverse transcriptase polymerase chain reaction ; nd , not done .
all specimens listed were collected 127 days after onset of the case - patient s illness with the exception of breast milk , which was collected 131 days after illness onset .
postdelivery placenta also underwent immunohistochemical ( ihc ) staining ; there was no ihc evidence of sars - cov .
on the basis of previous reports from hong kong , sars infection can be associated with critical maternal illness , spontaneous abortion , or maternal death ( 9,12 ) .
we have described a serious sars - associated illness that necessitated mechanical ventilation in a pregnant case - patient .
her pregnancy was also complicated by placenta previa and gestational diabetes two conditions that she was at increased risk of developing because of advanced maternal age ( 13 ) .
however , at the time of delivery , clinical specimens from the infant were not available for sars - cov testing .
all healthcare workers involved in the delivery and subsequent care of the infant have remained healthy .
however , serologic testing for sars - cov infection was not performed on these persons .
the infant was delivered by cesarean section with contact , droplet , and airborne precautions in place ( i.e. , staff wore fit - tested n95 respirators and the cesarean section took place in a negative - pressure operating room ) .
since sars - cov was not detected in specimens collected at delivery and the patient delivered months after her illness onset , it is not clear if such precautions were necessary .
however , other patients have demonstrated viral shedding in feces ( 14,15 ) and peritoneal fluid ( 16 ) , suggesting that sars - cov may be present in other body fluids and hence , transmission during vaginal and cesarean deliveries is plausible .
the presence , in this case , of sars - cov antibodies in cord blood and breast milk raises the issue of whether sars - cov infection during pregnancy results in passive immunity .
serial serologic testing of newborn clinical specimens and breast milk may provide a better understanding of the natural history of the fetal and newborn immune response to sars - cov infection during pregnancy .
this report , in conjunction with the reports from hong kong ( 9,12 ) , provides an initial view of the spectrum of illness and outcomes associated with pregnancy - related sars - cov infection . a variety of factors might contribute to this range of outcomes ( e.g. , timing of sars - cov exposure during pregnancy ; use of steroids , ribavirin , or both ; differences in host immune response ; the presence of coexisting conditions ) .
more comprehensive epidemiologic and clinical summaries about the course of other sars - affected pregnancies and long - term follow - up of infants are needed to fully define the pregnancy - related risks of this infection .
data on larger numbers of pregnant women infected with sars - cov may help refine infection - control strategies and provide a sound basis for clinical guidelines to manage future cases . | we report a laboratory - confirmed case of severe acute respiratory syndrome ( sars ) in a pregnant woman .
although the patient had respiratory failure , a healthy infant was subsequently delivered , and the mother is now well .
there was no evidence of viral shedding at delivery .
antibodies to sars virus were detected in cord blood and breast milk . | Case Report
Conclusions |
renewable chemical fuels can be synthesized through solardriven electro , photo , and thermochemical splitting of co2 and h2o.1 the latter approach utilizes the entire spectrum of concentrated solar radiation as hightemperature process heat for the production of co and h2 ( syngas ) via metal oxide redox cycles.1b , 2 a critical drawback of this approach is the inert gas consumed to lower the partial pressure of oxygen ( po2 ) for shifting the thermodynamic equilibrium of the reduction step to lower temperatures.3 this , in turn , requires separation of o2 from the product gases for recycling the inert carrier gas and closing the material cycle.3a , 4 the separation of o2 has been a requirement in a variety of commercial applications such as oxycombustion , autothermal gasification of carbonaceous feedstock , and o2 removal to avoid catalyst passivation by o2 in fuel cells and when deoxygenating biofuels to make these more akin to petroleumderived fuels.5 industrially , o2 can be separated from air by pressure swing adsorption ( psa ) using zeolites and carbon molecular sieves , by ceramic mixed ionicelectronic conducting ( miec ) membranes,6 and by cryogenic distillation .
psa and miec membranes can not produce highpurity inert gas,6b and separating o2 from gas mixtures at low po2 using membranes relies on a stripping gas with even lower po2 .
these separation technologies further require an input of electrical work ranging from 100 to 350 kwh per metric ton o2,6 , 7 which penalizes the solartofuel energy conversion efficiencies .
since solar thermochemical cycles inherently suffer from heat losses , it would be beneficial to utilize an oxygen separation technology driven by waste heat .
thermochemical solidstate o2 separation ( tssos ) using metal oxide redox materials such as cu2o / cuo,3a , 8 mn3o4/mn2o3,8 and coo / co3o4
8 , 9 utilizes lowgrade process heat and does not require electricity .
tssos has the potential to separate and concentrate o2 at low po2 via temperatureswing.3a the current stateoftheart tssos redox material , cu2o , has a maximum oxygen exchange capacity ( , i.e. , the difference in the oxygen nonstoichiometry between reducing and oxidizing conditions ) of about 200 mmol o2 per mol cu2o , exchanged at approximately 10 mol o 2 min g cu 2o
when cycled between 11201450 k.3a we show below that cu2o can not be employed with lowgrade process heat at 600900 k. with the aim of augmenting the o2 exchange capacities and rates of tssos redox materials for a more energyefficient o2 separation process that utilizes lowgrade solar thermal energy at lower temperatures , such as waste heat from solar fuel production processes , we evaluate perovskites that offer high o2 conductivities and a stable crystal structure over a large range of oxygen nonstoichiometry.6a , c , 10 the o2 exchange capacity characterizes the tradeoff between high energy conversion efficiencies at low temperature during the endothermic reduction and high rates and extend of the oxygen separation process at high oxide reduction temperatures . for a perovskite with abo3 stoichiometry where a and b are metal cations in twelve and sixcoordinated interstices the tssos redox cycle can be represented by equations ( 1 ) and ( 2):(2 )
( 1)equation image
( 2)equation image
conceptually , o2 is stripped from a gas mixture at low po2 through oxidation of the perovskite at low temperatures .
this yields as an output of the oxidation step an inert gas with a lowered po2 while concentrated o2 is evolved from the solid at an elevated temperature and increased po2 through partial reduction of the metal oxide . in principle , these reactions are analogous to the electrochemical oxygen reduction and evolution reactions ( orr / oer ) , where the bonding of o / oh or oh / ooh reaction intermediates to the catalyst surface controls the catalytic activity of the electrode surface.10 , 11 the ideal catalytic activity of a surface is determined by an intermediately strong bonding of the key reaction intermediates , which facilitates coverage of the surface with reactants and desorption of products from the surface , as described by the sabatier principle.12 analogously , we hypothesize that metal oxide redox materials for removal of o2 from gas mixtures with a lower po2 than the po2 during the extraction of o2 from the solid can be characterized with an intermediately strong binding of the lattice oxygen .
to test this hypothesis , we screened the redox energetics of binary metal oxides across the periodic table using experimentbased thermochemical data.13 figure 1 a shows the free energy ( g ) of the oxidation and reduction reactions for 32 solid metal oxide and six metal / metal oxide pairs14 versus the thermochemical oxide stability .
a ) free energy of the oxide oxidation at 600 k and oxide reduction at 900 k ( g
rxn ) versus the enthalpy of the oxide reduction at 298 k ( h
red ) .
b ) the limiting free energy of the redox cycle ( g
rxn , lim ) versus h
red .
the colored compositions were examined experimentally , with blue and red marked materials limited by the oxide reduction and oxidation , respectively , and purple marking materials that facilitate a redox tradeoff .
c ) dftmodels of the oxidized and reduced srcoo3 and srcoo2.5 surface , representatively for strontium cobaltite .
the analysis utilizes the enthalpy of the oxide reduction at room temperature as a descriptor1012 of the correlated reaction energetics , which is equivalent to the amount of energy required to break metal
generally , either one of the two reactions is slightly more endergonic , thereby limiting the o2 exchange capacity .
figure 1 b shows the limiting free energy of a redox cycle near the intersection of both correlations . as indicated by the volcanoshaped curve , the ideal redox material binds oxygen strongly enough to oxidize the oxide at relatively low temperatures
stronger than the ag / ag2o reference but weakly enough to reduce the oxidized redox material at moderately higher temperatures
the ideal metal oxide compositions are where these effects balance , located near the top of the volcano curve
. ideally , this region corresponds to negative free energies for both reactions . for the temperatures chosen in our analysis , this can be achieved with rare materials such as rh2o / rho or toxic materials such as pbo / pb3o4.15 generally , the volcanolike shape of this correlation is due to the fact that the amount of energy absorbed for breaking metal oxygen bonds during the reduction step correlates with the amount of heat liberated when forming these bonds during the oxidation step .
thus , as shown in figure s1 , the location of the volcanotop can be determined from only computing the reduction enthalpy as the entropy of o2 gas participating in either reaction is the same for all specific redox couples and as entropic contributions of the solids introduce significant deviation from these correlations only at significantly higher temperatures when approaching melting and boiling points .
to tailor inexpensive and nontoxic metal oxides , we calculated the free energy of oxygen vacancy formation ( g
v[o ] ) using density functional theory ( dft ) for twelve perovskites that have attracted attention for solidoxide fuel cells,6a , 10 air separation,6c and solarthermal applications.16 stoichiometric abo3(010 ) and oxygendeficient abo2.5(010 ) facets ( a = sr , ba , or la ; and b = mn , co , ni , or cu ) were modeled ( figure 1 c ) , using the gridbased projectoraugmented wave ( gpaw ) and atomic simulation environment ( ase ) electronicstructure code.17 figure 1 b shows the thermochemical stability and the reaction energetics as calculated from the scaling of g
v[o ] and the redox energetics of the bulk oxides ( see the supporting information).18 the analysis predicts an ideal o2 exchange capacity for srcoo3 , relative to too strong and too weak oxygen binding for bamno3 and bacoo3 , respectively .
we validated this descriptorbased design for metal oxide redox materials by means of dynamic o2 exchange experiments using srcoo3 , bamno3 , and bacoo3 , synthesized via the pecchini method3b and commercial ag2o and cu2o as reference materials .
the composition and surface morphology of all solids were characterized using hightemperature xray diffraction ( htxrd ) and scanning electron microscopy ( sem ) .
the o2 exchange capacity and exchange rates were determined by thermogravimetric analysis ( tga ) .
figure 2 a and b display dynamic tga runs for srcoo2.95 , bacoo2.58 , bamno2.94 , ag2o , and cu2o ( initial stoichiometries ) that were cyclically reduced at 900 k and 0.2 bar o2 ( simulating air ) and oxidized at 600 k and 0.035 bar o2 ( simulating the composition of the gas phase from reducing ceria for solardriven splitting of co2 and h2o19 ) . as expected , cu2o and ag2o oxidize and reduce strongly to cuo and ag , respectively , essentially without exchanging o2 reversibly .
compared to these reference materials , we find augmented o2 exchange capacities for the perovskites .
srcoo2.95 reaches a maximum o2 exchange capacity of 440.012 mmol o2 per mol srcoo2.95 and a maximum o2 exchange rate of 12.10.003 mol o 2 min g srcoo 2.95
whereas bacoo2.58 and bamno2.94 perform at much lower capacities of 3.40.015 and 0.50.015 mmol o2 per mol of perovskite and lower exchange rates of 0.80.003 and 0.040.005 mol o 2 min gperovskite
, respectively .
as predicted by dft , the performance of bacoo3 and bamno3 appears limited by reoxidation and reduction , respectively .
this theorybased screening of twelve perovskites identifies a wellknown composition , srcoo3 , that shows a significantly augmented performance for this novel application compared to the o2 exchange capacities and rates of the reference materials at the same conditions .
additionally , with an o2 exchange rate of 12.1 mol o 2 min gperovskite
, srcoo3 outperforms the stateoftheart cu2o / cuo cycle , which can not be used with lowgrade process heat at 600900 k and which has an o2 exchange rate of only 10 mol o 2 min g cu 2o
( for both , oxide reduction and oxidation ) at significantly higher and thereby economically less attractive temperatures of 11201450 k and comparable po2.3a
dynamic o2 exchange : tga runs of a ) ag2o and cu2o and b ) srcoo3 , bacoo3 , and bamno3. c ) lattice constants of srcoo3 , bacoo3 , and bamno3 derived from htxrd analyses of oxide reduction at 0.2 bar po2 ( empty circles ) and oxide oxidation at 0.035 bar po2 ( filled circles ) .
error bars are standard deviations within a 68 % confidence interval . to support that the tga data is indicative of reversible o2 exchange , figure 2 c shows the perovskite lattice constants computed using data from htxrd analysis .
although all perovskites exhibit thermal expansion upon heating , only srcoo3 shows a major difference in the lattice expansion at varied po2 , which can be attributed to the formation and filling of o vacancies.20 this , along with the electronic structure trends and the tga analysis , suggests that srcoo3 is particularly suitable as a redox material for tssos . to understand how the oxide composition controls the o2 exchange , figure 3 a plots g
v[o ] versus the dftcalculated bond length between the transition metal and the nearest o atom ( d
b o ) at abo2.5(010 ) .
generally , we observe stable o vacancies ( negative g
v[o ] values ) correlating with large d
b o values .
this is in agreement with the lattice expansion shown in figure 2 c , as the lattice expansion due to a higher chemical potential for oxygen in the gas phase decreases the bonding of oxygen in the solid , which , in turn , increases the length of the metal oxygen bond . although the slope of this correlation is essentially due to the metal at the bsite interstices , the absolute value of d
b o is governed by the metal at the a site , as demonstrated by the consistently higher d
b o values of the ba versus the sr compounds
however , the scatter of the correlation shown in figure 3 a suggests that the trends in the free energy of the o vacancy formation and in the metal oxygen bond length can not alone be rationalized with geometric arguments .
correlation of
g
v[o ] with a ) d
b o and b ) q
o ( empty , lightgray , and darkgray symbols mark srbo2.5 , labo2.5 , and babo2.5 , whereas circles , squares , diamonds , and triangles mark acuo2.5 , anio2.5 , amno2.5 , and acoo2.5 ) .
c ) charge density differences ( cdd ) of the marked surface after o vacancy formation relative to the stoichiometric surface and the reference gasphase o2 ( given at the height of the transition metal cation ) .
figure 3 b plots g
v[o ] versus the dftcalculated partial charge21 of oxygen ( q
o ) .
bacoo3 , for instance , accumulates less charge at the o anion than srcoo3 and bamno3 , which correlates with the facile reduction of bacoo3 .
generally , g
v[o ] scales with q
o , with the slope corresponding approximately to the ionization energy of oxygen , 13.62 ev per electron22 and the intercept reflecting entropic contributions to g
v[o ] and the reference chemical potential of oxygen in the gas phase ( see the supporting information ) .
the quality of this linear correlation suggests that the trend in the free energy of the o vacancy formation is controlled by the enthalpy of breaking metal
oxygen bonds , that is , by the quantity of electric charge transferred from the o atom to the lattice when forming the o vacancy .
this is illustrated with figure 3 c , which shows the difference in the charge density distribution due to o vacancy formation at srcoo3(010 ) and bamno3(010 ) .
although the partial charge of the o anion yielding the vacancy is approximately equal at both surfaces ( figure 3 b ) , the charge transfer from the bonding o 2p states to the 3d states of the transition metal is significantly higher at bamno3(010 ) relative to srcoo3 ( 010 ) .
we note that this analysis describes the width of the 2p3d gap for 3d transition metals at the b site of abo3 perovskites .
incorporating other metal cations into the b interstices , such as lanthanides with f states , may alter these trends due to a different entropycontrolled shape of the states that are accepting electrons when forming o vacancies . in summary ,
analogous to the enthalpy difference of bulk oxide reduction , we suggest that the charge transfer from o 2p to bsitemetal 3d states explains the higher o2 exchange capacity of srcoo3 versus bamno3 at the atomic scale . in accord with charge transfer controlling the formation of o vacancies at the oxide surface , our sabatier analysis employs the enthalpy of the bulk oxide reduction as a descriptor of the redox energetics . to demonstrate how this information can be used practically to predict the o2 exchange capacity of a metal oxide
, we have determined the o2 exchange capacity for five metal oxides as the difference of the oxygen nonstoichiometry at equilibrium between o2 evolution at 900 k and 0.2 bar po2 and o2 fixation at 600 k and 0.035 bar po2 ( figure 4 ) .
the plot shows that the o2 exchange capacity resembles the volcanoshaped trend of the limiting redox energetics .
relative to this trend across three orders of magnitude are minor deviations , such as for bacoo3 , which are presumably due to differences in surface morphology , crystal structure , and the computational versus experimental nonstoichiometry ( see the supporting information ) .
this demonstrates how computing the enthalpy of the oxide reduction from first principles allows predicting the o2 exchange capacity of metal oxides .
predicting the o2 exchange capacity and the limiting free energy of solidstate o2 separation from the enthalpy of the oxide reduction at 298 k and 1 bar .
although the present article focuses on the thermodynamics of o vacancy formation , the kinetics of conducting these vacancies from and to the surface are of equal importance when designing metal oxides for solidstate o2 separation .
the defect chemistry of several perovskite families , including la2nio4+ with a relatively high mobility of o interstitials , has been investigated previously.23 in these perovskites excess oxygen is incorporated as interstitial o or o anions with anion frenkel pairs being the predominant intrinsic lattice defects.23 oxygen transport may be anisotropic23 or isotropic , such as in sr0.75y0.25coo2.625.24 a low frenkel energy may yield high o vacancy concentrations , in prbaco2o5.5 for instance,25 with an ordered sublattice of the a cations ensuring high oxygen ion mobility.25 similarly , dft was used previously to predict and understand oxygen conduction trends in metal oxides.18 these and other studies23 , 26 outline the prospects of an advanced understanding of oxygen conduction for designing advanced redox materials .
based on the sabatier principle applied to the bonding of lattice oxygen atoms , we implemented a descriptorbased design principle for predicting the o2 exchange capacity of metal oxides and perovskites in particular .
the computations were validated through dynamic o2 exchange experiments using ag2o , cu2o , and three perovskites and rationalized based on the compositiondependent bond geometry and charge transfer during the formation of o vacancies at the metal oxide surface .
srcoo3 was identified as an ideal material for solardriven thermochemical separation of o2 . in a broader context
, the presented principles may also aid the design of oxygen conductors for related applications , such as solidoxide fuel cells and air separation using dense ceramic membranes .
thermochemical equilibrium calculations to guide the design of redox materials , the thermochemical equilibrium of binary bulk metal oxides , o2 , and their reduction products was determined at the specified po2 and temperatures from tabulated freeenergy data , with an absolute accuracy of 110 kj mol.13 per convention , negative free energy differences mark exergonic reactions . at the computed conditions , the correlations of g and h
red for the metal oxide oxidation and reduction have average relative errors of 12.4 % and 17.6 % , respectively .
these values increase with increasing temperature due to entropic contributions and indicate that the provided volcano plot could be employed for identifying materials active for thermochemical solidstate o2 separation .
electronic structure calculations
twelve perovskite surfaces were modeled using dft , performed with the gpaw code.17b , c exchangecorrelation interactions were treated by the revised perdew burke
ernzerhof ( rpbe ) functional.27 atomic configurations were handled in ase.17a a fermi dirac smearing of 0.1 ev was used to achieve convergence , and the structure optimization results were extrapolated to 0 k. the linesearch broyden fletcher
shanno ( bfgs ) algorithm was employed to optimize the atomic geometries until the maximum force was less than 0.05 ev .
the utility of dft+u methods for surface calculations is not determined in general and was found unnecessary for many metal oxides.18 , 28 here , for all dft calculations the generalized gradient approximation ( gga ) was used without a hubbard u term as we found previously that the hubbard u correction did not improve the description of surface reactivity with the employed models.18 the cubic bulk structures of abo3 compositions consisted of one metal atom ( sr , ba , or la ) at the twelvecoordinated asite interstices , one metal atom ( mn , co , ni or cu ) at the sixcoordinated bsite interstices , and three oxygen atoms that were allowed to optimize their positions ( relax ) .
the bulk structures had periodic boundary conditions in all directions and were modeled using a kpoint sampling of 444 .
compositions containing mn , co , or ni were modeled using spinpolarized calculations and , to avoid reminiscent stress in the calculations , the lattice constants were chosen as the dftcalculated bulk lattice constants .
table s1 provides a summary of the lattice constants and magnetic moments along with a discussion of the accuracy of the employed dft methods that predict the lattice constants within 0.363.89 % of experimental values .
the aoterminated abo3(010 ) facet was chosen for modeling the perovskite surfaces as this facet was identified as being the thermodynamically most stable surface of cubic perovskites for various compositions.18 the surface models consisted of one upper abo3(010 ) layer that was allowed to relax and one lower abo3(010 ) layer constrained to the bulk geometry .
the surfaces were periodically repeated in the directions parallel to the surface and were modeled with 10 of vacuum perpendicular to the surface .
the partial charge density was determined for all atoms contained in the surface models through bader decomposition.21 to model the perovskite surfaces at different o vacancy concentrations , one third of the stoichiometric lattice oxygen in the upper surface layer was removed while the oxygen concentration in the lower surface layer was maintained .
reduced and oxidized surface models with a2b2o5(010 ) and a2b2o6(010 ) stoichiometry , marked with the conventional abo3(010 ) and abo2.5(010 ) notation , respectively
. the free energy of forming o vacancies ( g
v[o ] ) at the surface was computed as follows [ eq .
( 3)]:18 , ( 3 )
( 3)equation image
where g
v , g
s , and go
are the free energies of the perovskite surface with the o vacancies , the stoichiometric surface and the reference energy of the liberated lattice oxygen [ taken as the energy difference of stable h2o and h2 in the gas phase , see eq .
( 3 ) in the supporting information ] , such that negative free energies indicate exergonic reactions .
the formation of o vacancies as computed corresponded to formation of one monolayer o vacancies equivalent to an oxygen nonstoichiometry of =0.5 in abo2.5 ( figure 1 c ) .
details on converting the dftcomputed electronic energy to gibbs free energy at 298.15 k and 1.013 bar , the reference energies , and scaling relations18 to estimate the bulk formation energies are provided in the supporting information .
the error of dftcomputed adsorption energies ( employed as a descriptor of surface reactivity , comparable to the energy of forming surface o vacancies in this work ) was estimated previously to be 0.08 ev.29
perovskite synthesis
three perovskites , namely srcoo3 , bacoo3 , and bamno3 , were synthesized using a modified pecchini method , employing stoichiometric amounts of mn(no3)24 h2o ( alfa aesar , 98 % ) , sr(no3)2 ( alfa aesar , 98 % ) , co(no3)2 ( alfa aesar , 97.7 % ) , ba(no3)2 ( alfa aesar , 99 % ) , c2h6o ( alcosuisse , 96.1 % ) , and c6h8o7 ( fluka , 99.5 % ) .
the solid products were ground using mortar and pestle , uniaxially pressed into pellets ( 10 metric tons , 6 and 25 mm in diameter ) , and sintered in air at 1473 k for 5 h ( srcoo3 and bacoo3 ) or in pure o2 at 1273 k for 48 h ( bamno3 ) . to achieve the desired oxidation state ,
the sintered srcoo3 was ground , fully immersed in naclo ( migros , < 5 % in h2o ) , washed with deionized water , and subsequently dried for at least 2 h at 473 k. ag2o ( merck , 99 % ) and cu2o ( johnson matthey alfa , 99.5 % ) were used as reference materials .
solidstate analysis xrd and htxrd were performed in the bragg brentano geometry using cuk radiation ( 2080 2 , 0.06 min scan rate , 45 kv/20 ma output , panalytical / xpert mpd / dy636 , philips ) .
the original oxygen content of the perovskites was srcoo2.95 , bacoo2.58 , and bamno2.94 before the redox cycling , as determined using tga ( sta 409/c/3 , netzsch ) of the complete reduction of the metal oxides in 5 % h2 in ar at 823 k and 1 bar . to estimate changes in the lattice constants ,
the perovskites were reduced by heating from 600 to 900 k in 100 k steps at 0.2 bar po2 and thereafter oxidized by cooling from 900 to 600 k in 100 k intervals at 0.035 bar po2 .
the morphology of all materials was analyzed using sem ( 15 kv accelerating voltage , tm1000 , hitachi ) and is shown in detail in the supporting information .
thermochemical cycling experiments starting materials ( 0.1 g ) were placed in an al2o3 crucible supported with an al2o3 rod on the microbalance of the tga ( 0.1 g ) .
the materials were thereafter exposed to a gas flow ( constant flow rate of 200 ml min at 273 k and 1 bar ) with specified po2 , which was adjusted by mixing o2 ( 99.5 % , messer ) and n2 ( 99.999 % , carbagas ) using three electronic mass flow controllers ( mfc400 , netzsch ; accuracy 1 % , precision 1 ml min ) .
the mass change of the samples was recorded during two consecutive redox cycles with oxide reduction at 900 k and 20 vol % o2 and oxide oxidation at 600 k and 3.5 vol % o2 , respectively ( 1 k ) .
heating and cooling was performed at + 10 k min and 10 k min , respectively . to correct for buoyancy
, blank runs were performed using the same measurement conditions employed for the experimental runs . the oxygen exchange capacity ( dimensionless )
was defined as the difference in the oxygen nonstoichiometry of the metal oxides after the oxide reduction (
red ) and after the oxide oxidation (
oxi):(4 )
( 4)equation image
where m
red and m
oxi are the metal oxide mass ( in g , determined using tga ) , after the oxide reduction , and after the oxide oxidation , and mo is the molar mass of oxygen ( in g mol).the uncertainties of the oxygen exchange capacities and rates were estimated using error propagation from the accuracies of the experimental analysis . | abstractseparation and concentration of o2 from gas mixtures is central to several sustainable energy technologies , such as solardriven synthesis of liquid hydrocarbon fuels from co2 , h2o , and concentrated sunlight .
we introduce a rationale for designing metal oxide redox materials for oxygen separation through thermochemical pumping of o2 against a po2 gradient with lowgrade process heat .
electronic structure calculations show that the activity of o vacancies in metal oxides pinpoints the ideal oxygen exchange capacity of perovskites .
thermogravimetric analysis and hightemperature xray diffraction for srcoo3 , bacoo3 and bamno3 perovskites and ag2o and cu2o references confirm the predicted performance of srcoo3 , which surpasses the performance of stateoftheart cu2o at these conditions with an oxygen exchange capacity of 44 mmol o 2 mol srcoo 3
1 exchanged at 12.1 mol o 2 min1 g1 at 600900 k. the redox trends are understood due to lattice expansion and electronic charge transfer . | Introduction
Results and Discussion
Conclusions
Experimental Section |
his vision , leadership , and hard work established the procedure of using the liver as a site for successful islet transplantation .
the transplants normalized glycemia in rats previously made diabetic by streptozotocin injection ( 2 ) .
as he and his colleagues began publishing manuscripts , it did not take long for the surgical community to apply this technique to humans ( fig .
this proved to be a much more ambitious task than first imagined . transporting pancreata isolated from brain - dead donors on life support , the inherent delays in islet isolation , the presence of autoimmune disease in the recipients , and the need to use powerful immunosuppressive drugs with significant side effects all presented significant barriers .
islets are removed from a donated pancreas by collagenase digestion , followed by purification to separate the islets from exocrine tissue .
islets are then infused by gravity into a catheter lodged in the hepatic portal vein .
blood flow within the vein carries the islets into the liver tributaries where they lodge within the sinusoids and establish vascular connections .
najarian and sutherland at the university of minnesota transplanted islets in nondiabetic recipients who were their own donors and reported reproducible successes beginning in 1980 ( 4 ) .
the patients had various forms of chronic , unrelentingly painful pancreatitis and each underwent a total pancreatectomy for pain relief and nutritional rehabilitation .
rather than dispose of the patient 's resected pancreas , the clinical researchers used it to make a crude extract of islets that was returned to the operating room within 2 h. the islets were infused over 30 min into the patient 's liver while hepatic portal venous pressure was monitored .
a summary of the results reported that if over 300,000 autoislets could be transplanted , the success rate of preventing diabetes for more than 2 years was 74% ( 5 ) .
the autoislet experience was very encouraging because it proved that islet transplantation in nondiabetic humans was feasible .
the stage for future successes in alloislet transplantation in patients with type 1 diabetes was set .
case reports and small patient series ' revealed evidence for function of islets after transplantation and brief or partial improvement in glycemic control ( 611 ) .
one vexing variable was the irony that one of the drugs important generally for transplantation success , cyclosporine , had inhibitory effects on -cell function ( 1219 ) .
oddly , this became the pattern for other important immunosuppressive drugs ( 3 ) , especially glucocorticoids ( 14,18,2022 ) .
this led to the use of a glucocorticoid - free immunosuppressive regimen developed by shapiro et al .
( 1 ) , which in turn led to their now historic series of successful islet transplants in type 1 diabetic patients .
another key aspect of the edmonton approach was to use an average of two sequential islet transplants to establish normoglycemia . over time , however , the successes enjoyed by the edmonton group began to slowly diminish . in the year 2000 , the first seven patients who were insulin free had normal or nearly normal levels of glucose and a1c . over 2 years
the series had grown to 17 patients , 14 ( 86% ) of whom were c - peptide positive and 11 of whom remained insulin free , although two were using oral hypoglycemic agents ( 23 ) . by 2005 ,
the edmonton series had grown to 66 patients , 85% of whom were c - peptide positive but only 15% of whom were insulin - free ( 24 ) . the other 85% , who were again using insulin ,
were reported to be using less than they had been pretransplantation . using these data , the median time to a return to insulin therapy
the immune tolerance network ( itn ) had undertaken a multicenter trial of alloislet intrahepatic transplantation using the edmonton protocol .
the goal was to establish the fidelity with which nine centers in canada , the u.s . , and
this consortium reported in 2006 ( 25 ) that , of 36 type 1 diabetic subjects studied , 44% achieved the primary end point of insulin independence with adequate glycemic control 1 year after islet transplantation .
it is noteworthy that previous experience with islet transplantation at the various research sites was strongly associated with success in achieving the primary end points . of the 18 subjects , 12 ( 67% )
were successful at 1 year post - transplant at sites where 4 or more patients had been transplanted in the preceding 2 years .
only 4 of 18 subjects ( 22% ) were successful at sites where there was a history of less than four transplantations previously .
this outcome clearly signaled that the procedure itself has a steep learning curve . at this time , it remains more of a research procedure than one that is generally applicable to treating patients with type 1 diabetes . during
the past decade other groups reported results that were generally in agreement with the edmonton and itn outcomes . as early as 2000 , oberholzer et al .
( 11 ) reported that of 13 of 13 alloislet recipients had measureable blood c - peptide levels for at least 3 months , as did 7 of 11 recipients at 6 months and 5 of 8 recipients at 1 year post - transplant .
( 26 ) used the edmonton protocol and reported success rates of 79% for insulin independence at 1 year and 43% at 18 months for 16 recipients .
toso et al . ( 27 ) used sequential kidney followed by islet transplantation and the edmonton protocol .
of 8 patients transplanted , insulin - free status was achieved in all for at least 3 months , and 5 of 11 were insulin - free after an average of 24 months ( 1134 ) .
( 28 ) reported that of six recipients , two were insulin - free at 24 months .
( 29 ) reported seven patients who underwent the islet after kidney transplantation approach ; two achieved insulin - independence and six had persistent graft function at 1 year .
( 30 ) compared islet transplantation alone ( ita ) with islet after kidney ( iak ) transplantation .
they observed partial function ( c - peptide positivity ) at 24 months in four of eight and six of six recipients in the ita and iak groups , respectively , as well as insulin - independence at 24 months in four of eight and five of six , respectively .
these reports from groups in various parts of the world using more or less the same edmonton protocol , some with the added feature of studying islet after kidney transplantation , generally achieved similar results reported by the edmonton and itn groups .
the overall conclusion of these studies is that islet transplantation can be considered largely successful at 1 year with a decline thereafter to an average success rate ( insulin - independence and at least nearly normal glycemia ) of 50% at various times during the 2nd year , and appreciably less at 5 years post - transplant .
do persistently measureable c - peptide levels combined with either a return to less intense insulin therapy and less hypoglycemia or , alternately , a state of insulin - independence but not completely normal a1c levels both represent successes , partial successes , partial failures , or failures ?
the analogy of whether a glass is half full or half empty seems unavoidable in an analysis of success versus failure in alloislet transplantation . the answer to this question is in the eye of the beholder . to the skeptic 's eye
, the procedure looks interesting , but is replete with problems . given the current nearly normal life expectancy of people with type 1 diabetes who are well managed with insulin - based treatment , an invasive procedure with a 50% chance of success for only 12 years does not seem very useful . to the optimist 's eye , a different conclusion might be reached .
certain patients with type 1 diabetes are difficult to manage medically and have a very poor quality of life because of recurrent hypoglycemia and rapid development of chronic complications secondary to chronic hyperglycemia .
the diabetes care and complications trial ( 31 ) established that maintenance of a1c levels less than or equal to 7% is mandatory for minimizing complications . a procedure , such as alloislet transplantation , which shows promise of providing c - peptide positivity , nearly normal a1c levels , and strikingly fewer episodes of hypoglycemia for as long as 5 years , can not be easily dismissed ( 24,3233 ) .
furthermore , initial reports have appeared claiming that islet recipients have macro- and microvascular as well as quality of life benefits from restoration of c - peptide secretion even if a1c levels are not normalized ( 3436 ) .
an objective approach to evaluating the progress of islet transplantation is to compare its history to that of pancreas transplantation .
the procedure of pancreas transplantation , originated at the university of minnesota , struggled greatly in its early days with high patient mortality and failure of grafts by 6 months ( 37 ) .
however , a return to the canine lab to reshape protocols and the advent of cyclosporine changed things dramatically . in the ensuing decades , a gratifying increase in pancreas survival and decrease in patient mortality took place . at its current zenith of success ,
pancreas transplantation enjoys virtually no patient mortality attributable to the surgery and an average of 80% success 3 years post - transplantation ( 38 ) .
2 ) , one sees that islet transplantation might not being doing quite as poorly as one might at first think .
pancreas allograft survival at 15 months post - transplant in the third epoch of this procedure ( 19841987 ) was precisely that of islet transplantation in its first successful years of 20002005 , namely , 50% .
comparison of rates of progress of alloislet transplantation versus pancreas transplantation as therapy for type 1 diabetes .
the first alloislet transplant to be reported as successful appeared in 1980 , 20 years earlier than the edmonton series was reported . the first pancreas transplant to be reported as successful appeared in 1966 , 18 years earlier than the 19841987 series was reported . at 15 months post - transplant ,
the edmonton series of alloislet transplants for the years 20002005 compared favorably with the success rate of pancreas transplants for the years 19841987 .
a major deficiency of virtually all published studies of islet transplantation is the lack of a suitable control group of medically managed patients . while one might argue that islet transplantation improves the health and lives of islet recipients , this is not the central issue .
such an argument begs the question of whether the complicated and expensive procedure of islet transplantation performs better than standard insulin - based therapy . without a randomized , nontransplanted control group
patients who choose islet transplantation do so because they view their quality of life as poor , most often because of recurrent hypoglycemia .
it is not always clear from published reports how objectively the diagnosis of recurrent hypoglycemia was made or how vigorously such patients underwent a period of insulin - based management by skilled diabetologists .
consequently , one can not be certain of the claim that partially failed islet transplantation leads to the use of less insulin and less hypoglycemia on a cause - effect basis .
it could just as easily be that patients who enter transplant programs come under close clinical scrutiny by interested diabetologists who begin managing them more skillfully .
the sole study that did include a medically managed control group was reported by thompson et al .
( 36 ) . in this study , 44 patients were candidates for islet transplantation ; 27 became islet recipients whereas 17 continued on medical management .
the group receiving transplants were reported to have less likelihood of progression of retinopathy than the medically managed patients .
first , although the two groups had comparable durations of diabetes , no randomization procedure was used to determine which patients would be transplanted , and , second , a1c levels at entry into the study were significantly lower in the transplant group .
nonetheless , this is a valuable study that illustrates the possibility for randomized studies to ascertain whether islet transplantation accrues additional benefits compared with intensive medical management .
with few exceptions , published reports of islet transplantation in type 1 diabetes have not adequately assessed the level to which endogenous insulin secretion is restored by the procedure . in most instances , basal levels and sometimes glucose - stimulated responses of c - peptide from recipients post - transplant are provided .
prior to transplant , only meager laboratory data about functionality of the islets to be transplanted are generally obtained .
typically , islets are stimulated with low and high concentrations of glucose in static incubations , and data are reported as a secretory index ( insulin or c - peptide responses to the higher glucose concentration divided by the response to the lower glucose concentration ) .
this practice at best relates only to whether the islets are alive or dead and imparts little about their degree of functionality .
consider , for example , that a fold response ( usually 1.0 or a doubling is considered acceptable ) to the higher glucose challenge will be dramatically affected by the baseline value .
the lower the denominator used to divide the numerator , the more exaggeratedly high the quotient will look . what is needed is assessment of glucose - induced insulin secretion by either static or perifusion protocols .
the former uses several glucose concentrations so that a half - maximal effective concentration can be calculated ; the latter uses multiple samples during perifusion so that assessment of first- and second - phase responses to glucose stimulation can be obtained .
these approaches would provide much more sophisticated information about the donor islets used for human transplantation and would go a long way in interpreting the clinical outcomes obtained in recipients .
the usefulness of more intensive functional studies can be easily appreciated from the autoislet experience in humans . in this scenario ,
the number of islets transplanted correlates very well with the magnitude of insulin responses to several stimulii ( 39 ) . reported outcomes in the current literature have come from programs that made heroic efforts to procure pancreata as quickly as possible and then produce islet preparations that were as pure as possible .
this involved exposure of islets to collagenase for variable periods of time followed by extensive centrifugation in cold temperatures .
the 50% that are recovered after purification by cold centrifugation are , not surprisingly , often damaged .
the method of counting viable islets afterward may or may not include damaged islets that are not totally dead .
it seems very likely that the number of healthy , transplanted islets is significantly overestimated , meaning the recipients may not have received the stated goal of 6,000 healthy islets / kg body mass . transplanting 6,000 islets
/ kg body mass delivers roughly 420,000 islets per person , 4050% the number of islets in a pancreas of a nondiabetic human .
this number is similar to that contained in the remaining pancreatic segment in hemipancreatectomized donors and in the recipient of a hemipancreas , and is adequate to maintain normoglycemia for many years ( 40,41 ) .
an important difference is that islets used for autoislet transplantation are not purified and undergo much less stress pretransplantation .
one answer to the alloislet problem may be to eliminate the cold centrifugation step ( 42 ) .
cold centrifugation minimizes the total tissue mass used to infuse islets into the liver , which is thought to lessen the likelihood of complications such as hepatic portal hypertension or lobar infarction .
however , this may be a case of too much caution because autoislet transplantation , in which there is no purification , has no significant history of either complication .
lacy 's group originally recommended the liver , based on their rodent studies , and thereafter it was the site traditionally used for human auto- and alloislet transplantation .
however , not only is the liver the site where ingested environmental toxins accumulate , but it is also where orally administered immunosuppressive drugs that are toxic to -cells are concentrated .
drug concentrations in hepatic portal venous blood are two- to threefold greater than in systemic circulation ( 43,44 ) and reach concentrations that inhibit -cell function in vitro ( 3,1219 ) .
this and other complications of currently available immunosuppressive drugs ( infection , potential cancer ) demand that the search for less toxic drugs continue unabated so that the islet , as well as pancreas , transplantation approach can become more clinically acceptable .
3 ) , although they do respond normally to other stimuli , such as arginine .
this has been demonstrated in human autoislet recipients ( 45,46 ) , human alloiset recipients ( 47,48 ) , and animals ( 49,50 ) .
a likely explanation is that glucose flux within the liver comes into contact with -cells on the periphery of transplanted islets .
this is especially relevant during hypoglycemia when glycogenolysis is stimulated by catecholamines and central nervous system inputs .
the intimate contact between intrahepatic glucose flux and -cells in the islet allograft abrogates the hypoglycemic signal delivered to the transplanted -cells from systemic blood coursing through the hepatic artery .
this hypothesis was tested in animals that received alloislet transplantation in hepatic and nonhepatic sites and thereafter underwent insulin - induced hypoglycemic challenges before and after prolonged fasting ( 50 ) . in all sites except the liver ,
when the intrahepatically transplanted animals underwent prolonged fasts and hepatic glycogen depletion , the initially absent glucagon responses to hypoglycemia were recovered ( fig .
4 ) . refeeding the animals to replete liver glycogen caused the glucagon response to hypoglycemia to disappear once again .
nondiabetic control subjects have plasma glucagon responses to insulin - induced hypoglycemia during a stepped hypoglycemic clamp .
this response is absent in patients with type 1 diabetes as well as patients who have received successful intrahepatic alloislet or autoislet transplantation .
restoration of absent glucagon responses to hypoglycemia from intraheptic islets in rats after prolonged fasting .
liver glycogen depletion caused by prolonged fasting results in restoration of the glucagon response to hypoglycemia .
this abnormality in -cell function does not occur in fed animals if islets are placed into nonhepatic sites .
it is very important to retain -cell responses to hypoglycemia in humans who undergo islet transplantation , return to insulin therapy , and become once again at risk for hypoglycemia .
it is established that glucagon responses to hypoglycemia from the native pancreas of type 1 diabetic patients is defective because of absent signaling from neighboring -cells .
alloisets transplanted in nonhepatic sites will respond to hypoglycemia and will protect the patient with a partially successful transplant who uses insulin to manage hyperglycemia . what alternate sites might be considered ?
preclinical literature supports consideration of celiac artery , intravenous access to lung , intrapancreas , intramuscular , subcutaneous , thymus , testis , intracisterna magna , omental pouches , bowel surfaces , peritoneal cavity , spleen , bone marrow , and kidney capsule ( 50,5254 ) .
i believe the use of liver should be reconsidered and a new look should be given to other sites first examined in 1972 but passed over for human use .
with few exceptions , published reports of islet transplantation in type 1 diabetes have not adequately assessed the level to which endogenous insulin secretion is restored by the procedure . in most instances , basal levels and sometimes glucose - stimulated responses of c - peptide from recipients post - transplant are provided .
prior to transplant , only meager laboratory data about functionality of the islets to be transplanted are generally obtained .
typically , islets are stimulated with low and high concentrations of glucose in static incubations , and data are reported as a secretory index ( insulin or c - peptide responses to the higher glucose concentration divided by the response to the lower glucose concentration ) .
this practice at best relates only to whether the islets are alive or dead and imparts little about their degree of functionality .
consider , for example , that a fold response ( usually 1.0 or a doubling is considered acceptable ) to the higher glucose challenge will be dramatically affected by the baseline value .
the lower the denominator used to divide the numerator , the more exaggeratedly high the quotient will look . what is needed is assessment of glucose - induced insulin secretion by either static or perifusion protocols .
the former uses several glucose concentrations so that a half - maximal effective concentration can be calculated ; the latter uses multiple samples during perifusion so that assessment of first- and second - phase responses to glucose stimulation can be obtained .
these approaches would provide much more sophisticated information about the donor islets used for human transplantation and would go a long way in interpreting the clinical outcomes obtained in recipients .
the usefulness of more intensive functional studies can be easily appreciated from the autoislet experience in humans . in this scenario ,
the number of islets transplanted correlates very well with the magnitude of insulin responses to several stimulii ( 39 ) .
reported outcomes in the current literature have come from programs that made heroic efforts to procure pancreata as quickly as possible and then produce islet preparations that were as pure as possible .
this involved exposure of islets to collagenase for variable periods of time followed by extensive centrifugation in cold temperatures .
the 50% that are recovered after purification by cold centrifugation are , not surprisingly , often damaged .
the method of counting viable islets afterward may or may not include damaged islets that are not totally dead .
it seems very likely that the number of healthy , transplanted islets is significantly overestimated , meaning the recipients may not have received the stated goal of 6,000 healthy islets / kg body mass . transplanting 6,000 islets / kg
body mass delivers roughly 420,000 islets per person , 4050% the number of islets in a pancreas of a nondiabetic human .
this number is similar to that contained in the remaining pancreatic segment in hemipancreatectomized donors and in the recipient of a hemipancreas , and is adequate to maintain normoglycemia for many years ( 40,41 ) .
an important difference is that islets used for autoislet transplantation are not purified and undergo much less stress pretransplantation .
one answer to the alloislet problem may be to eliminate the cold centrifugation step ( 42 ) .
cold centrifugation minimizes the total tissue mass used to infuse islets into the liver , which is thought to lessen the likelihood of complications such as hepatic portal hypertension or lobar infarction .
however , this may be a case of too much caution because autoislet transplantation , in which there is no purification , has no significant history of either complication .
lacy 's group originally recommended the liver , based on their rodent studies , and thereafter it was the site traditionally used for human auto- and alloislet transplantation .
however , not only is the liver the site where ingested environmental toxins accumulate , but it is also where orally administered immunosuppressive drugs that are toxic to -cells are concentrated .
drug concentrations in hepatic portal venous blood are two- to threefold greater than in systemic circulation ( 43,44 ) and reach concentrations that inhibit -cell function in vitro ( 3,1219 ) .
this and other complications of currently available immunosuppressive drugs ( infection , potential cancer ) demand that the search for less toxic drugs continue unabated so that the islet , as well as pancreas , transplantation approach can become more clinically acceptable .
3 ) , although they do respond normally to other stimuli , such as arginine .
this has been demonstrated in human autoislet recipients ( 45,46 ) , human alloiset recipients ( 47,48 ) , and animals ( 49,50 ) .
a likely explanation is that glucose flux within the liver comes into contact with -cells on the periphery of transplanted islets .
this is especially relevant during hypoglycemia when glycogenolysis is stimulated by catecholamines and central nervous system inputs .
the intimate contact between intrahepatic glucose flux and -cells in the islet allograft abrogates the hypoglycemic signal delivered to the transplanted -cells from systemic blood coursing through the hepatic artery .
this hypothesis was tested in animals that received alloislet transplantation in hepatic and nonhepatic sites and thereafter underwent insulin - induced hypoglycemic challenges before and after prolonged fasting ( 50 ) . in all sites except the liver ,
when the intrahepatically transplanted animals underwent prolonged fasts and hepatic glycogen depletion , the initially absent glucagon responses to hypoglycemia were recovered ( fig .
4 ) . refeeding the animals to replete liver glycogen caused the glucagon response to hypoglycemia to disappear once again .
nondiabetic control subjects have plasma glucagon responses to insulin - induced hypoglycemia during a stepped hypoglycemic clamp .
this response is absent in patients with type 1 diabetes as well as patients who have received successful intrahepatic alloislet or autoislet transplantation . reproduced from ref .
restoration of absent glucagon responses to hypoglycemia from intraheptic islets in rats after prolonged fasting .
liver glycogen depletion caused by prolonged fasting results in restoration of the glucagon response to hypoglycemia .
this abnormality in -cell function does not occur in fed animals if islets are placed into nonhepatic sites .
it is very important to retain -cell responses to hypoglycemia in humans who undergo islet transplantation , return to insulin therapy , and become once again at risk for hypoglycemia .
it is established that glucagon responses to hypoglycemia from the native pancreas of type 1 diabetic patients is defective because of absent signaling from neighboring -cells .
alloisets transplanted in nonhepatic sites will respond to hypoglycemia and will protect the patient with a partially successful transplant who uses insulin to manage hyperglycemia . what alternate sites might be considered ?
preclinical literature supports consideration of celiac artery , intravenous access to lung , intrapancreas , intramuscular , subcutaneous , thymus , testis , intracisterna magna , omental pouches , bowel surfaces , peritoneal cavity , spleen , bone marrow , and kidney capsule ( 50,5254 ) .
i believe the use of liver should be reconsidered and a new look should be given to other sites first examined in 1972 but passed over for human use .
a valid argument against islet transplantation as a treatment for type 1 diabetes is the undeniable arithmetic that not nearly enough pancreas donors exist to treat patients with type 1 diabetes , let alone all people with type 1 and type 2 diabetes , especially in the face of the current diabetes epidemic . does this mean we have been wasting our time and resources by studying islet transplantation ?
there will always be diabetic patients who need -cell replacement by transplantation of islets or the pancreas .
one group of patients comprises those with rapid development of secondary complications despite optimal medical care .
another group is made up of patients with the neurological disorder of autonomic insufficiency , which is accompanied by a 50% death rate within 5 years of diagnosis .
they are clearly candidates because successful pancreas transplantation converts this death rate from 50 to 10% .
our experiences with islet transplantation have taught us lessons that will be important for the use of -cell surrogates , be they stem cell derivatives or modified cell lines .
we have learned about culturing cells , isolating islets and -cells , identifying safe and physiological sites for transplantation , avoiding immunosuppressive drugs that are toxic to -cells , meeting environmental needs for physiological -cell function , and selecting appropriate patients for -cell replacement .
this is important information to use as we continue to meet the challenge of creating better means of controlling hyperglycemia and avoiding its complications .
we just need to continue on with new scientific work until the transplantation glass is successfully filled . | alloislet transplantation for the treatment of type 1 diabetes enjoyed highly favorable status in the first half of the last decade but declined in favor during the second half .
in this perspective , i will briefly review the literature published in this area from 2000 to 2010 for the purposes of extracting lessons we have learned , considering whether the procedure should be deemed a partial success or a partial failure , and offering several strategies to improve alloislet transplantation outcomes in the future . in the end ,
i hope to strike a positive note about where this procedure is going , and how it will be applied to establish insulin independence in patients with type 1 diabetes . | Lessons learned.
Partial failure or partial success?
Strategies to top off the half-full glass
Fully evaluate islet function before and after transplantation.
Improve methods of islet preparation.
Consider nonhepatic sites for transplantation.
Take full advantage of -cell function in transplanted islets.
Bridge to the future. |
it can occur in every organ of the body , including the gall bladder , gastrointestinal tract , skin , appendix , kidney , and lung .
caesarean section endometriosis is a rare event , with an incidence of 0.03 - 0.47% .
we , hereby , report a case of endometriosis in a caesarean section scar , which was initially misdiagnosed as a desmoid tumour .
a 30-year - old woman presented with a gradually increasing painful abdominal lump for the past six years . the pain was not associated with menstruation .
ultrasound revealed a large firm mass lesion in the lower anterior abdominal wall in the midline extending up to the pubic region and showing hypoechoic and isoechoic components .
ct scan revealed a 10 8 5 cm ill - defined heterogeneous soft tissue density mass lesion in the anterior abdominal wall , infraumbilical region predominantly epicentred at the right rectus abdominis muscle with extensions into opposite rectus muscle , skin , and subcutaneous tissue [ figure 1 ] . based on the clinical and radiological findings ,
ct scan revealing a 10 8 5 cm ill - defined heterogeneous soft tissue density mass lesion in the anterior abdominal wall involving rectus abdominis muscle , skin , and subcutaneous tissue ( arrows ) grossly , the specimen measured 13.2 12.3 5.5 cm .
it was a soft tissue mass covered with skin , along with a part of the rectus muscle .
the skin surface showed scarring [ figure 2 ] . on the cut section , multiple cystic spaces filled with haemorrhagic fluid were evident [ figure 3 ] .
the endometrial glands and stroma were seen infiltrating into the abdominal musculature and adipose tissue .
foci of haemorrhage and chronic inflammatory infiltrate were also seen [ figures 4 and 5 ] .
based on the above findings , a diagnosis of abdominal wall endometriosis ( awe ) was made .
gross appearance of the abdominal mass covered with skin cut section of the specimen showing multiple cystic spaces ( arrows ) , along fibro - fatty tissue microscopy revealing endometrial tissue ( arrows ) embedded in abundant desmoplastic stroma ( arrow heads ) ( h and e , 100 ) photomicrograph revealing endometrial tissue ( arrows ) , adjacent abdominal fat ( arrow heads ) , and chronic inflammatory infiltrate ( asterix ) ( h and e , 100 ) following wide local excision , the patient was administered danazol 100 mg twice daily for initial six months . after nine months of follow - up , the patient is doing well and is symptom free .
endometriosis is characterised by the presence of histologically normal endometrial tissue outside the uterine cavity , usually the pelvis .
the most common extrapelvic location of endometriosis is the abdominal wall , particularly surgical scars related to gynaecologic or obstetric surgery .
the incidence following hysterectomy is 1.08 - 2% , whereas after caesarean section , the incidence is 0.03 - 0.4% .
awe has also been reported after amniocentesis , hypertonic saline solution abortion , and laparoscopy .
an occasional case of awe in the absence of previous surgery has also been reported .
various theories have been proposed for endometriosis :
retrograde spread of collections of endometrial cells during menstruationblood , lymphatic , or iatrogenic spreadmetaplasia of the pelvic peritoneal cellsimmune system dysfunction and autoantibody formation
retrograde spread of collections of endometrial cells during menstruation blood , lymphatic , or iatrogenic spread metaplasia of the pelvic peritoneal cells immune system dysfunction and autoantibody formation scar endometriosis is believed to be the result of direct inoculation of the abdominal fascia or subcutaneous tissue with endometrial cells during surgical intervention and subsequent stimulation by estrogen . in different series , the time interval between operation and presentation has been shown to vary from three months to seven years .
the non - cyclic nature of pain has occasionally been reported , which may explain why it is clinically often misdiagnosed as in the present case .
most patients also present with a palpable mass at the site of maximum tenderness in the region of the surgical scar .
on sonography , these lesions appear hypoechoic , vascular , or solid , with some cystic changes .
the radiological findings are non - specific and a wide spectrum of disorders presenting as a mass should be considered in the imaging differential diagnosis .
these include neoplasms such as sarcoma , desmoid tumour , lymphoma , or metastasis , as well as non - neoplastic causes such as a suture granulomas , ventral hernia , haematoma , or abscess .
therapeutic options for awe are pharmacologic therapy with hormonal agents such as progestogens , oral contraceptive pills , and danazol ; or it may be surgical excision . the success rate of medical therapy has been reported to be low , offering only temporary alleviation of symptoms often followed by recurrence after cessation of the drug . moreover , due to side effects such as amenorrhoea , weight gain , hirsutism , and acne , compliance is unlikely . wide surgical excision , therefore , is the treatment of choice .
mesh replacement may also be done along with this . to conclude , awe is difficult to diagnose clinically and radiologically .
a high index of suspicion is recommended when a woman presents with postoperative painful abdominal lump . | endometriosis is a common gynaecological condition that usually presents as an abdominal lump .
it can be a diagnostic dilemma and should be considered as a differential diagnosis for lumps in the abdomen in females .
we discuss a case of abdominal wall endometriosis following caesarean section , which was misdiagnosed as a desmoid tumour . | INTRODUCTION
CASE REPORT
DISCUSSION |
immunoglobulin ( ig ) g4-related sclerosing disease is an immunoglobulin deposition disease characterized by infiltration of organs by igg4-positive plasma cells .
manifestations include retroperitoneal fibrosis , dacryoadenitis , sialadenitis , thyroiditis , pneumonitis , pancreatitis , sclerosing cholangitis , tubulointestinal nephritis , prostatitis , and hypophysitis .
regardless of organ involvement , biopsy with review of histopathology and immunohistochemical staining for igg4 are essential for diagnosis .
herein we describe a case of igg4-related sclerosing disease that involved the left pelvocaliceal system and extended to the level of the left ureter , thereby causing left hydronephrosis .
a 70-year - old man was transferred to kyung hee university hospital at gangdong for further evaluation of left hydronephrosis .
he had recently visited a private clinic because of dyspepsia , and abdominal ultrasonography had shown left hydronephrosis .
his laboratory data showed a white blood cell count of 4.410/mm , hemoglobin of 14.3 g / dl , a platelet count of 164k , a serum creatinine level of 1.1 mg / dl , and an erythrocyte sedimentation rate of 6 mm / h . the results of a liver function test and electrolyte values were within the normal range .
urine analysis showed a white blood cell count of 0 - 1 and a red blood cell count of 0 - 1 .
an abdominal computed tomographic ( ct ) scan revealed an enhanced soft tissue mass infiltrating the left renal pelvis of the left kidney and extending to the level of the left mid ureter , causing left hydronephrosis ( fig .
a chest ct scan revealed a small , well - defined nodule in the left lower lobe , an enlarged right hilar lymph node , enlarged interlobar lymph nodes , and an enlarged left paraesophageal lymph node ( fig .
there were adhesions between the hard mass lesions and the left ureter around the ureteropelvic junction .
the histological examination was negative for malignancy but showed soft tissue mixed lymphocytic infiltration with germinal centers ( fig .
igg4 immunostaining revealed numerous cells positive for igg and more than 50 igg4-positive cells per high - powered field ( fig .
the patient 's serum igg level was increased to 1,839 mg / dl ( normal range , 870 to 1,700 mg / dl ) .
the patient was initially treated with 40 mg of oral prednisolone daily for 2 weeks with a subsequent decrease in the dosage by 5 mg every 2 weeks .
he was maintained on steroid therapy for 3 months , and the follow - up ultrasonography showed minimal hydronephrosis .
we plan to keep treating the patient with prednisolone therapy and to perform regular follow - up abdominal ct scans .
lately , igg4-related sclerosing disease has been proposed to describe the group of autoimmune disorders involving connective tissue . prominent infiltration of igg4-positive plasma cells in affected tissues is a characteristic feature of this disease .
an increasing number of sclerosing diseases are recognized as part of the disease spectrum , such as autoimmune pancreatitis , sclerosing cholangitis , chronic cholecystitis , chronic sclerosing sialadenitis , retroperitoneal fibrosis , tubulointestinal nephritis , interstitial pneumonia , and lymphocystic hypophysitis . in our case , fibrotic tissues around the left kidney and ureter resulted in hydronephrosis .
a chest ct scan showed the left paraesophageal , right hilar , and both interlobar lymph nodes to be enlarged and the presence of a small nodule in the left lower lobe .
the abdominal ct showed an infiltrating soft tissue mass involving the left pelvocaliceal system and extending to the left midureter .
immunohistochemical staining of the specimen showed more than 50 igg4-positive cells per high - powered field .
histopathology and immunohistochemical staining for igg4 are the most valuable diagnostic tools in igg4-related systemic disease .
thus , further randomized trials are needed . in our case , although hydronephrosis was present , the patient had no signs of renal functional impairment .
if severe hydronephrosis and renal functional impairment are present , ureteral stent indwelling is needed . in periureteral igg4-related systemic sclerosing disease ,
biopsy should be regarded as a diagnostic option for diseases such as retroperitoneal fibrosis , retroperitoneal sarcoma , lymphoma , and retroperitoneal hematoma , which are difficult to differentiate with ct scans . also , the relationship between igg , igg4 , and perirenal masses must to be kept in mind .
urologists should be aware of the possibility of igg4-related disease in the differential diagnosis of a periureteral mass . | immunoglobulin ( ig ) g4-related sclerosing disease is a newly defined clinicopathological entity characterized by lymphoplasmacytic infiltration of igg4-positive plasma cells and varying degrees of fibrosis within affected tissues .
patients usually exhibit multisystem involvement and often respond well to steroid and immunosuppressive therapy .
this report presents a case of igg4-related sclerosing disease involving the ureter and kidney .
we hope to bring igg4-related sclerosing disease to the attention of urologists , because it is an uncommon disease that commonly responds to systemic corticosteroids . | INTRODUCTION
CASE REPORT
DISCUSSION |
spontaneous esophageal perforation ( boerhaave 's syndrome ) was first described by hermann boerhaave in 1724 ; it can easily prove fatal if the diagnosis is delayed more than 24 h after onset , although the mortality has improved recently .
it has been reported that chest x - ray , endoscopic examination , enhanced esophagography and computed tomography scan were available for rapid and precise diagnosis . however , treatment is often delayed because its diagnosis is confused .
the reason might be that the chief complaints , represented by chest pain , chest discomfort , fever , back pain or abdominal pain , are manifold in addition to the rarity of the disease . in cases diagnosed rapidly ,
such cases often combine septic shock and the indication for surgery is still disputable taking into consideration the patient 's tolerance and surgical stress .
we present a case of spontaneous esophageal perforation in a 68-year - old man treated with emergent surgery 96 h after onset who was discharged in good health 86 days after emergent surgery .
we successfully treated the patient using thoracic drainage and jejunostomy , and report this case with reference to the literature .
the patient was a 68-year - old man who vomited after consuming alcohol , followed shortly thereafter by epigastralgia .
vital signs were as follows : blood pressure 160/100 mm hg , pulse rate 105 beats/ min , respiratory rate 32 breaths / min , body temperature 38.3c and spo2 95% ( by mask at 3 l / min ) .
blood and laboratory data were as follows : white blood cell count 5,900/l , red blood cell count 411 10/l , hemoglobin 12.3 g / dl , neutrophils 81.7% , platelets 9.7 10/l , total protein 6.1 g / dl , albumin 2.4 g / dl , blood urea nitrogen 34 mg / dl , creatinine 1.2 mg / dl , creatine phosphokinase 111
iu / l , total bilirubin 0.6 mg / dl , na 142 meq / l , cl 108 meq / l , k 4.2 meq / l , c - reactive protein 40.2 mg / dl , and procalcitonin 12.5 ng / ml . chest x - ray showed left pleural effusion and cardiomegaly ( fig .
1b ) , but his general condition deteriorated and the patient was diagnosed with sepsis .
a 5-cm - long perforation was identified at the left side of the lower esophagus .
the left thoracic cavity was rinsed with 10 l of saline , and thoracic drainage was performed with chest tubes .
orally administered contrast medium was seen from the perforated esophagus as a fistula on postoperative day 20 ( pod20 ) ( fig .
the lesion of esophageal perforation was covered with regenerated epithelium on endoscopic observation on pod72 .
peroral intake was started on pod76 and the patient was discharged from hospital on pod86 .
recent numerous advances in surgical and nutritional techniques , powerful and selective antibiotics , and better postoperative intensive care have all contributed to significant improvements in the mortality and morbidity associated with esophageal perforation . however , standard or personalized treatment is now controversial because of the variety of disease seriousness .
delayed diagnosis has been considered as one of the reasons why disease seriousness varies because the onset is nonspecific , with symptoms such as vomiting and severe chest or epigastric pain .
sepsis due to mediastinitis or pyothorax progresses rapidly and the mortality rate doubles if diagnosis is delayed beyond 24 h . in the present case ,
the patient complained of severe epigastric pain and dyspnea , and chest x - ray showed left pleural effusion and cardiomegaly .
therefore heart failure was initially suspected and drainage was delayed . in determining treatment , the general condition of the patient , the degree of contamination and the state of the esophagus around the perforation represent the most important factors .
a consensus has been reached that esophageal perforation should be repaired and thoracic and mediastinal drainage should be performed when diagnosis is achieved within 24 h [ 4 , 5 , 6 , 7 , 8 ] .
in contrast , the treatment for cases diagnosed more than 24 h after onset has not reached a consensus . to date , cases with esophagectomy or simple adequate drainage have been reported .
postoperative leakage reportedly occurs in 83% of such cases treated over 24 h after onset . in our case ,
the patient developed septic shock and perforation extending to the thoracic cavity , so we selected surgical drainage and feeding jejunostomy for postoperative enteral nutrition .
in addition to drainage , enteral nutrition reportedly enhances the immune function of the intestinal mucosal barrier against fatal infection [ 10 , 11 ] and has been linked to protection from sepsis or multi - organ failure [ 12 , 13 ] .
therefore , the authors believe that adequate drainage and early enteral nutrition in cases such as the present one could be minimally invasive while providing great benefit for spontaneous esophageal perforation with delayed diagnosis , except that it took a longer period until oral intake could be started . in conclusion , it is unnecessary to say that rapid and precise diagnosis is important , and minimal invasive and adequate treatment based on the patient 's condition can rescue in situations of spontaneous esophageal perforation . | spontaneous esophageal perforation is relatively uncommon , but carries a high mortality rate if diagnosis or treatment is delayed .
we report the case of a 68-year - old man with spontaneous esophageal perforation who was successfully treated over 96 h after onset by thoracic drainage and jejunostomy for enteral nutrition .
he vomited after drinking alcohol , soon followed by epigastralgia .
heart failure was suspected on admission to another hospital .
spontaneous esophageal perforation was diagnosed 48 h after admission .
chest tube drainage was performed , but his general condition deteriorated and he was transferred to our hospital .
emergent surgery was performed and esophageal perforation combined with pyothorax and mediastinitis was identified on the left side of the lower esophagus .
the left thoracic cavity was rinsed and thoracic drainage was performed .
feeding jejunostomy was performed for postoperative enteral nutrition .
effective drainage and sufficient nutrition management appear extremely valuable in treating spontaneous esophageal perforation . | Introduction
Case Report
Discussion
Disclosure Statement |
physical activities can preserve brain function and improve
blood flow and oxygen transfer2 as well as
slow the loss of hippocampal formational tissue in the aging brain .
because of its unique three - dimensional movement , horseback riding has been applied as a
physical therapy to stimulate movement to maintain balance3 .
one of its effects is to transfer movement to the pelvis , producing
an effect very similar to pelvic tilting4 .
in addition , horseback riding exercises parts of the body that are not used by other
frequently performed exercises , including agonistic muscles , which are deep muscles that
contract and relax to maintain balance5 . psychological research on horseback
riding exercise indicates it decreases depression in
teenagers with emotional disorders6 as
well as improves teenagers self - esteem and self - control7 .
however , there is little information about horseback riding exercise as a physical therapy
and even less information about whether such exercise is effective for elderly people or
indeed how it may affect them .
therefore , this study investigated the effects of horseback
riding exercise on the background electroencephalogram ( eeg ) of elderly people who performed
horseback riding for 8 weeks .
all
participants or their guardians provided informed consent prior to participation . before the
start of the study , the purpose and methods were explained in detail to the participants .
the participants were divided into 2 groups : a horseback riding exercise group and a control
group ( n = 10 each ) .
riding was performed for 15 minutes , 3 times per week
for 8 weeks .
the post - exercise tests were performed over a period of 8 weeks in the same
manner as pre - study tests .
we investigated the background eegs , specifically the relative alpha power index , in both
groups . for the exercise ,
we used jeju horses from the i rehabilitation center - affiliated
horse riding course ; these horses are normally used to provide riding experiences to
visitors and are therefore healthy , accustomed to their surrounding environment , well
trained , tame , and have stable strides .
all participants wore protective gear including
safety helmets , vests , and boots . at the beginning of the tests ,
the warm - up exercises were performed at a walking speed , which
was the slowest speed ( 110 m / min ) . for this study ,
the slowest walking speed was maintained
for the participants safety and comfort . in the exercise ,
an instructor led a horse by the
bridle while another assistant supported a participant by holding him or her by the leg
prevent them from falling from the horse and provide assistance as needed .
quantitative
electroencephalography can be used to diagnose neurological changes in the brain ; it
constitutes an electric signal due to an active change in the brain that can capture the
electrical flow between neurons through electrodes attached to the surface of the head8 .
we collected the eeg data using a
computerized polygraph ( polyg - i , laxtha inc .
, korea ) and telescan , which is a real - time
analysis program .
the eeg electrodes were attached to the surface of the head of the
participants as they were seated in a comfortable reclining chair . to reliably capture data ,
the same researcher attached the electrodes and operated the testing instruments .
body and
head movements were controlled as much as possible , and the participant kept their eyes
closed for 3 minutes while resting in order to minimize the interruption of waves caused by
eye movement .
the eeg signals were analyzed for 60120 seconds , excluding the first and last
60 seconds when the test might have been influenced by external environments .
the background
eeg data were analyzed on the basis of a monopolar derivation from 8 points on the surface
of the head ; the 8 electrodes were attached to fp1 , fp2 , f3 , f4 , t3 , t4 , p3 , and p4
according to the international 1020 electrode system .
the saved data were transformed into
frequencies using a fast fourier transform , which expresses the quantitative relationship
between eeg frequencies and their intensity .
power spectrum analysis was subsequently
applied , and the band - to - band power was calculated .
the relative alpha power
( 813 hz/450 hz ) , which emerges in a state of relaxation and rest , was subdivided into the
relative slow alpha power ( 811 hz/450 hz ) and the relative fast alpha power
( 1113 hz/450 hz ) and analyzed .
the
pre- and post - intervention data were compared within each group and between groups by using
paired t - tests independent t - tests , respectively .
there were no significant differences in the general characteristics between groups ( p >
0.05 ) ( table 1table 1.general characteristics of participants ( n = 20)categoryhorseback riding(n=10)control(n=10)age ( years)69.5 3.269.7 3.5height ( cm)157.6 8.7158.5 6.8weight ( kg)58.8 7.959.2 6.7bmi
bmi : body mass index after the horseback riding program , the relative slower alpha power was analyzed ; it
appeared to be active in the t3 and p4 domains but was suppressed in the fp1 , fp2 , f3 , f4 ,
t4 , and p3 domains ( table 2table 2.relative slow alpha power before and after the interventionpre - interventionpost - interventionhorseback ridingfp10.321 0.1470.308 0.177fp20.337 0.1500.326 0.148f30.339 0.1400.337 0.151f40.341 0.1590.319 0.164t30.288 0.1310.314 0.119t40.254 0.1540.212 0.096p30.369 0.1460.367 0.147p40.345 0.1970.351 0.166controlfp10.243 0.1370.288 0.177fp20.246 0.1350.277 0.176f30.251 0.1410.302 0.181f40.237 0.1530.279 0.201t30.216 0.1160.225 0.131t40.164 0.1410.210 0.155p30.291 0.1200.309 0.150p40.277 0.1510.325 0.180data are
moreover , after the horseback riding program , the relative faster alpha
power appeared to be active in all domains of the horseback riding exercise group but
suppressed in all domains of the control group ( table
3table 3.relative fast alpha power before and after the interventionpre - interventionpost - interventionhorseback ridingfp10.055 0.0330.058 0.033fp20.060 0.0340.061 0.034f30.059 0.0280.067 0.038f40.057 0.0250.062 0.039t30.060 0.0250.073 0.031*t40.054 0.0190.055 0.029p30.088 0.0720.098 0.059p40.078 0.0570.093 0.049*controlfp10.048 0.0260.045 0.028fp20.047 0.0300.045 0.031f30.058 0.0370.050 0.034f40.051 0.0420.046 0.035t30.059 0.0370.050 0.034t40.040 0.0240.039 0.018p30.064 0.0420.054 0.038*p40.054 0.0370.048 0.027data are mean sd . *
. there was a significant difference between the horseback riding exercise and
control groups with respect to the f3 domain ( p < 0.05 ) ( table 4table 4.comparison of relative fast and slow alpha powerhorseback ridingcontrolrsafp10.013 0.1240.025 0.093fp20.020 0.1370.012 0.131f30.006 0.1330.037 0.114f40.020 0.1520.043 0.136t30.013 0.1120.018 0.075t40.040 0.1500.031 0.113p30.000 0.0920.018 0.122p40.026 0.1830.018 0.137rfafp10.000 0.0530.013 0.034fp20.000 0.0530.006 0.025f30.020 0.0410.019 0.040*f40.007 0.0590.000 0.036t30.007 0.0250.013 0.050t40.007 0.0450.006 0.044p30.007 0.0250.000 0.051p40.013 0.0350.000 0.051*p < 0.05 , rsa : relative slow alpha power , rfa : relative fast alpha power ) .
p < 0.05 * p < 0.05 , rsa : relative slow alpha power , rfa : relative fast alpha power
during acute graded maximal exercise , the alpha wave power is reportedly decreased in the
frontal and temporal lobes8 , 9 .
alpha wave power is an index of stable emotional status or
mental health ; in this regard , alpha wave power appears to be more closely associated with
mental stress than exercise intensity , which is physically recognized .
the present study
evaluated the changes in background eeg , specifically alpha wave power , which indicates
stability and relaxation10 , 11 .
the relative slow alpha power was analyzed before and after the horseback riding exercise
program ; the results show it increased equally in all brain wave domains in the control
group . in the horseback riding exercise group , the relative slow alpha power was active in
the t3 and p4 domains but suppressed in the fp1 , fp2 , f3 , f4 , t4 , and p3 domains .
after the
horseback riding exercise program , the relative fast alpha power was increased in all
domains of the horseback riding exercise group .
horseback riding exercise is considered to enhance
concentration and comfort by activating the brain waves in all domains .
the relative alpha
power by the motor learning process is reported to increase in the fz , cz , oz , c3 , c4 , t3 ,
and t4 domains during learning through exercise imagination but is decreased in those
observing and performing behaviors12 , 13 .
after the learning process , the relative
alpha power differs significantly in participants who observe and actually perform behaviors
compared to those who perform exercise imagination in the pz , oz , c4 , t3 , and t4 domains ,
indicating the brain activation level is higher during exercise learning through behavior
observation and actual performance than imagination exercise learning . in the present study , there were small differences in the attachment points of the 8 brain
wave channels , background eeg , and evoked eeg .
however , the results corroborate the notion
that learning through exercises helps increase the relative alpha power .
the main limitation
of this study is its lack of generalizability owing to the relatively small sample as well
as the fact that the daily life habits aside from the horseback riding exercise program were
known only through personal interviews .
therefore , individual life habits may have affected
the results . accordingly , further studies with more subjects and better evaluation methods
are required to confirm the results . moreover , further studies should investigate the
psychological , cognitive , and physical changes as a result of horseback riding .
finally ,
comparative studies involving different age groups and patients suffering from differing
diseases and conditions should be performed . | [ purpose ] this study investigated the effect of horseback riding exercise on the
background electroencephalograms of elderly people who performed horseback riding for 8
weeks .
[ subjects ] twenty elderly people were divided into the horseback riding exercise
and control group ( n = 10 each ) . [ methods ]
the exercise was performed for
15 minutes , 3 times per week for 8 weeks .
electroencephalograms were analyzed .
post - exercise evaluation was performed after 8 weeks .
[ results ] after the horseback riding
exercise , the relative slower alpha power index was active in the t3 and p4 domains but
suppressed in the fp1 , fp2 , f3 , f4 , t4 , and p3 domains .
moreover , the relative faster
alpha power index was active in all domains of the horseback riding exercise group but was
suppressed in all domains of the control group .
there was a significant difference between
groups in the f3 domain .
[ conclusion ] the alpha power index increased significantly after
horseback riding exercise , suggesting the exercise improved background
electroencephalogram . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
adaptive immunity is based on the recognition of specific molecular structures , named epitopes , by either antibodies / b cell receptors or t cell receptors .
antibodies and b cell receptors bind a wide variety of structures , including proteins and carbohydrates . in the case of protein ligands , antibodies can recognize either a series of contiguous residues ( linear epitopes ) or a set of residues encoded in disparate regions of the protein sequence and brought together in the three dimensional structure of the protein ligand ( discontinuous epitopes ) .
t cells recognize a complex between mhc molecules ( named hla in humans and h-2 in mouse ) and , in most cases , a peptidic epitope of 816 residues in length [ 1 , 2 ] .
t cell responses are a key component of adaptive immunity . in concert with antibody responses , cd8 t cells , recognizing class i binding epitopes , and cd4 t cells , recognizing their class ii counterparts , are key players in immunity to viruses and bacteria . in the case of allergic reactions ,
cd4 t cell responses play a key role in pathogenesis both directly and indirectly through the regulation of antibody responses of the ig e class .
accurate measurement of b and t cell responses is a valuable tool to study autoimmunity , allergies , immunity to pathogens , and host - pathogen interactions and assist in the design and evaluation of t cell - based vaccines and immunotherapies [ 58 ] .
accordingly , a large number of studies have been devoted to defining b and t cell epitopes , a process that has been facilitated by an ever - increasing expansion and refinement of experimental methods . as immune investigations proceed over time ,
alternatively , large - scale epitope identification can reveal hundreds of potential epitopes [ 912 ] .
the immune epitope database ( iedb ) [ 13 , 14 ] is a freely available resource that serves as a repository of experimentally derived immune epitope information available in the peer - reviewed published literature , as well as from direct submission from nih - niaid funded large - scale epitope identification studies .
the iedb content covers a broad range of indications , to include infectious diseases ( excluding hiv ) , allergies , transplantation , and autoimmune disease and a similarly broad range of hosts , including humans , nonhuman primates , mice , and livestock , amongst others .
the database , as of october 2014 , hosts experimental data related to 121,812 different peptidic epitope structures .
this large amount of information might , in some cases , pose a challenge for the identification and selection of appropriate sets of epitopes for use in specific contexts .
thus , it is clearly desirable , given the ever - growing body of information contained in the iedb , to develop tools to enable the efficient generation of sets of validated reference epitopes for any antigenic source of interest . while an epitope , according to classic definitions , is any structure capable of interacting with t and b cell receptors , in practice the consensus in the scientific community is that certain types of assays identify the most relevant and validated epitopes . in the context of antibody reactivity , by way of example , epitopes identified on the basis of x - ray structures of ag / antibody complexes , biological activity , and in vivo assays or recognized by ige in the case of allergens are considered more biologically relevant than linear epitopes recognized in elisa assays and elicited by peptide immunization . in the case of t cell reactivity , again by way of example , multimer / tetramer staining assays or readouts based on ics or elispot assays , are preferred over older assay platforms such as thymidine incorporation following multiple in vitro restimulations with peptides .
these high quality assays have been selected based on our experience and judgment ; however , in the web tool developed , the user will be capable of customizing it by his / her needs , that is , select only epitopes derived from neutralization or elisa assays or any other desired selection . indeed , for many applications , it is desirable to study t cells ex vivo , without manipulation .
this is because manipulations , such as in vitro expansion , are known to profoundly change the phenotype and characteristic of the t cells [ 1517 ] , thus questioning the physiological relevance of some of the experimental observations .
however , direct ex vivo detection of human t cell responses is often difficult , largely because the immune response in any individual may target many different epitopes , and different individuals typically recognize unique epitope repertoires .
simultaneous use of many different epitopes as a pool might represent a powerful approach to detecting t cell responses , because even if the frequency of t cells recognizing each individual epitope may be below the limit of detection , a pool of a large number of epitopes ( i.e. , responses ) might pass the limit of detection .
an important consideration in the definition of reference sets of epitopes is how to factor the number of individual donors or experiments in which a given structure is reported to elicit a positive response , and particularly if this validation is provided in multiple independent studies .
for example , different studies often report on essentially the same epitope but utilize different nested , truncated , or frame - shifted version of the same sequence , leaving uncertainty on how to combine the data or which particular version of the epitope to select for testing .
clarification of a general approach for combining data from such disparate studies would greatly facilitate the generation of nonredundant sets of epitopes . in the present study
, we have attempted the definition of an automated process to generate reference sets of high quality epitopes for various disease indications .
the resulting tool , made available to the scientific community , provides a standardized and reproducible platform to automatically extract and process relevant data from the iedb without the need of complex analysis and judgment calls from the user . at the same time
, the tool also offers flexibility to enable the end user to design sets meeting specific user - defined criteria .
we have also analyzed the data currently available in the iedb , to determine how many sets of pathogen or autoantigen specific epitopes could be identified on the basis of the data available to date .
the web page application is written in php / html code with a mysql connection that allows communication between the database and the user interface .
the first was based on the type of assays used to characterize the epitopes and the second on the frequency by which each epitope was recognized . regarding the assay type scoring system for mhc class i or class ii epitopes , in our selection we included epitopes defined by multimer / tetramer staining , elispot , and ics assays .
we arbitrarily associate a numerical parameter value of 3 , 2 , and 1 to these assay types , respectively .
each of these assay types can be used in either an ex vivo or in vitro configuration . to provide that ex vivo assays are always ranked higher , we assigned an ex vivo configuration a value of 4 , and an in vitro configuration a value of 1 , and calculated a final assay score by multiplying the assay type and configuration values . in the case of b cell epitopes , x - ray structure , biological activity , and in vivo assays
were assigned a score of 1 , as well as epitopes that present ige ; for these epitopes , the effector origin was not taken in consideration . since each epitope
can be associated with multiple records , each describing different assays and thus with different assay scores , the highest assay score , reflecting the highest level of validation reached for that epitope , was selected and carried forward . in terms of scoring each epitope on the basis of the frequency by which it was recognized , we utilized a previously described response frequency ( rf ) score .
the rf score is calculated as(1)rrn , where n is the total of subjects tested and r is the number of positive responses . the square root is a correction factor , approximating one standard deviation for the number of responding donors .
there is no information of subjects tested , the epitope will be assigned r = 1 , n = 1 if the assay outcome is positive and r = 0 , n = 1 if it was negative
. the rf score ( rfs ) also takes into account all data for a given epitope across all publications .
we decided to use an older and simpler rf definition , as compared to the one used in , as it is more suitable to filter epitopes based on their rfs .
as an example , consider an epitope 1 with positive responses ( p ) = 1 and total subjects ( t ) = 1 ; the new definition gives a rfs = 1 ( 0.041.00 ) whereas the older version gives an rfs = 0 .
in contrast with epitope 2 with p = 8 and t = 11 , the new version gives rfs = 0.73 ( 0.43 : 0.92 ) and the older rfs = 0.47 . in order for the rfs to reflect the difference between epitopes 1 and 2 in terms of the total number of subjects , the latter
should be assigned a higher score to be used for ranking and filtering the data , which at the end benefits the user giving simpler and useful results .
both the assay score and rf score are calculated for each epitope and provided in the results .
for mhc class i epitopes , it is generally observed that a length of about 811 residues is optimal for t cell recognition and use in assays .
because of the structure of the class i binding groove , distinct class i sequences typically represent unique epitopes , even if they are nested within a longer sequence that is also recognized by t cells .
accordingly , for the present study , we have not subjected class i epitopes of nested or overlapping character to further processing . for mhc class ii epitopes ,
however , optimal epitopes are usually longer than the minimal t cell recognized 9-mer core . in general ,
peptides of varying length but that carry the same core may all be similarly active and/or recognized by the same t cell specificity .
thus , many of the epitope structures contained in the iedb for class ii epitopes are redundant , nested or largely overlapping .
for this reason , it is desirable to devise strategies to reduce the complexity of class ii epitope sets . here
, we developed a clustering algorithm to generate consensus sequences or cluster of epitopes , an illustration of such a process can be found in table 1 . in order to solve this problem ,
then , taking the highest ranked peptide as starting sequence , we move down the ranked list aligning the sequences to find nested or overlapping epitopes by at least 9 residues . for this approach , we only consider identical matches over the region of overlap and identical nested peptides ; given this definition , mismatches will be treated as separate epitopes .
when a nested peptide is found , we will keep only the larger peptide and calculate a new rf score using the sum of all responded and tested subjects per epitope in the cluster . for overlapping epitopes , a consensus epitope or cluster will be generated combining the sequences , if the cluster length is up to 20 residues . in these cases ,
the rf score will be calculated as a new rf score as in the nested case . for the assay type scoring system ,
the highest ranked assay and application of all the assays associated with the set of nested epitopes will be considered . in the case of donors with latent tuberculosis infection ( ltbi ) , leukapheresis or whole unit blood samples from 10 adults
were obtained from the university of california , san diego , antiviral research center ( avrc ) clinic .
donors were classified as ltbi based on positive quantiferon - tb gold in - tube ( cellestis ) , as well as a physical exam and/or chest x - ray that was not consistent with active tuberculosis . because dengue virus ( denv ) prevalence is low in the san diego area ,
to obtain denv seropositive samples , anonymous blood donations from healthy adults were obtained by the national blood center , ministry of health , in the area of colombo , sri lanka .
plasma of the associated donation was tested for serology using the flow - based u937+dc - sign neutralization assay ( conducted at the university of north carolina , chapel hill ) as previously described [ 11 , 20 ] . because tb prevalence is low in the colombo area ,
all samples were collected and used following guidelines from the institutional review boards ( irb ) of lji and the medical faculty , university of colombo ( serving as national institutes of health - approved irb for genetech research institute ) .
15-mer peptides were synthesized as crude material on a small ( 1 mg ) scale by mimotopes ( victoria , australia ) and/or a and a ( san diego ) .
pbmcs were purified by density gradient centrifugation ( ficoll - hypaque , amersham biosciences ) from 100 ml of leukapheresis sample or 450 ml of whole blood , according to manufacturer 's instructions .
cells were cryopreserved in liquid nitrogen suspended in fetal bovine serum ( gemini bio - products ) containing 10% dimethyl sulfoxide .
10 cells / well ) were incubated with peptide pools ( 1 g / ml ) for 2 hrs . brefeldin a ( 1 g / ml ) ( bd bioscience ) was added to the mixture and incubated for another 4 hours ( i.e. , a total of 6 hrs with peptide ) .
cells were then washed and stained for cell surface markers using anti - cd3-alexa fluor 700 ( ucht1 ) , anti - cd8-v500 ( rpa - t8 ) ( both from bd biosciences ) , anti - cd4-apc efluor780 ( rpa - t4 ) , anti - cd45ra - efluor450 ( hi100 ) , and anti - ccr7(cd197)-percpcy5.5 ( g043h7 ) ( all three from affymetrix ebioscience ) for 30 mins on ice .
cells were then washed , fixed with 4% paraformaldehyde , blocked with human sera , and stained for intracellular ifn using anti - ifn-fitc ( 4s.b3 , affymetrix ebiosciences ) .
the frequency of cells responding to the tb / denv - specific peptides was quantified by determining the total number of gated subset+ and cytokine+ cells and background values subtracted ( as determined from the medium alone control ) using flowjo software ( tree star ) .
as a preliminary step towards deriving sets of reference epitopes associated with preferred validated assays , we processed the data contained in the iedb relating to t cell epitopes .
as of october 2014 , a total of 28370 epitopes are associated with positive results in at least one t cell assay . as an example of filtering strategies ,
we first considered only peptidic epitopes associated with infectious agents and allergies ( figure 1 ) and initially focused on bacteria , viruses , and nonhuman eukaryotes as epitope sources . next , we only considered data in which humans were the host of the immune response and for which data was available to allow specifically characterizing responses as either class i or class ii restricted . in the case of class i , we further considered only epitopes of 811 residues , and in the case of class ii , only peptides of 1320 residues were considered . the next step in our process was to filter the results further by selecting epitopes that have been tested in high quality assays .
this is possible because the iedb curates the specific assays that are used to define and characterize the specific epitopes reported in the literature or provided to the database by direct submission .
while obviously any desired assay set could be used , here we selected for inclusion the multimer / tetramer staining , elispot and ics assays .
this assay - based filtering resulted in a final total of 6345 epitopes , 2512 and 3833 for class i and class ii epitopes , respectively ( figure 1 ) , representing about 20% of the initial 28370 epitopes .
we surveyed the epitope data in the iedb in terms of the species and antigens of provenance ( epitope sources ) .
the relative population of these categories in the iedb as of the 2009 date was discussed by davies et al . .
in table 2 , we list 43 categories for which at least ten class i or class ii epitopes were identified for viruses / bacteria ( table 2(a ) ) , nonhuman eukaryotes ( table 2(b ) ) , and autoantigens ( table 2(c ) ) .
the table also lists b cell epitopes as identified and discussed further below . as a result of the processes described above , we generated sets of epitopes for the various categories .
as an example , table 3 details the 21 epitopes defined for the parvoviridae human class i category .
in addition to the epitope sequence , source organism and protein , and mhc restriction , the iedb epitope i d is provided to facilitate retrieval of additional information from the iedb pertaining to the epitope .
finally , the response frequency , assay score , the types of assay , and effector origin ( ex vivo or in vitro ) , utilized to derive the assay score , are also given .
all epitope tables from the categories shown in table 2 can be found in the supplemental file
having established the conceptual framework for selection of epitope sets , we next expanded our applicability .
autoimmune epitopes are identified by the fact that both source antigen and host organism are the same ( e.g. , both the t cells and the epitope are originated from a human source ) . as listed in table 2(c ) , all autoimmunity epitope categories relevant to a given disease are condensed into a single category . a protein tree functionality available on the iedb and a list of the most common antigens associated with each disease were used to extract autoimmune epitopes .
the identified categories for which at least ten class i or class ii epitopes were available are listed in table 2(c ) , along with the number of epitopes contained in each category .
we considered expanding the scope of the study to also select epitopes recognized by species other than humans . in this case , the second most frequently represented host species is mouse .
accordingly , an option was created in the web application ( next section ) to allow selection of murine epitopes .
the number of murine epitopes identified is listed as a separate column in table 2 .
finally , we also expanded our analysis to allow selection of b cell / antibody epitopes . in this case
, we set a 5 to 20 residue size window and initially selected x - ray structure , biological activity , and in vivo assays as most biologically relevant .
in addition , we included induction of ige subclass responses , as this type is most relevant in the context of allergic diseases .
the number of b cell epitopes identified accordingly is also listed as a separate column in table 2 .
finally , we developed a tool , which will be hosted by the iedb as an additional link in the search results page and will be part of the next iedb update release in fall 2015 .
this tool allows generation of specific epitope sets following the default criteria described above but also allows users to customize the generation of novel sets .
the main interface automatically obtains query results from iedb searches and adds them to the query parameters of the tool .
there , the user may choose to generate sets of class i , class ii , antibody linear , or antibody discontinuous epitopes , relating to either human or mouse as the host organism from which the epitopes are derived .
because of the flexibility of the design , additional host organisms may be included , provided sufficient data becomes available in the literature .
an advanced options webpage can be accessed from the main page , and a sample screen shot of this option is shown in figure 2(b ) . in the advanced options
, the user can set their custom rf score threshold and assay score threshold , rank the results using the rf score or assay score , include the clustering tool , filtering by determined alleles ( known mhc allele ) , and set the minimum and maximum size if the epitopes .
also , a number of additional assays , generally considered as being less rigorous , are provided , allowing the user to set custom parameters .
every field in the advance options dynamically changes to fit the default values for a given query . for example , if the user selects mhc class ii epitopes , the web tool will set the clustering field to
to experimentally validate the usefulness of the tool we decided to synthesize some of the actual peptide sets identified by the tool and experimentally test them for recognition by human t cell responses .
one of the main challenges for testing large pools for t cell recognition is that ex vivo t cells assays require relatively substantial amounts of each epitope ( in the 10.1 microgram / ml range ) , compounded by the fact that peptide solubility in solvents most widely applicable to preparing peptide stock solutions , such as dmso , is usually limited to about 2040 mg / ml . since solvents like dmso are toxic in cellular assays at concentrations above 0.5% , this would seem to effectively limit the number of peptides that can be safely included in a pool to about 20
. however , in many cases the solubility of one peptide is not drastically influenced by the presence of other peptides ( especially if the sequences , isolectric point , and general solubility are different ) .
for this reason , we predicted that it might be possible to make pools of peptides already dissolved in a solvent like dmso , mix the solutions , and relyophilize the pool of pools .
indeed , we routinely find these sequentially lyophilized pools , once resuspended , to be much more soluble than the individual components .
accordingly , we synthetized a set of 207 ebv human cd8/class i epitopes , identified by the default setting described above ( supplemental table 1a ) .
in addition , we also synthetized a set of 92 cd8/class i epitopes derived from denv virus , obtained by selecting only peptides with rf > = 0.01 ( supplemental table 1b ) , and a set of 86 epitopes cd4/class ii epitopes derived from mycobacterium tuberculosis , based on an rf score > = 0.1 and being recognized ex vivo ( supplemental table 1c ) .
peptides corresponding to these three sets of epitopes were pooled and tested with human pbmc as a source of t cells .
for these experiments we selected pbmc from 5 individuals infected with denv virus and likely uninfected with tb ( see methods for details ) and pbmc from 5 ltbi individuals and likely uninfected with denv .
because of the high incidence of ebv infection worldwide , we assumed that most if not all individuals tested would be latently infected with ebv .
pbmc were stimulated with the denv cd8 pool , mtb cd4 pool , and ebv cd8 pool .
after ex vivo stimulation , the ifn response was measured by ics ( figures 3(a)3(d ) ) . the gating strategy for these experiments
representative plots of the responses for both cd4 and cd8 t cells are shown for a ltbi donor ( figure 3(b ) ) and a denv donor ( figure 3(c ) ) .
the ifn responses induced by each epitope pool in the relevant cd4/cd8 compartments for all donors tested are summarized in figure 3(d ) . in the case of the mtb cd4 pool ,
ex vivo cd4 but not cd8 t cell responses were seen in the ltbi ( p = 0.004 ) and denv donors .
the reactivity was lower in the denv donors than in the ltbi donors ( p = 0.03 ) .
this lower , but detectable , reactivity is explained by the fact that the denv seropositive individuals are vaccinated against mtb with m. bovis bcg and are likely exposed to nontuberculous mycobacteria . in the case of the denv cd8 pool ,
cd8 t cell responses , but not cd4 , were detected in 3 out of 5 denv donors .
this ex vivo reactivity in 3 out of 5 donors is in line with what was detected in previous studies .
as expected , no cd4 responses were seen to the cd8 denv pool , as also low cd8 and cd4 t cell reactivity was noted in the mtb donors with the denv cd8 pool . finally , as also expected , in both cohorts reactivity was detected against the ebv cd8 pool in cd8 but not in cd4 t cells ( p = 0.004 for ltbi and p = 0.05 for denv ) .
we devised a strategy that allows automatically filtering datasets to select epitopes of appropriate size , defined restriction , and assay type for use in characterizing responses to specific indications . while querying the iedb database
can also generate these sets , a certain degree of complexity in the queries and the setting of multiple parameters would be necessary . in our application ,
the epitope sets are automatically generated , while the user is still enabled to change the default settings to generate validated epitope sets matching specific criteria .
we further identified which epitope categories are supported by current iedb data , and found that reference epitope sets could be produced for 43 categories with data currently available in the iedb .
the number of such categories , broadly based on previous epitope classification work , is undoubtedly destined to grow , allowing an ever more comprehensive study of immune responses in a broad variety of experimental systems . to further illustrate the broad applicability of the approach
, we also extended our work to address autoimmune epitopes , b cell epitopes , and murine epitopes .
while these actual epitope sets are provided as tables within the paper , we implemented a web application to automatically generate epitope sets , based on the fact that the iedb content is rapidly growing and new epitopes are added to the iedb in each of its biweekly updates .
we plan to continuously gather feedback on this web application from the scientific community , and to implement changes and modifications through the main iedb website . finally , to illustrate applicability in an actual experimental setting , we selected and synthesized peptide sets corresponding to ebv , denv , and mtb epitopes .
the experimental testing of these epitope sets demonstrated the applicability of these sets as a valuable resource to allow detection of t cell responses ex vivo .
as such , these sets of epitopes represent potentially valuable resources for diagnostic purposes , as well as for further detailed characterization of the immune response to specific targets of immunological interest . | accurate measurement of b and t cell responses is a valuable tool to study autoimmunity , allergies , immunity to pathogens , and host - pathogen interactions and assist in the design and evaluation of t cell vaccines and immunotherapies . in this context
, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of t and b cells .
however , the ever - growing information contained in the immune epitope database ( iedb ) and the differences in quality and subjects studied between epitope assays make this task complicated . in this study
, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the iedb . from the sets
generated , three epitope sets ( ebv , mycobacteria and dengue ) were experimentally validated by detection of t cell reactivity ex vivo from human donors . furthermore , a web application that will potentially be implemented in the iedb was created to allow users the capacity to generate customized epitope sets . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions |
renal cell carcinoma , which accounts for approximately 3% of all adult cancers , has a tendency to metastasize , commonly to the lungs , bones , liver , adrenal glands and brain .
metastases to the breast from extramammary tumours are uncommon and metastatic renal cell carcinoma to the breast is extremely rare accounting for 3% of the cases .
we report metastases to the breast and the difficulty in diagnosis . thus far , this is the first case reported as in most cases ; the metastasis to the breast is part of systemic metastases .
the patient is a 67 year old lady , diagnosed with right renal cell carcinoma in 2002 for which she underwent right radical nephrectomy . histological examination showed a clear cell type .
regular follow - ups with computed tomography of the abdomen were negative for 5 years . in march 2007
clinical examination revealed a 2 2 cm right breast swelling and a vague retro - areolar mass 1 1 cm with an enlarged ipsilateral axillary lymph node .
mammogram ( figure 1 ) revealed a dense spiculated mass at right upper outer quadrant and a retroareolar mass which is associated with clustered micro - calcifications suggestive of malignancy .
ct thorax and abdomen showed a speculated hyper - dense lesion in the right breast and there was no evidence of local recurrence of rcc or other organ metastasis .
mammogram : dense speculated mass at the right upper outer quadrant and a retro - areolar mass with clustered mocrocalcification ultrasound : irregular mass with acoustic shadow at right 9 o'clock and another well - defined solid mass with irregular margin at 3 o'clock position . in view of cytological findings , mammogram and absence of distant metastases which suggested a possibility of either multicentric breast carcinoma or metastatic renal cell carcinoma , we proceeded with right mastectomy and axillary clearance .
the diagnosis of metastatic renal cell carcinoma to the breast with one lymph node involvement was made .
the diagnosis of metastatic renal cell carcinoma to the breast with one lymph node involvement was made .
it is represented by nearly up to 2% of all breast swelling which account for nearly 400 cases that have been reported in the literature .
malignant melanoma , leukaemia and lymphoma are the most common among all malignancies that have been described metastasizing to the breast .
8 cases presented as the initial sign of metastasis from rcc while the rest occur after nephrectomy done for rcc .
the cases presented are usually found 1 to 18 years following nephrectomy for rcc [ 3 , 4 ] . in the case presented , the metastasis occurred 5 years after nephrectomy .
the survival rates for the patient with renal cell carcinoma , 5-year and 10-year post nephrectomies were 95% and 91% for pt1 ; 80% and 70% for pt2 ; 66% and 53% for pt3a ; 52% and 43% for pt3b ; and 43% and 42% for pt3c , respectively .
the most common extra mammary metastasis in mammography finding was a well circumscribed mass with increased density but without speculation , calcifications or other signs that characterize the majority of primary carcinomas .
micro calcifications are unusual and are mostly associated with metastatic carcinoma of the ovary , in which psammoma bodies may be abundant . however , in our study , the case presented with microcalcifications as shown in the mammography study along with dense speculated mass at ruq area .
this produce suspicious of primary breast carcinoma with metastatic disease from rcc as a provisional diagnosis .
treatment for metastasis from rcc after a long - term cancer - free survival is removal of the tumour bloc in total followed with immunotheraphy , targetted therapy and chemotherapy .
this is based on a clinical trial indicating that metastasectomy rcc ( mrcc ) whereby solitary and fully resectable lung metastasis , together with adequate performance status , then the treatment of choice should be metastasectomy .
( i do not understand this paragraph from the linguistic point of view ) best outcomes are seen with single site of rcc metastasis to lung where 50% 5 years survival is seen compared with 19% , in patients who had more than one site of cancer removed . usually , rcc metastasis to other tissue is diagnosed after tissue biopsy and most of the time , cytological biopsy is enough to confirm the diagnosis
, the fine needle aspiration cytology ( fnac ) which was repeated twice suggests metastatic disease ( the presence of malignant cells with predominant eosinophillic cytoplasm with minority of them having cytoplasmic clearing cells ) but can not exclude primary breast carcinoma . due to this limitation ,
definitive diagnosis is important such as to prevent unnecessary mastectomy in extramammary tumour such as rcc metastasis .
treament for metastasis from rcc after a long - term cancer - free survival is removal of the tumour bloc in total followed with immunotheraphy , targetted therapy and chemotherapy .
alpha interferon and interleukin ( il)-2 are the two most frequently used types of immunotherapy in metastatic renal cell cancer .
unfortunately , long - term results of clinical trials indicate that only approximately 15% of patients with advanced renal cell carcinoma have an anticancer response when treated with high - dose interleukin 2 and interferon . in the presented case ,
we have proceeded with mastectomy and axillary clearance after full discussion with the patient and the family as cytological and mammogram study were strongly suspicious of breast carcinoma with no evidence of recurrence of rcc and patient opted for mastectomy .
careful assessment and adequate tissue examination is paramount to confirm the diagnosis . resection of isolated metastasis from rcc is feasible and can achieve long term relapse free survival . | breast is an uncommon and rare site for metastasis .
primary and secondary tumors of the breast need to be differentiated as management is different .
we present a 67 year old female patient with two breast lumps and an axillary lymph node , 5 years after nephrectomy for renal cell carcinoma ( rcc ) .
mammogram report showed a dense spiculated mass at right upper outer quadrant and a retroareolar mass associated with clustered micro calcifications .
fine needle cytology and trucut biopsy were inconclusive .
computer topography ( ct ) abdomen did not show evidence of rcc recurrence .
after discussion with the patient , she underwent mastectomy with axillary clearance and the final histopathology report was consistent with metastasis from rcc .
the management of this case is discussed . | Introduction
Case report
Histological section (
Discussion
In summary |
the tumor was named aggressive due to its characteristically slow and insidious growth as well as carrying a moderate - to - high risk of local relapse .
usually , it presents as a vulval polyp clinically and is diagnosed only on histopathology .
it is a rare local mesenchymal tumor of unknown etiology usually affecting vulva , perineal region , buttocks , or pelvis of women in reproductive age .
considering its nature of aggression and chance of local relapse , appropriate management and long - term follow - up are necessary to diagnose early recurrence .
no single modality of treatment of recurrence has been found to be of proven benefit till now .
radiotherapy and chemotherapy have been used as adjunctive therapies but are unlikely to be useful as it has few mitotic activity . despite the availability of many options of treatment , recurrence of aa
a 40-year - old female para 2 presented with a swelling on the right labia majora with a duration of 3 years growing slowly throughout and increased in size for 6 months [ figure 1 ] . pedunculated large , partly encapsulated growth from vulva there was no history of any vulval discharge , bleeding , sexual difficulty , or pain , except a sensation of weight hanging while standing .
menstrual cycles were regular with a normal flow . local examination revealed a well - circumscribed pedunculated fleshy polypoidal mass measuring 18 cm 10 cm .
ultrasonography ( usg ) of the swelling revealed heterogeneous hyperechoic areas with peripheral vascularity , thick echoes , and nonvascular central areas .
computed tomography ( ct ) scan of pelvis revealed no pelvic disease inside and had similar findings of the mass like usg . with a clinical diagnosis of a vulvar fibroepithelial polyp or lipofibroma , she underwent local excision of the tumor with ligation of the stalk [ figure 2 ] .
the cut surface revealed a glistening , gelatinous , and soft homogeneous appearance [ figure 3 ] . on histopathology ,
the tumor was composed of spindle- and stellate - shaped cells scattered in a myxoid background [ figures 4 and 5 ] .
, there would be spindle cells in fatty background with minimum of vessels unlike aa .
there were many variable - sized thin - walled and thick - walled vascular channels in the histopathological specimen , which were diagnostic of aa .
a 6 monthly follow - up has been done for more than 2 years now without any specific therapy for the prevention of recurrence , yet showing no sign of any relapse so far .
cut open specimen of angiomyxoma showing homogeneous white area high power picture of angiomyxoma , ( h and e , 40 ) immunohistochemistry picture of angiomyxoma , cd34 ( low power ) postoperative picture of vulva after resection of angiomyxoma
the aa tumor commonly presents as an asymptomatic mass in the genital area of women in their reproductive life , but is occasionally reported in men ( male - to - female ration being 1:6 ) . the term aggressive denotes its propensity for local aggression and recurrence after excision .
clinically , aa may misdiagnosed as bartholin cyst , lipoma , labial cyst , gartner duct cyst , etc .
superficial angiomyxoma , angiomyofibroblastoma , cellular angiofibroma , and smooth muscle tumors also need to be considered in the differential diagnosis of a polypoidal mass in the perineum .
in addition , aa has thick - walled vessels , which are less numerous than thin - walled vessels in angiomyofibroblastoma .
on ct scan , these tumors have a well - defined margin with attenuation less than that of muscle .
the attenuation on ct and high signal intensity on magnetic resonance imaging ( mri ) are likely to be due to the presence of loose myxoid matrix and high water content of aa .
usually , this tumor does not metastasize , but there are reports of multiple metastases in women treated initially by excision and ultimately succumbing to it .
this hormonally responsive tumor is believed to arise from specialized mesenchymal cells of the pelvic perineal region or from the multipotent perivascular progenitor cells , which often display variable myofibroblastic and fibroblastic features .
immunohistochemically , most aa express different combinations of estrogen and progesterone receptors , vimentin , desmin , smooth muscle actin cd34 , and cd44 , but all are invariably negative for s-100 , carcinoembryonic antigen , and keratin .
recent cytogenetic and molecular studies have revealed a variety of genetic alterations , involving the chromosome 12 , in the region 12q13 - 15 . a gene in this region , called high - mobility group protein isoform i - c ( hmgi - c ) , which encodes protens involved in the transcriptional regulation , appears to have a role in the pathogenesis of this tumor .
detection of inappropriate hmgi - c expression using the immunoperoxidase technique with anti - hmgi - c antibody may potentially be a useful marker for microscopic residual disease .
unlike most aas , our case was completely encapsulated without any breach in continuity and without any projections into the neighboring tissues .
this may be the reason why there was no recurrence in the past 2 years or so .
considering her very poor economic status and the hpe report , we decided not to start any preventive therapy , i.e. , gnrh agonist , etc .
, in her case . on h and e staining , the tissue resembled a typical aa , and immunohistochemically , it showed positive for cd34 , desmin , and vimentin , hence proving beyond any doubt that it was a case of vulval aa .
our patient required no additional treatment or any investigations postoperatively till date and has been asymptomatic and enjoying good health . despite this fact , aa is notorious for local recurrence in approximately 70% of the cases after a period of 2 years postoperatively , and it has been reported even 20 years after surgery as well .
proposed the following guideline for treating aa : ( 1 ) complete excision of the lesion when possible , avoiding mutilating surgery , ( 2 ) adjunct therapy using arterial embolization and/or hormonal treatment needed in case of partial resection of the tumor , and ( 3 ) radiotherapy is reserved for cases that are resistant to embolization and/or hormonal therapy and still symptomatic .
there are no specific guidelines for postoperative management of vulvar aa ; however , due to high recurrence rate and potential morbidity associated with undiagnosed recurrences , several authors recommend a periodic evaluation with physical examination and mri up to 15 years after excision .
this case report illustrates the challenges that a physician might face when dealing with a vulvar mass which may be an aa .
though it is a rare entity , it should always be considered , especially when it is an insidious painless lesion , particularly in premenopausal women in their third to fourth decades of life .
all relevant hematological and radiological studies including mri or ct scan should help in reducing the number of misdiagnosed cases of aa preoperatively .
once its anatomical location and extension , if any , are defined , any vulvar tumor particularly aacan be optimally treated by surgical excision only , while avoiding any mutilating surgery .
if complete resection is possible under the circumstances , one should expect lowest recurrence rate .
aa is rarely life - threatening , and therefore one can afford to have a partial resection when high operative morbidity is anticipated .
irrespective of treatment modalities instituted postsurgery , it is evident that aa requires close and long - term follow - up . | aggressive angiomyxoma is a rare , locally invasive mesenchymal tumor predominantly presenting in women of reproductive age and also having a moderate - to - high risk for local relapse .
hence , it needs to be differentiated from other mesenchymal tumors occurring in this region .
we present here a case of a 40-year - old female presenting with a large , fleshy , pedunculated mass on the right labia majora . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest |
esophageal cancer ( ec ) is one of the most fatal types of cancer wordwide , with 455,800 new cases and 400,200 deaths in 2012 .
esophageal squamous cell carcinoma ( escc ) is the predominant type in china , in contrast to the predominance of esophageal adenocarcinoma in the western countries , which covers more than 90% of all ec cases .
surgery remains the treatment of choice for localized cancer ; however , the prognosis is still poor . in recent years
previous studies have shown that a number of inflammatory biomarkers , such as c - reactive protein ( crp ) , neutrophil to lymphocyte ratio ( nlr ) , and platelet to lymphocyte ratio ( plr ) , were associated with prognosis in several types of cancer , including ec .
recently , the systemic immune - inflammation index ( sii ) is a useful prognostic indicator for patients with small cell lung cancer and hepatocellular carcinoma . to the best of our knowledge ,
however , no studies regarding sii in patients with ec are available . in this study , therefore , we aimed to determine the prognostic value of sii in patients with resectable escc .
a total of 298 patients with resectable escc were included in the current retrospective study from jan 2005 to dec 2008 .
the eligibility criteria were included : ( 1 ) escc diagnosed by histopathology ; ( 2 ) curative surgery for localized disease ( stage i iii ) ; ( 3 ) without neoadjuvant treatment ; ( 4 ) without any form of acute and/or chronic inflammatory disease ; and ( 5 ) preoperative serum laboratory examination were obtained before surgery within 1 week .
the counts for neutrophil , lymphocyte , and platelet were taken within 1 week prior to surgery .
the sii was defined as follows : sii = neutrophil platelet / lymphocyte . in the current study , a cancer - specific survival ( css )
chicago , il ) and r 3.2.3 software ( institute for statistics and mathematics , vienna , austria ) .
a web - based r software ( cutoff finder ) was used to determine the optimal cut - off value for sii .
cutoff finder is a freely available web application that can be accessed using an arbitrary web browser ( http://molpath.charite.de/cutoff ) .
a nomogram model was also established and the predictive accuracy was evaluated by harrell 's concordance index ( c - index ) . in the current study , p
statistical analyses were conducted with spss 17.0 ( spss inc . , chicago , il ) and r 3.2.3 software ( institute for statistics and mathematics , vienna , austria ) .
a web - based r software ( cutoff finder ) was used to determine the optimal cut - off value for sii .
cutoff finder is a freely available web application that can be accessed using an arbitrary web browser ( http://molpath.charite.de/cutoff ) .
a nomogram model was also established and the predictive accuracy was evaluated by harrell 's concordance index ( c - index ) . in the current study , p <
of the total number of patients , 38 ( 12.8% ) were women and 260 ( 87.2% ) were men .
the mean values for neutrophil , platelet , lymphocyte , and sii were 4.54 1.92 ( 10/l ) , 225 74 ( 10/l ) and 750 482 ( 10/l ) , respectively .
additionally , correlation analyses showed that sii was negatively correlated with lymphocyte ( r = 0.362 , p < 0.001 ) and positively correlated with neutrophil ( r = 0.620 , p < 0.001 ) and platelet ( r = 0.607 , p <
sii was negatively correlated with lymphocyte ( r = 0.362 , p < 0.001 ; a ) and positively correlated with neutrophil ( r = 0.620 , p < 0.001 ; b ) and platelet ( r = 0.607 , p < 0.001 ; c ) .
the optimal cut - off value for sii was 410 ( 10/l ) according to the cutoff finder ( fig .
patients then were divided into 2 groups using the sii cut - off of 410 ( 10/l ) .
there were 81 ( 27.2% ) patients with sii 410 ( 10/l ) and 217 ( 72.8% ) patients with sii > 410 ( 10/l ) .
the relationships between sii and clinical characteristics were shown in table 1 . in the current study ,
the sii was significantly associated with tumor length ( p = 0.006 ) , tnm stage ( p = 0.012 ) , nlr ( p < 0.001 ) , plr ( p < 0.001 ) , and crp ( p < 0.001 ) .
the vertical line designates the optimal cut - off point with the most significant ( log - rank test ) split .
the cut - off value was 410 with a sensitivity of 73.6% and a specificity of 47.1% .
patients with sii 410 ( 10/l ) had a significantly better 5-year css than patients with sii > 410 ( 10/l ) ( 51.9% vs 24.0% , p <
, we revealed that sii was also significantly associated with css based on the tnm stage ( fig .
in multivariate analyses , sii was an independent prognostic factor in patients with resectable escc ( p = 0.027 ) .
however , nlr ( p = 0.661 ) or plr ( p = 0.064 ) were not significant independent predictors for css ( table 3 ) . in the current study , we used the crp , a well - known inflammatory indicator , in multivariate analyses .
our result revealed that crp was still an independent prognostic factor in patients with escc ( p < 0.001 ) .
meier css curves stratified by sii . patients with sii 410 ( 10/l ) had a significantly better 5-year css than patients with sii > 410 ( 10/l ) ( 51.9% vs 24.0% , p <
the predictive value of sii was significant in patients based on the tnm stage ( i stage : 61.8% vs 35.0% , p = 0.005 ; b ) , ( ii stage : 48.3% vs 27.1% , p = 0.028 ; c ) , and ( iii stage : 38.9% vs 17.8% , p = 0.045 ; d ) .
css = cancer - specific survival , sii = immune - inflammation index , tnm = tumor node metastasis .
to predict the risk for patients with escc , a novel nomogram model was established by sii , crp , and tnm stage combined with age and sex ( fig .
4 ) . it can predict the probability of death for patients with escc . in the current study ,
to the best of our knowledge , this is the first report to demonstrate the prognostic role of sii in patients with resectable escc . in this study
, we revealed that sii was an independent significant predictive factor ( p = 0.027 ) .
we performed a cutoff finder based on r software to verify to optimal cut - off value and conclude that 410 ( 10/l ) may be the optimal cut - off value for sii in predicting css in patients with resectable escc .
in addition , the current study was also the first attempt to establish a predictive nomogram model to improve predictive accuracy based on sii .
, we analyzed the prognostic role of sii in escc patients without neoadjuvant treatment mainly because neoadjuvant therapy ( chemotherapy and/or radiation ) will have an important impact on the inflammation .
previous studies have shown that nlr and plr were independent predictors of survival in various cancers , including ec . however , due to the inconsistent results , the prognostic values of nlr and plr in ec remain controversial .
recently , we conducted a meta - analysis revealed that high levels of nlr were significantly correlated with poor survival in patients with ec . in the current study
however , we demonstrated that plr , but not nlr , was an independent prognostic factor in patients with resectable escc .
sii was initially performed to indicate the host inflammatory status in patients with resectable hepatocellular carcinoma .
then , another study confirmed that sii was superior to nlr and plr as an independent factor for patients with small cell lung cancer . in the current study , we revealed that sii was a significant predictive indicator in patients with resectable escc ( p = 0.027 ) .
however , nlr ( p = 0.661 ) or plr ( p = 0.064 ) were not significant independent predictors for css .
moreover , we used a cutoff finder based on r software to verify the optimal cut - off value , which was 410 ( 10/l ) compared to 330 ( 10/l ) and 1600 ( 10/l ) in previous studies .
it is well know that nomogram could establish a simple graphic representation of a statistical predictive model . in the current study , therefore , we attempt to establish a predictive nomogram model to predict the probability of the death risk for resectable escc patients based on sii , crp , and tnm stage combined with age and sex . the nomogram performed well in predicting css by the c - index ( 0.68 ) .
second , we excluded patients who had neoadjuvant chemotherapy and/or radiotherapy , which may have influenced our results .
in addition , our study revealed that sii is an independent predictive factor in patients with escc ; however , it should be kept in mind that the sensitivity and specificity looks like not very high . moreover
, the c - index showed that the nomogram model has a good accuracy but it is not perfect indicating that there is still room for improvement .
therefore , further studies are needed to illuminate the relationship between sii and prognosis in patients with resectable escc .
in summary , our study showed that sii is still a useful prognostic indicator for patients with resectable escc .
we conclude that 410 ( 10/l ) may be the optimal cut - off value for sii . | abstractthe aim of the study was to determine the prognostic role of systemic immune - inflammation index ( sii ) in patients with esophageal squamous cell carcinoma ( escc).a total of 298 escc patients were enrolled in the current retrospective study .
the sii was calculated by the formula : neutrophil platelet / lymphocyte .
the optimal cut - off value was calculated by the cutoff finder .
univariate and multivariate analyses were evaluated for cancer - specific survival ( css ) .
additional , we also established a nomogram model to predict the prognosis for patients with escc.the optimal cut - off value was 410 109/l for sii .
patients with sii 410 ( 109/l ) had a significantly better 5-year css than patients with sii > 410 ( 109/l ) ( 51.9% vs 24.0% , p < 0.001 ) .
multivariate analyses revealed that sii was a significant independent predictive indicator ( p = 0.027 ) .
a nomogram could be more accuracy for css for patients with escc ( c - index : 0.68).the sii is a useful independent prognostic indicator for patients with resectable escc . | Introduction
Materials and methods
Statistical analysis
Results
Discussion
Conclusion |
briefly , two cases , both with meningitis , occurred in young adults , who recovered .
the remaining three cases , two with bacteremic pneumonia and one with septicemia , were in elderly patients , who died .
however , the towns they lived in were quite distant , and the patients were admitted to different hospitals in different periods .
serotyping of the isolates by slide agglutination was performed by using polyvalent and monovalent antisera to capsular serotypes a through f ( difco laboratories , detroit , mi ) .
briefly , in a first round of pcr , primers to the ompp2 gene were used to confirm the h. influenzae species , while primers directed to the bex region confirmed capsulation . a second round of pcr with primers directed to the cap e specific region ( 6 ) generated an expected product of 1,350 bp .
biotypes were assigned by determining indole production , urease level , and ornithine decarboxylase activities .
mics of ampicillin were determined by e - test ( ab biodisk , solna , sweden ) .
susceptibilities to trimethoprim / sulfamethoxazole , ceftazidime , chloramphenicol , azithromycin , aztreonam , ciprofloxacin , imipenem , and tetracycline were tested by disk diffusion assay .
both the e - test and the disk diffusion assay were performed by using haemophilus test medium , as recommended by the national committee for clinical laboratory standards ( nccls ) ( 7 ) .
production of -lactamase was detected by the cefinase disk test ( bbl , becton - dickinson , sparks , md ) .
restriction fragment length polymorphism analysis by pfge was conducted as described ( 5 ) , except for digestion of genomic dna , performed by using smai ( 20 u ) or apai ( 20 u ) restriction enzymes ( new england biolabs , wilbury way hitchin , u.k . ) .
the copy number of cap e locus was determined by southern blot analysis , using as probe the 1,350-bp amplicon obtained by pcr of a prototype type e strain .
since the kpni and smai sites flank the cap locus of encapsulated h. influenzae strains , the copy number of the locus can be estimated by the size of the restriction fragment obtained after digestion of the chromosome with these enzymes ( 8) .
restriction fragments were separated by pfge as described ( 5 ) , transferred to nylon membranes , and hybridized with the probe .
labeling of the probe and hybridization reactions were obtained by using the ecl kit ( amersham pharmacia biotech , little chalfont , u.k . ) .
although no data are available on the size of cap e locus , on the basis of results obtained on hib strains , the dna fragment for a single - copy strain was expected to be approximately 2728 kb .
in fact , the kpni / smai fragment includes the cap locus , whose size is 18 kb in hib strains , plus additional segments ( about 10 kb ) upstream and downstream of the cap region ( 8) .
strains with two or more copies of the cap b locus featured fragments of increased size ( 45 kb , 63 kb , 81 kb , 99 kb ) ( 8) .
hie , haemophilus influenzae type e ; csf , cerebrospinal fluid ; pfge , pulsed - field gel electrophoresis ; m , male ; f , female . by pcr
, all isolates exhibited the type e capsular genotype . by slide agglutination method , performed at the national reference laboratory ,
four isolates were designed as type e , and one was misidentified as nontypable . at the regional level , only one of the five pcr - positive strains had been recognized as type e , two had been identified as nontypable , and two had not been typed . by biotyping ,
four isolates were classified as biotype iv , and one as biotype i ( patient no .
mics of ampicillin ranged from 0.125 g / ml to 0.25 g / ml , indicating that all isolates were susceptible to this antibiotic ; none produced -lactamase . as assessed by disk diffusion assay , all isolates were also susceptible to trimethoprim / sulfamethoxazole , ceftazidime , chloramphenicol , azithromycin , aztreonam , ciprofloxacin , imipenem , and tetracycline .
pfge with smai restriction enzyme digestion generated not well - resolved profiles , as several very close fragments of 194145 kb were obtained ( data not shown ) .
following apai digestion , profiles were easier to compare ( figure ) : three isolates ( patients no . 3 , 4 , and 5 ) shared an indistinguishable pattern ( pattern 1 ) ; one isolate ( patient no .
1 ) showed a profile closely related to pattern 1 but with two band differences ( pattern 1a ) .
2 appeared clearly different from the others ( pattern 2 ) , according to criteria reported by tenover et al .
all hie isolates were unrelated to invasive hib strains circulating in italy ( 9 ) ( figure ) .
according to epidemiologic data , patients with strains that showed an indistinguishable pfge pattern did not appear to share any common risk factor .
southern hybridization with the probe for the cap e gene after pfge identified a single fragment of 20.5 kb in each isolate tested , suggesting the presence of one copy of the cap locus ( table 2 ) .
pulsed - field gel electrophoresis patterns of apai - digested chromosomal dnas of haemophilus influenzae isolates .
lanes 15 , h. influenzae type e ( hie ) isolates , respectively , from patient nos . 15 ; lane 6 , h. influenzae type b ( hib ) strain belonging to one of the subclones endemic in italy ( 9 ) ; m , ladder pulsed - field gel marker with molecular weights indicated in kilobases ( kb ) at the right .
the isolates in lanes 3 , 4 , and 5 showed indistinguishable profiles ( pattern 1 ) ; the isolate in lane 1 was closely related to pattern 1 ( pattern 1a ) , whereas the isolate in lane 2 was clearly different from pattern 1 ( pattern 2 ) .
although studies on nasopharyngeal carriage of h. influenzae have shown the presence of serotype e as colonizer ( 1113 ) , few studies have described serious infections attributable to this capsular type ( 1417 ) .
our results suggest that hie may cause either fatal infections in elderly patients with underlying disease or meningitis in adults whether or not underlying conditions are present . in italy , these cases of hie infection are the first reported . despite limited data on the frequency of non type b invasive disease before 1997 , our results are based on 4 years of laboratory - based active surveillance on a large population sample . since no type e strains were detected in the first 2 years of this surveillance , this clustering is likely due to the emergence of invasive hie disease .
alternative explanations might include improved laboratory confirmation methods ( however , the proportion of meningitis cases without an identified etiologic agent did not decrease from 1998 to 2001 ) or improved reporting in the last 2 years of surveillance ( however , no changes to the system were made , and quality indicators , such as proportion of isolates sent to the reference laboratory , remained stable throughout the 4 years ) .
an unequivocal method of assigning capsular type is required to monitor infection attributable to uncommon serotypes , especially since , as in some hie isolates , expression of capsular polysaccharide is not sufficient to be detected by slide agglutination ( 18 ) . in our study ,
pcr capsular typing allowed the identification of an additional hie strain that had been misidentified by slide agglutination .
the prevalence of ampicillin resistance in both -lactamase positive and negative h. influenzae isolates has increased worldwide . neither -lactamase production nor intrinsic resistance was detected among our hie isolates , confirming the low incidence of ampicillin - resistant h. influenzae strains from invasive disease in italy ( 5,9 ) .
four of the five isolates , found in two neighboring regions , appeared to represent a unique clonal group with two subtypes .
this clustering of most strains in one pfge pattern might be explained with the clonal population structure of encapsulated h. influenzae previously observed in italy ( 5,9 ) , but further studies are needed to clarify this point .
moreover , since the type e isolates analyzed in this study had no close genetic relationship to the invasive hib strain circulating in italy , their derivation from type b isolates by capsular switch is unlikely .
h. influenzae pfge typing has a stronger discriminatory power than biotyping ( 19 ) . in our study , however , in the isolates assigned to the same clonal group by pfge , one was classified as biotype i , and the others were biotype iv .
these findings suggest that biotyping could be a useful adjunct to pfge when typing hie isolates . in our study , all five hie isolates contained a single copy of the cap locus , suggesting that they did not possess unusual virulent traits related to the capsule .
since the size of the restriction fragment obtained was smaller than expected on the basis of cap b locus size , a possible explanation may be that the size of region 2 of cap e locus is smaller than that found in hib strains .
alternatively , the dna flanking the cap e locus may differ from that found in hib strains ; therefore , the segments upstream and downstream from the cap e region would be smaller .
both the laboratory - based active surveillance of invasive h. influenzae disease and pcr capsular genotyping could have improved the capability to detect cases attributable to serotypes other than b. nevertheless , our data suggest the emergence of invasive hie disease among the adult population in italy and underline the need to closely monitor infection caused by non | we describe the first reported cases of invasive type e haemophilus influenzae disease in italy .
all five cases occurred in adults .
the isolates were susceptible to ampicillin and eight other antimicrobial agents .
molecular analysis showed two distinct type e strains circulating in italy , both containing a single copy of the capsulation locus . | The Study
Conclusions |
recent advances in endoscopic resection techniques , such as endoscopic submucosal dissection ( esd ) , allow for en bloc curative resection of early gastric cancers ( egcs ) with a minimal risk of lymph node metastasis ( lnm ) , regardless of tumor size or presence of submucosal fibrosis .
esd has several merits over gastrectomy : it leads to a more accurate histopathological diagnosis , it is a minimally invasive procedure , and it demonstrates a high rate of curative resection and a low rate of local recurrence .
thus , esd has been accepted as a standard and safe treatment modality for egcs in eastern asia including korea and japan [ 1 - 4 ] .
esd for egcs with absolute and expanded indications is accepted as a safe treatment strategy , according to recent long - term follow - up studies on esd for egcs in eastern asia
. however , the results for esd of egcs with specialized histologies are somewhat different from those of egcs with common histologies .
furthermore , there have been few reports on the safety and outcomes of esd for egcs with specialized histologies .
here , i summarize the outcomes of esd for egcs with two rare specialized histologies on the basis of my experience .
it demonstrates well - differentiated structures and its histopathologic characteristics are epithelial projections which are scaffolded by a central fibrovascular core . until now , the biological behavior and prognostic significance of pac are unclear due to the rare incidence of pac . at present ,
pac is classified into intestinal - type adenocarcinoma using the lauren classification , and as a differentiated - type adenocarcinoma using the japanese classification of gastric carcinoma .
however , it is reported that pac has higher rates of liver metastasis and lnm , and a lower 5-year overall survival rate than non - papillary gastric adenocarcinomas , such as tubular adenocarcinoma . considering that
pac has a more aggressive nature compared with other gastric adenocarcinomas , an inevitable question occurs whether it could be treated under the same esd indication criteria as tubular adenocarcinomas .
it is unlikely that the same esd indication criteria can be rationally applied to gastric adenocarcinomas with and without considerable pac components [ 10 - 12 ] . according to data from my hospital ( pusan national university hospital , busan , korea )
, 49 patients underwent surgery for egc with pac between january 2005 and may 2013 .
the lnm rate was 7.1% ( 1/14 ) in mucosal cancers , which was higher than that in differentiated - type mucosal cancers ( 0.4% ) , but similar to that in undifferentiated - type mucosal cancers ( 4.2% to 7.3% ) .
when the current esd indication criteria were applied to these 49 patients , 17 patients met the esd indication criteria , as follows : six patients in the absolute indication criteria ( mucosal cancer 20 mm in size without ulcerative findings ) and 11 patients in the expanded indication criteria ( four patients , mucosal cancer > 20 mm in size without ulcerative findings ; two patients , mucosal cancer 30 mm in size with ulcerative findings ; and five patients , minute [ < 500 um from the muscularis mucosa ] submucosal cancer 30 mm in size ) . among the 17 patients with egc who met the esd indication criteria , two patients ( 11.8% ) had lnm ( table 1 ) . during the same period ,
24 patients having egc with pac underwent esd as a primary treatment at the same hospital . in the pre - esd diagnostic work - up
, 10 patients met the absolute esd indication criteria and 14 patients met the expanded esd indication criteria . in the final post - esd histopathologic examination , 13 patients ( 54% ) achieved an out - of - esd result , nine had lymphovascular invasion or deep submucosal invasion , three had mucosal cancer > 30 mm in size with ulcerative findings , and one had minute submucosal cancer > 30 mm in size . of these patients ,
nine ( 37.5% ) underwent additional gastrectomy with lymph node dissection for non - curative resection .
the frequency of additional gastrectomy in patients with pac is higher compared to the previously reported frequency of additional surgery after esd ( 2.1% to 14.6% ) .
therefore , considering the higher frequency of lnm and additional surgery associated with this technique , esd should be more carefully performed for egcs with pac , even in cases with suspected esd indications after the pre - esd work - up , compared with other differentiated - type adenocarcinomas .
gastric carcinoma with lymphoid stroma ( gcls ) is a rare histological variant of gastric cancer , according to the 2010 world health organization classification system , and it accounts for 1% to 4% of all gastric carcinomas .
typically , the characteristic histopathologic features of gcls include poorly developed tubular structures and prominent lymphoid infiltrates in non - desmoplastic stroma
. however , there are no standardized diagnostic criteria and the histolopathological diagnosis for gcls is obscure .
gcls has distinct clinicopathologic characteristics and it is generally accepted that patients with gcls have a favorable prognosis with a low lnm rate .
it has been reported that an increase in the number of tumor - infiltrating lymphocytes , which are reflective of the host s immune response to tumor cells , is significantly associated with reduced metastasis and improved survival .
however , gcls has histopathologically poorly developed tubular structures , and , therefore , it tends to be regarded as undifferentiated - type adenocarcinoma in the esd indication criteria . according to the data from my hospital , out of 59 patients with egc with gcls who underwent surgery between january 2007 and december 2014 , two patients ( 3.4% ) demonstrated lnm .
the lnm rates are 0.0% ( 0/9 ) in mucosal cancers with gcls and 4.0% ( 2/50 ) in submucosal cancers with gcls , respectively .
in particular , the lnm rate in submucosal cancers with gcls ( 4.0% ) was significantly lower than that in submucosal cancers with differentiated - type and undifferentiated - type adenocarcinoma ( 19.4% , 140/722 ) ( fig .
1 ) . egc with gcls is a specific type of gastric cancer that has a favorable outcome , and is associated with a very low lnm rate despite deep submucosal invasion .
therefore , patients with egc with gcls , including those with deep submucosal invasion , may be good candidates for esd as like well - differentiated neuroendocrine tumor .
however , further investigation is needed to establish new recommendation criteria for esd for egcs with gcls .
the current esd indications for egcs are based on the differentiation degree of the tumor .
although egc with pac is one of differentiated - type adenocarcinomas and so treated according to the same esd indication criteria as for other differentiated - type adenocarcinomas , the lnm rate under the current esd indication criteria was higher compared with other differentiated - type cancers . furthermore , more than half of the patients undergoing esd for egc with pac ultimately came to achieve out - of - esd indication . on the other hand , egc with gcls
this outcome is associated with a very low lnm rate in patients having egc with gcls , even in patients with deep submucosal invasion .
this suggests that patients with egc with gcls , even those with deep submucosal invasion , may be potential candidates for esd .
although large - scale prospective multi - center studies with longer follow - up periods will be required to determine the optimal esd criteria for these tumors , clinicians should be aware of these rare disease entities . | endoscopic submucosal dissection ( esd ) enables en bloc curative resection of early gastric cancers ( egcs ) with a negligible risk of lymph node metastasis ( lnm ) .
although esd for egcs with absolute and expanded indications is safe , the results differ between egcs with specialized and common histologies .
egc with papillary adenocarcinoma is a differentiated - type adenocarcinoma . at present , it is treated with esd according to the same criteria as other differentiated - type adenocarcinomas .
the lnm rate under the current indication criteria is high , and over half of the patients who undergo esd as a primary treatment for egc with papillary adenocarcinoma achieve an out - of - esd result .
gastric carcinoma with lymphoid stroma in egc has a low lnm rate and a favorable outcome , despite deep submucosal invasion .
patients with this gastric cancer subtype may be good candidates for esd , even with deep submucosal invasion .
large - scale prospective multi - center studies with longer follow - up periods are needed to set proper esd criteria for these tumors .
clinicians should be aware of these disease entities and esd should be more carefully considered for egcs with papillary adenocarcinoma and gastric carcinoma with lymphoid stroma . | INTRODUCTION
PAPILLARY ADENOCARCINOMA
GASTRIC CARCINOMA WITH LYMPHOID STROMA
CONCLUSIONS |
there is clear evidence for the use of a protective ventilation protocol in patients with acute respiratory distress syndrome ( ards ) .
the hallmark ards network ( ardsnet ) trial of 2000 showed a significant mortality benefit in patients ventilated with a protective ventilation strategy with low tidal volume ( vt ) of 6 ml / kg of ideal body weight ( ibw ) and plateau pressure of 30 cm h2o or below .
ventilator induced lung injury ( vili ) through large vts , volutrauma , high airway pressures , barotrauma , and sheer stress from repetitive opening and closing of alveoli , atelectotrauma , can cause lung damage in normal lungs .
protective ventilation strategies for patients with noninjured lungs remains controversial and large randomized studies are needed .
a number of studies have shown that protective ventilation is beneficial in patients at risk of developing ards .
for instance in patients who have undergone a primary physiological insult such as critically ill patients with sepsis , pneumonia , trauma or high risk surgical patients requiring intensive care unit ( icu ) care postoperatively . in these patients
, it is sensible to implement a protective ventilation strategy to minimize the risk of developing ards .
a multicenter randomized trial in 2013 showed improved clinical outcomes with fewer respiratory complications in patients ventilated with a lung - protective ventilation strategy undergoing major abdominal surgery . in an observational study gajic et al .
they concluded that high vts ( > 6 ml / kg ibw ) were associated with the development of acute lung injury ( ali ) . in another study by gajic et al .
, over 3000 icu patients without ards were mechanically ventilated for 48 h or more .
high vts were associated with ards with an odds ratio of 2.6 for a vt over 700 ml .
performed a randomized trial comparing traditional and protective vts in 152 critically ill - patients .
the trial was stopped early as more patients in the conventional group developed ali ( 13.5% vs. 2.6% p = 0.01 ) .
we recommend a protective ventilation strategy in critical care patients who require mechanical ventilation for over 48 h and have or are at risk for ards .
the aim of this audit was to assess compliance with a safe ventilation strategy for mechanically ventilated patients on icu with or at risk of ards .
the mechanical ventilation protocol set out by ards clinical network was used as the standard for both audits in the management of patients requiring mechanical ventilation .
these recommend a vt of 6 ml / kg of ibw and a plateau airway pressure of 30 cm h2o .
the aim of this audit was to assess compliance with a safe ventilation strategy for mechanically ventilated patients on icu with or at risk of ards .
the mechanical ventilation protocol set out by ards clinical network was used as the standard for both audits in the management of patients requiring mechanical ventilation .
these recommend a vt of 6 ml / kg of ibw and a plateau airway pressure of 30 cm h2o . the survey was carried out alongside the initial audit .
the aim of this audit was to assess compliance with a safe ventilation strategy for mechanically ventilated patients on icu with or at risk of ards .
the mechanical ventilation protocol set out by ards clinical network was used as the standard for both audits in the management of patients requiring mechanical ventilation .
these recommend a vt of 6 ml / kg of ibw and a plateau airway pressure of 30 cm h2o . the survey was carried out alongside the initial audit .
data were collected prospectively for both phases of the audit over an 8-week period , data collection were anonymous and only nonidentifiable information was used .
the initial audit was carried out from december 2010 to january 2011 , and the re - audit was carried out from january 2012 to february 2012 .
all critical care patients who were mechanically ventilated for over 48 h and who were at risk of developing ards were included in the audit .
risk factors for ards included : sepsis , trauma , high - risk surgery , pneumonia , aspiration , and blood transfusion .
exclusion criteria were : patients ventilated for < 48 h , patients ' breathing spontaneously and patients ' using noninvasive ventilation .
data collection for each patient included : admission diagnosis , age , sex , height , and ibw .
patients ' height was measured on admission and recorded on the icu chart ; this along with the patient 's sex , was used to calculate ibw [ table 1 ] . a reference sheet with
ibw calculated using this formula was available for a range of heights at each icu bed space .
ibw was then used to calculate a vt of 6 ml / kg as part of a protective ventilation strategy .
ibw calculated using a patient 's sex and height in the following formula ventilation parameters collected included mean daily vt , plateau airway pressure and the lowest partial pressure of arterial oxygen ( p)/fraction of inspired oxygen ( f ) ratio ( p / f ratio ) for each ventilated day recorded .
the p / f ratio is used as an indicator of disease severity in ards .
data were collected on a spreadsheet using microsoft excel for windows and analyzed using statistical package for the social sciences ( spss ) .
student 's t - test was used for continuous data and fischer 's exact test for categorical data . an anonymous survey in the form of a short questionnaire
was carried out prospectively over a 2-day period in january 2011 alongside the initial audit .
the questionnaire was distributed to a random sample of 30 nurses working at our critical care unit .
questions assessed their understanding regarding definitions of ards , and their knowledge of how to calculate ibw and the ideal vt for patients with or at risk of ards . following the initial audit , an education program targeted at junior doctors and icu nurses
education focused on the importance of using safe ventilation parameters in patients at risk of ards , how to calculate ibw and deliver vt 's of 6 ml / kg of ibw at the bedside .
these displayed recommended ventilation parameters including ibw for a range of heights and ideal vt 's for a range of ibw 's .
data were collected for a total of 141 ventilated days in the first audit and 125 ventilated days in the re - audit .
there were no differences between the two audit cohorts at baseline [ table 2 ] .
overall mean vt 's was 9.5 ml / kg of ibw in the initial audit compared to 6.6 ml / kg ibw ( p < 0.0001 ) following the implementation of the intervention [ figure 1 ] . no difference was seen in mean plateau airway pressures between the two phases of the audit ( 29.5 cm h2o vs. 30.2 cm h2o ) .
the lowest mean p / f ratio for the initial and re - audit were 131 and 173 mmhg , respectively ( p < 0.0002 ) [ table 2 ] .
outcome measures included 30 days mortality , which was the same in both cohorts , 5 ( 35.7% ) patients died in each cohort [ table 2 ] .
demographics and data between the two audit groups mean tidal volumes between the two audits .
the nursing survey was distributed to 30 nurses , of whom 24 ( 80% ) responded .
about 16 ( 67% ) of the nurses in the survey were a band five , 4 ( 17% ) were band six , and 3 ( 13% ) were a band seven .
20 ( 83% ) responders knew the meaning of ards , and 22 ( 92% ) used the hospitals ventilation protocol for patients with or at risk of ards .
the majority of nurses in the survey 16 ( 70% ) stated they were aware of how to calculate a safe vt for patients ' with ards .
10 ( 42% ) of the nurses surveyed knew the ideal vt for patients with ards .
this completed audit cycle demonstrated a significant improvement in clinical practice through the implementation of a simple education program aimed at junior doctors and nursing staff through increased awareness surrounding a protective ventilation strategy on icu .
the standards of this audit were to comply with a protective ventilation strategy for patients at risk of ards , in accordance with evidence based guidelines set out by ardsnet .
the initial and re - audit both met the standards for pressure limited ventilation as part of a protective ventilation strategy ; however , the initial audit failed to meet standards for a protective volume ventilation strategy .
patients were ventilated with large vt 's similar to conventional ventilation strategies of 10 ml / kg / ibw . following implementation of interventions , the re - audit demonstrated a significant improvement in compliance to this standard .
there is good evidence from both animal and human studies to suggest that a high vt strategy can cause volutrauma and is associated with a significant risk of vili .
lower vt of 6 ml / kg improves clinical outcome in patients with and at risk of ards .
this evidence is further enhanced by a randomized controlled trail by pinheiro de oliveira et al .
this suggests that high vt may induce lung injury in mechanically ventilated patients ' with normal lungs .
reasons for high vt include poor nurse education on how to implement a safe ventilation protocol in these patients as highlighted in the survey and a failure to recognize patients at risk of ards amongst intensive care staff .
many of the junior doctors were foundation level or medical core trainees with limited icu experience . the lowest
mean p / f ratio was significantly lower in the initial audit than the re - audit ; this may have contributed to higher observed vts in this group .
larger vt observed in patients with lower p / f ratios suggests that these patients may be particularly difficult to ventilate using a safe ventilation strategy .
the nursing survey identified an educational need amongst nursing staff regarding ards and appropriate ventilation strategies for these patients .
most responders claimed to use the ards ventilation protocol and were aware of how to calculate a safe vt for these patients .
the majority of responders did not know the ideal vt value of 6 ml / kg , without this knowledge , the ventilation protocol can not be implemented .
our survey found that most nurses had not undergone formal training on ards and its management , and when questioned , most felt they would benefit from further training .
there are many challenges to the effective implementation of a protective ventilation strategy on icu .
this audit demonstrated that simple interventions in combination with a targeted education program can improve compliance with a safe ventilation protocol .
these changes have improved our management of patients at risk of , and with established ards at the bedside .
we hope that a simple education program can be implemented across other icu 's to help optimize the management of patients with or at risk of ards . a mandatory half day training session on ards and lung - protective ventilation for critical care nursing staff and regular teaching on ards for junior doctors at the start of each rotation . more consultant led focus on safe ventilation strategy during ward rounds .
this should include a vt prescription box on the bedside chart of every patient , to be filled out on the daily consultant ward round .
ventilation strategy to be assessed as part of the protocol when setting up ventilation for a new icu admission , and to be recorded in the notes with a sticker . increased tolerance of permissive hypercapnia .
a mandatory half day training session on ards and lung - protective ventilation for critical care nursing staff and regular teaching on ards for junior doctors at the start of each rotation .
this should include a vt prescription box on the bedside chart of every patient , to be filled out on the daily consultant ward round .
ventilation strategy to be assessed as part of the protocol when setting up ventilation for a new icu admission , and to be recorded in the notes with a sticker . increased tolerance of permissive hypercapnia .
a mandatory half day training session on ards and lung - protective ventilation for critical care nursing staff and regular teaching on ards for junior doctors at the start of each rotation .
this should include a vt prescription box on the bedside chart of every patient , to be filled out on the daily consultant ward round .
ventilation strategy to be assessed as part of the protocol when setting up ventilation for a new icu admission , and to be recorded in the notes with a sticker . increased tolerance of permissive hypercapnia . | there is clear evidence for the use of a protective ventilation protocol in patients with acute respiratory distress syndrome ( ards ) .
there is evidence to suggest that protective ventilation is beneficial in patients at risk of ards .
a protective ventilation strategy was implemented on our intensive care unit in critical care patients who required mechanical ventilation for over 48 h , with and at risk for ards .
a complete audit cycle was performed over 13 months to assess compliance with a safe ventilation protocol in intensive care .
the ards network mechanical ventilation protocol was used as the standard for our protective ventilation strategy .
this recommends ventilation with a tidal volume ( vt ) of 6 ml / kg of ideal body weight ( ibw ) and plateau airway pressure of 30 cm h2o . the initial audit failed to meet this standard with vt 's of 9.5 ml / kg of ibw .
following the implementation of a ventilation strategy and an educational program , we demonstrate a significant improvement in practice with vt 's of 6.6 ml / kg of ibw in the re - audit .
this highlights the importance of simple interventions and continuous education in maintaining high standards of care . | Introduction
None
Aim
Methods
Results
Discussion
None
Recommendations |
recent years were associated with substantial development of enteral nutrition , and particularly , home enteral nutrition ( hen ) . for the last ten to 20 years , hen has been fully reimbursed in many european countries which has contributed to increased popularity of this treatment modality [ 8 , 13 ] .
also , in poland , hen has been developed and is reimbursed by the national health fund ( nhf ) since 2007 . according to the regulations of nhf ,
however , enteral feeding is reimbursed only if there is no possibility of oral nutrition , which excludes oral supplementation or sip feeding included in the espen definition of enteral feeding .
enteral nutrition is required in all patients who are unable to cover their energetic and nutritional requirements by means of normal ingestion despite , at least partially , retained function of the gastrointestinal tract .
potential advantages of hen in comparison to traditional hospital - based enteral nutrition include shorter hospitalization , lower direct costs of therapy and lower risk of secondary malnutrition - associated complications . according to sparse literature data , children constitute a minority among patients receiving hen .
published epidemiologic data on the administration rates of enteral / parenteral home nutrition is very limited and available only in a few countries [ 2 , 3 , 9 , 15 ] . in turn
, reliable data on the administration rates of hen is required to assess potential benefits associated with this treatment modality along with types and rates of associated complications .
additionally , it can be helpful in confronting requirements for this type of procedure with available resources .
finally , actual data is necessary for planning prospective studies of therapeutic outcomes of enteral nutrition .
constantly updated registries constitute the most optimal source of reliable data on hen service availability and utilization rates . despite their obvious advantages ,
only a few such national registries exist , however [ 2 , 3 , 7 , 15 ] .
since it was reimbursed and thus available , the dynamic increase in the number of patients who were offered hen services by polish medical centers was observed during recent years .
however , still there are some regions with no specialized centers offering hen services for children .
therefore , the aim of this first nationwide study was to assess the availability of pediatric hen services in poland in order to identify potential areas that should be improved .
this study was based on a retrospective 1-year analysis of hen services that were offered to polish children in 2010 .
all public healthcare services in poland are covered by the regional branches of the same funder , national health fund .
therefore , all 16 polish provinces have the same hen reimbursement criteria and procedures . according to these regulations ,
each healthcare provider is responsible for qualifying patients to nutritional treatment , training patients ( or their parents in the case of underage subjects ) in hen procedures , providing diets and all related devices , follow - up visits and all other necessary medical services . in january 2010 , the questionnaire , developed by the polish society for clinical nutrition of children and previously tested for reliability and validity ,
was sent to all regional centers providing pediatric hen services in poland ( n = 14 ) . in each center ,
the results of the survey were analyzed with an aid of statistica 8 ( statsoft ) package .
the analysis included the number of pediatric patients who received hen on january 1st 2010 and december 31st 2010 , their demographic characteristics and geographical distribution .
the prevalence rate of hen was expressed as the number of children receiving this type of service per one 1 million of the inhabitants in the analyzed region .
furthermore , the distributions of indications and methods of enteral nutrition administration were analyzed , along with the potential reasons of withdrawal from the hen program .
the number of children receiving hen in 2010 increased by 21% ( from 433 to 525 patients ) compared to january 1st 2010 . within the study period ,
73 patients were withdrawn from the hen program due to various reasons ( table 1 ) , and 165 new subjects were qualified to the enteral feeding ( 31.43%).table 1exclusion criteria from polish pediatric hen program in 2010criterionnumberlack of satisfactory outcome1complications associated with enteral nutrition2lack of patient s acceptance7lack of caregivers acceptance7satisfactory weight gain20patient s death17other19total73 exclusion criteria from polish pediatric hen program in 2010 the fraction of children receiving hen increased substantially in 2010 , from 11.34 per 1 million inhabitants on january 1st to 13.75 per 1 million on december 31st .
marked differences were observed in terms of geographical distribution of this parameter , from zero ( in two provinces ) to up to 30 pediatric patients per 1 million inhabitants ( fig .
1geographic distribution of polish children receiving hen in 2010 geographic distribution of polish children receiving hen in 2010 most patients receiving hen ( n = 419 , 79.8% ) were supervised by specialized pediatric centers , while the remainder ( n = 106 , 20.2% ) were handled by medical centers serving both children and adults .
median age of patients receiving hen in 2010 was 6 years ( range : 9 months18 years ) .
in most cases , hen was prescribed due to neurological disorders ( n = 337 , 64.2% ) .
other indications for hen are summarized in table 2 . in most cases ( n = 450 , 85.71% ) , enteral nutrition was administered by means of gastrostomy ( table 3).table 2indications for enteral nutrition in polish children receiving hen in 2010indicationnumberpercentagecerebral palsy16431.2encephalopathy529.9spinal muscular atrophy ( sma)448.4muscular dystrophy61.1other neurological disorders7113.5genetic syndromes9317.7short bowel syndrome10.2inflammatory bowel disease30.6other gastrointestinal diseases265.0cystic fibrosis203.8chronic renal failure152.9chronic liver failure10.2oncological diseases51.0malformation syndrome112.1congenital heart disease40.8metabolic disorders91.7total525100.0table 3methods of enteral nutrition administration in polish children receiving hen in 2010methodnumberpercentagenasogastric tube5911.2gastrostomy45085.7nasojejunal tube20.4jejunostomy40.8gastrojejunal tube101.90total525100.0 indications for enteral nutrition in polish children receiving hen in 2010 methods of enteral nutrition administration in polish children receiving hen in 2010
this first polish nationwide survey on hen prevalence rates among pediatric patients included data from all centers offering this type of specialized services . throughout 2010
nonetheless , the fraction of polish pediatric patients receiving hen ( 13.75 per 1 million inhabitants ) was still markedly lower compared to published data from other countries : 48.23 cases per 1 million in some regions of italy and 95.6 per 1 million in the united kingdom .
it should be remembered , however , that 2010 corresponded to already the fourth year of pediatric hen reimbursement / availability in poland .
one can assume this as one potential reason for both relatively low prevalence of hen services and dynamic increase in prescription rates of enteral feeding .
home enteral nutrition was not prescribed to children living in two out of 16 provinces , probably due to the lack of centers offering hen services in these administrative regions of poland . in another five provinces ,
however , there were several provinces where markedly higher hen administration rates ( above 15 per 1 million inhabitants ) were reported .
similar regional differences in hen distribution in children were previously reported from spain and italy [ 3 , 15 ] .
one should confer this highly variable hen administration rates in poland to the prevalence rates of pediatric disorders that usually require enteral nutrition in respective regions .
any discrepancies between these two measures would suggest potential regional deficiency of hen services and eventually substantiate funding new pediatric centers .
our survey documented that neurologic disorders constituted principle indication for enteral nutrition in polish children ( 64.2% ) .
also , in other european countries , chronic neurologic disorders predominate among indications for hen , but the fractions of pediatric patients receiving this treatment modality due to neurological reasons are lower : 28.23% in spain , 35% in france , and 50% in italy .
. revealed substantial change in the distribution of indications for hen administered in french children . in 2000 , chronic neurologic disorders constituted the most frequent indication for hen , in contrast to 1989 when enteral nutrition was usually prescribed due to primary alimentary disorders . compared to other european countries , our study revealed markedly lower percentage of pediatric patients who received hen due to respiratory and circulatory tract disorders , and chronic kidney or liver failure .
perhaps this underrepresentation of indications other than neurological and alimentary disorders resulted from recent educational activities of the polish society for clinical nutrition of children . during our certified courses , we focused on prescribing hen in chronic neurological and alimentary conditions .
the results of this study suggest , however , that during future courses provided by the society more attention should be paid to other disorders that may potentially require enteral nutrition .
recent evidence suggests that cystic fibrosis is a condition which definitely can benefit from enteral nutrition [ 6 , 10 , 12 , 16 , 17 ] .
in one study , cystic fibrosis patients corresponded to 23% of pediatric patients receiving enteral nutrition in france .
in contrast , our survey revealed only 20 ( 3.6% ) cystic fibrosis cases among children who were offered hen in 2010 .
taking into account rough estimates of cystic fibrosis prevalence in poland ( n = 1,500 ) , enteral nutrition was prescribed to only 1.3% of this group , hence to six - fold lower fraction compared to tube - feed cystic fibrosis cases managed in the united states . | published epidemiologic data on the administration rates of enteral / parenteral home nutrition is very limited .
the aim of this first nationwide study was to assess the availability of pediatric home enteral nutrition ( hen ) services in poland .
the questionnaire was sent to all regional centers providing pediatric hen services in poland ( n = 14 ) .
the analysis included the number of pediatric patients who received hen in 2010 , their demographic characteristics and geographical distribution .
furthermore , the distributions of indications and methods of enteral nutrition administration were analyzed , along with the reasons of withdrawal from the hen program .
the number and fraction of children receiving hen increased in 2010 , from 433 ( 11.34 per 1 million inhabitants ) on january 1st to 525 ( 13.75 ) on december 31st .
marked differences were observed in geographical distribution of this parameter , from zero to up to 30 pediatric patients per 1 million inhabitants .
median age of patients was 6 years ( range : 9 months18 years ) .
in most cases , hen was prescribed due to neurological disorders ( n = 337 , 64.2% ) , and administered by means of gastrostomy ( n = 450 , 85.71% ) .
this study revealed the dynamic development of pediatric hen services in poland but also documented their potential regional shortages . | Introduction
Materials and methods
Results
Discussion |
a recent us national institute on aging task force defined comorbidity as the co - occurrence of preexisting age - related health conditions ( eg , disability , anemia , impairments , urinary incontinence ) or diseases ( eg , diabetes , heart disease , hypertension ) in reference to an index disease ( eg , cancer , parkinson s disease , diabetes).1 adequate measurement and analytic control of comorbidity has long been recognized as a critical challenge in clinical epidemiology.2 the aforementioned task force has reviewed the methodology of measurement of comorbidity,3 including the nosology of disease classification4 and strategies to include disease severity in comorbidity scales.5 collapsing comorbid diseases into a single scale provides an index that is easy to comprehend and statistically efficient , which are the main advantages of an index over incorporating each disease into an analysis as an individual variable.6 a simple sum of the number of comorbid diseases treats each disease equivalently , thereby ignoring differences in the severity of the component diseases and differences in the severity of the disease state in different patients .
weighting schemes have been proposed and implemented to address each of these shortcomings.5,7 whether summing diseases included in the index or weighting them by severity , all comorbidity schemes inevitably misclassify study subjects with respect to the idealized true scale of comorbidity.3 the impact of this misclassification on the analytic results depends on whether comorbidity is the exposure of interest , study outcome , a confounder , or modifier.3 in most study populations , the prevalence of subjects with high comorbidity scores is low .
it is common , therefore , to combine subjects with some score above a threshold into a single open - ended category .
for example , the charlson index can theoretically range from 0 to 33 but was collapsed into categories of 0 , 12 , 34 , and 5 in its initial presentation.7 similar examples , particularly examples of collapsing the scores in the highest categories , are easy to find , even in this author s own work.8 the rationale for collapsing these categories is the same as the rationale for using an index of comorbid diseases : ease of comprehension and statistical efficiency .
the effect of collapsing these categories is also the same as the effect of collapsing disparate comorbid diseases : introduction of classification errors . in this paper , we show analytically and by synthetic example that collapsing the high - end categories of a comorbidity scale changes the estimate(s ) of effect(s ) of comorbidity and biases analyses that control for comorbidity as a confounder or analyze modification of an exposure s effect by comorbidity .
to depict the bias introduced by collapsing categories of a comorbidity scale , we created a scale with a strictly monotonically increasing risk of the outcome ( ri ) with each increase in the ordinal comorbidity scale ( indexed by i ) , and a strictly monotonically decreasing prevalence ( pi ) of the comorbidity value with each increase in the ordinal comorbidity scale .
table 1 depicts this synthesized data . while the data are synthetic , we note that the risks of an outcome in the scale categories and the prevalence of the scale categories correspond well with values one might observe .
for example , one might anticipate similar data if the population was an older population ( say 70-years - old and older ) , if the index of comorbidity was the charlson index , and the outcome was a three - year risk of death .
the number of persons with this value would be small , and the number of cases of some outcome within that category even smaller .
analysts might be tempted to collapse category i = 4 with category i = 3 , for example , to avoid sparse data problems or to improve the precision of the estimate of association in the highest comorbidity category .
the effect of this collapse is to set the risk for the combined category to a weighted average of the two individual categories . more generally , collapsing a set of the upper - end categories ranging from i = v to the maximum ( i = 4 , in this example ) generates a weighted average risk :
rv
4=i = v4pirii = v4pi table 2 depicts the risk ratios ( rrc = x vs c = 0 ) estimated from the synthetic data when the high - end categories are collapsed together .
the collapsed categories range from some value v , which can equal 1 , 2 , 3 , or 4 to the maximum ( 4 , in this example ) . setting v = 4
note that collapsing categories does not introduce a bias ; the estimate of risk and therefore risk ratio within each category is an accurate depiction of the effect in that category . with each additional combination , the risk in the highest category
becomes more heavily weighted with the low - risk comorbidity categories because these low risk categories are more prevalent .
when v = 1 , which corresponds to a comparison of any comorbidity ( collapsing categories 1 to 4 ) with no comorbidity ( category i = 0 ) , the risk ratio equals 12 .
this risk ratio is about five - fold lower than the risk ratio in the highest comorbidity category ( i = 4 , in which the risk ratio equals 60 ) and about five - fold higher than the risk ratio in the lowest category with any comorbidity ( i = 1 , in which the risk ratio equals 2.7 ) .
the risk ratio of 12 is not , in fact , a very good estimate of the effect of comorbidity in any of the most finely divided categories .
collapsing comorbidity categories can therefore diminish the ability to discern important patterns that are more apparent when categories are not collapsed .
that is , comorbid diseases are likely to be more prevalent among patients with high risk conditions ( eg , another health indicator such as frailty or disability ) and likely also to be related to the outcome under study ( eg , all - cause mortality ) .
a scale of comorbid disease is therefore often a potential confounder and a candidate for analytic adjustment . to examine the effect of collapsing comorbid categories when the comorbidity scale is used for analytic adjustment
, we postulated a second dichotomous variable ( e indexed by k = 0 or 1 within categories of the comorbidity scale ) whose association with the outcome is of primary interest .
we assumed that the prevalence of e = 1 depends on the category of the comorbidity scale , as depicted in table 3 .
we assumed , however , that the risk of the outcome did not depend on the category of e within strata of the comorbidity scale .
that is , after adjustment for the most finely divided comorbidity categories , the risk ratio associating e = 1 compared with e = 0 would be null ( rre = 1 vs e = 0 = 1 ) .
the crude risk in categories of e is the weighted average of the risks in table 1 , where now the weights correspond with the prevalence of comorbidity within category of e , as shown in table 3 .
that is
rk=i=14pi , kri , ki=14pi , k the risk equals 0.125 in e = 1 and 0.032 in e = 0 , which yields a crude rre = 1 vs e = 0 of 3.90 .
the substantial departure of this crude risk ratio from the true null association is entirely due to confounding by comorbidiy .
the relative risk due to confounding ( rrc ) , which equals the ratio of the crude and adjusted estimates , provides a measure of the direction and magnitude of this confounding , and in this case rrc = 3.9/1 = 3.9 . to resolve the confounding
, one can calculate the standardized risk ratio ( srre = 1 vs e = 0 ) , where the standard weights equal the prevalence of the comorbidity categories in e = 1 ( these weights are pi , 1 ) . that is :
srr = r1/i=14pi,1ri,0i=14pi,1when comorbidity categories are collapsed , the standardized risk in the denominator uses the weighted average risk in the collapsed category ( rv 4 , 0 , where the weights come from the unexposed group ) and the sum of corresponding weights in the exposed category ( sum from v to 4 of pi,1 ) .
srr=r1/i=1v1pi,1ri,0+i = v4pi , rrv 4,0i=14pi,1 the resulting srr is incompletely adjusted for confounding by comorbidity .
table 4 depicts the srre = 1 vs e = 0 and rrc for this scenario , and in a second scenario with the true srre = 1 vs e = 0 = 1.5 . in both cases , collapsing the upper - end categories of the comorbidity scales yields incomplete control for confounding by comorbidity .
the result is a bias of rrc toward the null , which can give rise to the appearance of an association between e and the outcome when the true association is null ( scenario 1 ) , can give rise to the appearance of a stronger association than is truly present ( scenario 2 ) , or can give rise to an underestimate of the true association if the true rrc < 1 and the true association between e and the outcome is causal .
often the analysis compares the effect of the exposure in those with the highest comorbidity category to the effect of the exposure in those with the lowest comorbidity category .
for example , one might calculate the interaction contrast ( ic),9 which measures the departure of risk in those with the high risk category of the exposure ( e = 1 ) and comorbidity ( i = 4 ) from the risk expected given ( a ) the independent effect of the exposure in those without comorbidity ( r0 , 1 r0 , 0 ) , ( b ) the independent effect of higher comorbidity in those without the exposure ( r4 , 0 r0 , 0 ) , ( c ) the risk in those with the low risk category of the exposure ( e = 0 ) and comorbidity ( i = 0 ) .
this concept simplifies to the risk difference in those with high comorbidity less the risk difference in those without comorbidity .
that is :
ic = r4,1(r0,1r0,0)(r4,0r0,0)r0,0 = ( r4,1r4,0)(r0,1r0,0 ) a second measure of interaction is the ratio of the risk ratios , which we will call effect measure modification ( emm ) . that is
:
emm = r4,1/r4,0r0,1/r0,0 when the highest categories of comorbidity are collapsed , however , r4,1 will be replaced with rv 4,1 and r4,0 will be replaced with rv 4,0 .
the result is an unpredictable bias in the estimates of the interaction between the exposure and comorbidity . in scenario 1 ,
therefore , ic must equal 0 and emm must equal 1 . as depicted in table 5 ,
the collapsed categories ( v < 4 ) all yield ic > 0 and emm > 1 , suggesting an interaction between e and comorbidity that does not exist . furthermore
, as v increases , the bias of ic decreases but the bias of emm increases . in scenario 2 , both the exposure and comorbidy affect the outcome . collapsing the comorbidity categories
can overestimate ic ( when v = 3 ) or underestimate ic ( when v 2 ) . on the other hand ,
the number of persons with this value would be small , and the number of cases of some outcome within that category even smaller .
analysts might be tempted to collapse category i = 4 with category i = 3 , for example , to avoid sparse data problems or to improve the precision of the estimate of association in the highest comorbidity category .
the effect of this collapse is to set the risk for the combined category to a weighted average of the two individual categories . more generally , collapsing a set of the upper - end categories ranging from i = v to the maximum ( i = 4 , in this example ) generates a weighted average risk :
rv
4=i = v4pirii = v4pi table 2 depicts the risk ratios ( rrc = x vs c = 0 ) estimated from the synthetic data when the high - end categories are collapsed together .
the collapsed categories range from some value v , which can equal 1 , 2 , 3 , or 4 to the maximum ( 4 , in this example ) . setting v = 4
note that collapsing categories does not introduce a bias ; the estimate of risk and therefore risk ratio within each category is an accurate depiction of the effect in that category . with each additional combination ,
the risk in the highest category becomes more heavily weighted with the low - risk comorbidity categories because these low risk categories are more prevalent .
when v = 1 , which corresponds to a comparison of any comorbidity ( collapsing categories 1 to 4 ) with no comorbidity ( category i = 0 ) , the risk ratio equals 12 .
this risk ratio is about five - fold lower than the risk ratio in the highest comorbidity category ( i = 4 , in which the risk ratio equals 60 ) and about five - fold higher than the risk ratio in the lowest category with any comorbidity ( i = 1 , in which the risk ratio equals 2.7 ) . the risk ratio of 12 is not , in fact , a very good estimate of the effect of comorbidity in any of the most finely divided categories . collapsing comorbidity categories can therefore diminish the ability to discern important patterns that are more apparent when categories are not collapsed .
. that is , comorbid diseases are likely to be more prevalent among patients with high risk conditions ( eg , another health indicator such as frailty or disability ) and likely also to be related to the outcome under study ( eg , all - cause mortality ) .
a scale of comorbid disease is therefore often a potential confounder and a candidate for analytic adjustment . to examine the effect of collapsing comorbid categories when the comorbidity scale is used for analytic adjustment
, we postulated a second dichotomous variable ( e indexed by k = 0 or 1 within categories of the comorbidity scale ) whose association with the outcome is of primary interest .
we assumed that the prevalence of e = 1 depends on the category of the comorbidity scale , as depicted in table 3 .
we assumed , however , that the risk of the outcome did not depend on the category of e within strata of the comorbidity scale .
that is , after adjustment for the most finely divided comorbidity categories , the risk ratio associating e = 1 compared with e = 0 would be null ( rre = 1 vs e = 0 = 1 ) .
the crude risk in categories of e is the weighted average of the risks in table 1 , where now the weights correspond with the prevalence of comorbidity within category of e , as shown in table 3 .
that is
rk=i=14pi , kri , ki=14pi , k the risk equals 0.125 in e = 1 and 0.032 in e = 0 , which yields a crude rre = 1 vs e = 0 of 3.90 .
the substantial departure of this crude risk ratio from the true null association is entirely due to confounding by comorbidiy .
the relative risk due to confounding ( rrc ) , which equals the ratio of the crude and adjusted estimates , provides a measure of the direction and magnitude of this confounding , and in this case rrc = 3.9/1 = 3.9 . to resolve the confounding
, one can calculate the standardized risk ratio ( srre = 1 vs e = 0 ) , where the standard weights equal the prevalence of the comorbidity categories in e = 1 ( these weights are pi , 1 ) . that is :
srr = r1/i=14pi,1ri,0i=14pi,1when comorbidity categories are collapsed , the standardized risk in the denominator uses the weighted average risk in the collapsed category ( rv 4 , 0 , where the weights come from the unexposed group ) and the sum of corresponding weights in the exposed category ( sum from v to 4 of pi,1 ) .
srr=r1/i=1v1pi,1ri,0+i = v4pi , rrv 4,0i=14pi,1 the resulting srr is incompletely adjusted for confounding by comorbidity .
table 4 depicts the srre = 1 vs e = 0 and rrc for this scenario , and in a second scenario with the true srre = 1 vs e = 0 = 1.5 . in both cases , collapsing the upper - end categories of the comorbidity scales yields incomplete control for confounding by comorbidity .
the result is a bias of rrc toward the null , which can give rise to the appearance of an association between e and the outcome when the true association is null ( scenario 1 ) , can give rise to the appearance of a stronger association than is truly present ( scenario 2 ) , or can give rise to an underestimate of the true association if the true rrc < 1 and the true association between e and the outcome is causal .
often the analysis compares the effect of the exposure in those with the highest comorbidity category to the effect of the exposure in those with the lowest comorbidity category .
for example , one might calculate the interaction contrast ( ic),9 which measures the departure of risk in those with the high risk category of the exposure ( e = 1 ) and comorbidity ( i = 4 ) from the risk expected given ( a ) the independent effect of the exposure in those without comorbidity ( r0 , 1 r0 , 0 ) , ( b ) the independent effect of higher comorbidity in those without the exposure ( r4 , 0 r0 , 0 ) , ( c ) the risk in those with the low risk category of the exposure ( e = 0 ) and comorbidity ( i = 0 ) .
this concept simplifies to the risk difference in those with high comorbidity less the risk difference in those without comorbidity .
that is :
ic = r4,1(r0,1r0,0)(r4,0r0,0)r0,0 = ( r4,1r4,0)(r0,1r0,0 ) a second measure of interaction is the ratio of the risk ratios , which we will call effect measure modification ( emm ) .
that is :
emm = r4,1/r4,0r0,1/r0,0 when the highest categories of comorbidity are collapsed , however , r4,1 will be replaced with rv 4,1 and r4,0 will be replaced with rv 4,0 .
the result is an unpredictable bias in the estimates of the interaction between the exposure and comorbidity . in scenario 1 ,
therefore , ic must equal 0 and emm must equal 1 . as depicted in table 5 ,
the collapsed categories ( v < 4 ) all yield ic > 0 and emm > 1 , suggesting an interaction between e and comorbidity that does not exist . furthermore
, as v increases , the bias of ic decreases but the bias of emm increases . in scenario 2 , both the exposure and comorbidy affect the outcome . collapsing the comorbidity categories
can overestimate ic ( when v = 3 ) or underestimate ic ( when v 2 ) . on the other hand ,
the common practice of collapsing the highest categories of comorbidity into a single category has the advantages of increasing the prevalence of subjects in the highest category of comorbidity , thereby improving the ease of comprehension and the statistical efficiency of the analysis .
the impact of this misclassification depends on how the comorbidity variable is used in the analysis .
when comorbidity is an exposure or predictor of the outcome in the analysis , then the misclassification changes the pattern of the outcome response as a function of the dose of comorbidity .
this result should be expected ; miscategorization of dose and in particular combining categories with dissimilar outcome risks yields misleading dose - response patterns.10 better analytic solutions are to collapse only adjacent comorbidity categories with similar risks6 or to use more sophisticated dose - response modeling , such as spline regression.10 the similarity of risks in adjacent categories is best left to judgment , perhaps informed by statistical testing , because of the poor power to detect important differences by statistical testing alone.11 when comorbidity is a candidate confounder in the analysis , then the misclassification biases the relative risk due to confounding toward the null ( assuming independent and nondifferential classification errors ) .
the result is residual confounding of the association between the exposure of interest and the outcome .
this result should also be expected ; independent and nondifferential misclassification of a confounder is known to yield residual confounding.12 importantly , misclassification resulting from crude categorization of even a covariate that has been well - measured on a continuous scale can result in substantial bias.13 as above , better analytic solutions are to collapse only adjacent comorbidity categories with similar risks,6 to use spline regression,10,14 or to include the comorbidity index as a single linear term in regression modeling.14 restricting the study sample to subjects with comorbidity scores below the threshold where category collapsing will improve comprehensibility and statistical efficiency is also an alternative , although this restriction may reduce the generalizability of study results.15 when comorbidity is a candidate modifier in the analysis , then the misclassification can give rise to the appearance of interaction when no interaction exists , can mask true interaction , and can bias the estimate of interaction.3 different combinations of these possibilities may appear depending on whether interaction is assessed as departure from additive or multiplicative effects , both of which have been proposed as important considerations in the examination of comorbidity.1,4 this result should also be expected ; independent and nondifferential misclassification of a modifier is known to affect analyses of interaction unpredictably.12 for most analyses of interaction , the best analytic solution is to restrict the analysis and inference to a category of comorbidity with uniform risk for the outcome .
the appeal of collapsing categories of comorbidity to facilitate interpretation and statistical analysis is often offset by misleading results . at a minimum
, analysts should assure the uniformity of outcome risk in collapsed categories before collapsing them .
often times , more appropriate analytic methods can achieve similar goals without sacrificing the validity of the study s results . | adequate control of comorbidity has long been recognized as a critical challenge in clinical epidemiology .
comorbidity scales reduce information about coexistent disease to a single index that is easy to comprehend and statistically efficient .
these are the main advantages of an index over incorporating each disease into an analysis as an individual variable .
many study populations have a low prevalence of subjects with high comorbidity scores , so it is common to combine subjects with some score above a threshold into a single open - ended category .
this paper examines the impact of collapsing comorbidity scores into these categories .
it shows analytically and by synthetic example that collapsing the high - end categories of a comorbidity scale changes the pattern of effect of comorbidity .
furthermore , collapsing the high - end categories biases analyses that control for comorbidity as a confounder or analyze modification of an exposure s effect by comorbidity .
each of these results specific to comorbidity scoring derives from more general epidemiologic principles .
the appeal of collapsing categories to facilitate interpretation and statistical analysis may be offset by misleading results .
analysts should assure the uniformity of outcome risk in collapsed categories , informed by judgment and possibly statistical testing , or use analytic methods , such as restriction or spline regression , which can achieve similar goals without sacrificing the validity of results . | Introduction
Methods and results
Collapsing categories changes the estimate of comorbiditys effect
Collapsing categories biases the relative risk due to confounding by comorbidity toward the null
Collapsing categories biases estimates of interaction unpredictably
Discussion |
pupil dilatation unresponsive to light stimuli and decreased constriction response to accommodation , with denervation supersensitivity to dilute cholinergic agents , is known as tonic pupil .
most tonic pupil cases are idiopathic , but among all known causes , trauma is the most frequent .
it has been described following ocular surgery ; there are two classic reports of tonic pupil following retinal detachment ( rd ) repair , one using an encircling band ( eb ) and another after an old electrocoagulation [ 2 , 3 ] , and one recent report after pars plana vitrectomy ( ppv ) .
we describe the onset of a tonic pupil in a child after ppv and eb for a rd . in this case ,
an 11-year - old boy attended our emergency department for decreased visual acuity ( va ) in his right eye ( re ) for the last week .
the patient had been a premature newborn , with 26 weeks and 800 g at birth , and he received a bilateral 360 argon photocoagulation for active retinopathy of prematurity ( rop ) .
va , at the emergency room , was hand movement in his re , with normal anterior segment .
fundoscopy showed a total rd with macula off , and an 180 inferior giant tear at the posterior edge of the 360 intense laser scar .
the left eye did not show any significant alteration , with an attached retina and a 360 peripheral laser scar .
he underwent a ppv and placement of a 2.5-mm eb and heavy silicone oil ( densiron ) endotamponade in his re . a mild infrared laser ( 810 nm )
, he developed a painless persistent corneal epithelial defect ( 4 4 mm ) in his re .
one month after the ppv , the patient was started on intense autologous serum treatment , achieving complete corneal epithelisation within five weeks . when postoperative dilating drops were discontinued a moderate mydriasis in his re , unresponsive to light , was observed ( fig .
four months later , pupil diameter of the re decreased and a slight anisocoria for right miosis was present ( fig . 2c ) .
one year after the ppv , corneal sensibility remains severely decreased and a moderate residual corneal scarring is still present .
pupil and accommodation defects have been described following extensive retinal laser treatment . the main explanation for this complication is thermal lesion of the short ciliary nerves , containing parasympathetic motor fibers , travelling through the choroid and suprachoroidal space .
infrared diode laser ( 810 nm ) produces a much deeper coagulation when compared to argon laser and affects the retinal pigment epithelium and choriocapillaris , and thus easily damages the nerves running in the suprachoroidal area .
there are few reports published in the literature , though , on tonic pupil following retinal surgery [ 2 , 3 , 4 ] .
report a case of a tonic pupil after eb with lamellar scleral resection and diathermy .
they attribute their clinical findings to a compromise of the short ciliary nerves as they pass around the globe. ebrahim et al .
recently published a case after ppv , argon endolaser and silicone endotamponade , performed under retrobulbar anesthesia .
they speculate that endolaser damaged the short ciliary nerves and that it is responsible for the tonic pupil .
it is surprising , though , that such a small amount of argon laser as described in their case can cause such a serious injury to the short ciliary nerves as to induce a tonic pupil . nevertheless , they did not rule out a possible causative effect of the retrobulbar anesthesia or even an idiopathic etiology of the case .
direct injury to the ciliary ganglion by the anesthetic needle can be another cause of tonic pupil onset .
there are , to our knowledge , no reports in the literature on this etiology .
this is not applicable to our case because the patient underwent surgery under general anesthesia . on the other hand
, direct compression of the ciliary ganglion exerted by the eb could be another plausible explanation in our case .
though we believe eb played a role in the development of the tonic pupil , this could be more related to short ciliary nerve damage .
eb is usually placed under the rectus muscles , in a much anterior position . in our case ,
the two long posterior ciliary nerves , branches of the nasociliary nerves , are responsible for the sensitivity of the iris , cornea and ciliary muscle .
experimental studies support that parasympathetic fibers travel with the long posterior ciliary nerves proximally , but arborice farther posteriorly near the posterior pole , where the choroid is thickest .
intraocular connections of the long and short ciliary nerves exist within or on the outer surface of the choroid .
this suggests that all innervation types can be damaged with an intense laser photocoagulation and that respecting horizontal retinal meridians during the treatment may not always help to preserve these nerves .
we hypothesize that there might also be a vascular etiology in the ciliary nerves lesion of our patient .
eb has been reported to induce relative ischemia to anterior ocular structures by means of venous obstruction or kinking of the long posterior ciliary arteries .
nevertheless , we can not rule out that the tonic pupil and corneal anesthesia may arise from the prematurity state and treatments the patient had previously received , especially after observing a slight hypoesthesia of the contralateral cornea . in conclusion
, we believe that a combination of different pathogenic factors have been involved in our case . thermal injury of the ciliary nerves , initially by the old argon rop laser and recently by the infrared diode laser for rd repair , combined to the hypothetical blood support deficiency induced by the eb may have resulted in a long ciliary nerves and parasympathetic denervation causing the permanent corneal anesthesia and the tonic pupil . | an 11-year - old boy presented with a total retinal detachment in his right eye .
he had a bilateral 360 argon laser treatment for an active retinopathy of prematurity performed after his birth .
he underwent an uneventful pars plana vitrectomy , encircling band , 810-nm diode endolaser and heavy silicone oil ( densiron ) endotamponade . a tonic pupil and abolition of corneal sensitivity , with a large epithelial defect ,
were observed during the postoperative period .
we discuss the possible etiopathogenic mechanisms of the long and short ciliary nerves damage , and the role that retinopathy of prematurity and retinal detachment laser treatment and the encircling band placement might have played in the development of the tonic pupil and the corneal anesthesia . | Introduction
Case Report
Comment
Disclosure Statement |
a 68-year - old women presented the chief complaint of severe left perioral and chin pain .
the patient had been diagnosed with phn in the left v3 area 15 months ago .
the left facial pain had continued for 1 year despite medical treatment . during this period
, 1,200 mg gabapentin , 300 mg phenytoin , 30 mg duloxetine , and 300 mg hydantoin per day were administrated but had little effect .
when the acute pain attack began , it continued for about 1 hour with eating difficulty .
the pain was aggravated by touching the left mandibular area , brushing her teeth , and was associated with talking .
we initially blocked the left mental nerve under us with a 10 - 12 mhz linear transducer ( vivid e ; general electronics , fairfield , ct , usa ) using a mixture of 2 ml 2% mepivacaine and 20 mg triamcinolone .
her pain severity 1 month after this intervention was 1 - 2/10 on the vas , and 80 - 90% on a pain relief scale . because pain relief was maintained continuously throughout the 2 , 4 , 6 , and 12 months follow - ups after us - assisted mental nerve block , no additional treatment including medication was provided .
a 45-year - old man visited our pain clinic with the chief complaint of right mandibular area pain .
the patient had been diagnosed with facial herpes zoster in the right v3 area 9 months previously .
the right facial pain had continued for 9 months despite medical treatment . during this period , 300 mg
pregabalin , 10 mg amitriptyline , and three 37.5 mg tramadol/375 mg acetaminophen combination tablets were administrated per os daily but had little effect .
the pain was aggravated by touching the right mandibular area , and palpation over the right mental foramen reproduced the pain .
we initially blocked the right mental nerve under us with a 10 - 12 mhz linear transducer using a mixture of 2 ml 2% mepivacaine and 20 mg triamcinolone .
this procedure decreased the pain immediately , and pain severity was 0 - 1/10 on a vas .
one month after this intervention , his pain relief was > 90% . because relief was maintained continuously throughout the 2 , 4 , 6 , and 12 months follow - ups after us - assisted mental nerve block , no additional treatment including medication was provided .
a 26-year - old women with phn in the left v3 area for 1 month visited our pain clinic for treatment by injection .
she had a cold , burning sensation on one her left teeth spreading to the left posterior ear .
her pain was aggravated by touching the left mandibular area , and palpation over the left mental foramen reproduced the pain .
we initially blocked the left mental nerve under us with a 10 - 12 mhz linear transducer using a mixture of 2 ml 2% mepivacaine and 20 mg triamcinolone .
when she returned to our pain clinic 1 month after the mental nerve block , the patient stated that the vas score had decreased from 9 to 4 for about 2 weeks following the mental nerve block but that her symptoms had then returned to their previous state .
therefore , we tried the same additional us - assisted left mental nerve treatment , but the outcome was the same with the earlier one .
the us - assisted mental nerve blocks were effective , but the effects were only maintained for 2 weeks , so we decided to perform prft .
after explaining the procedure , efficacy , and possible side effects of prft , the patient was placed in a supine position .
the transducer was applied transversely at the level of the second premolar , midway between the upper and lower borders of the mandible .
we scanned her mandible in the cephalad direction from the inferior border of the mandible and easily identified the mental foramen with a hypoechoic cleft ( fig .
a radiofrequency needle ( 10 cm ) insulated with a 5-mm active tip ( 22 g , smk - c10 ; radionics inc , burlington , ma , usa ) was advanced slightly via the mental foramen under us assistance ( fig .
2 ) . confirmation of the needle position inside the mental foramen was achieved under fluoroscopy . following negative aspiration ,
0.5 ml of radio - contrast agent ( omnipaque ge healthcare , cork , ireland ) was injected to confirm no vascular uptake through the mental branch of the inferior alveolar artery ( fig .
2 ) . sensory stimulation using a 50 hz , 0.3 - 0.5 v electrical current showed paresthesia over the mandibular area . after confirming the needle position
her pain severity was 1 - 2/10 on the vas 1 month after prft , and her pain relief scale was 80 - 90% . because this pain relief was maintained continuously throughout the 2 , 4 , and 6 months follow - ups after prft , no additional treatment including medication was provided .
a 68-year - old women presented the chief complaint of severe left perioral and chin pain .
the patient had been diagnosed with phn in the left v3 area 15 months ago .
the left facial pain had continued for 1 year despite medical treatment . during this period
, 1,200 mg gabapentin , 300 mg phenytoin , 30 mg duloxetine , and 300 mg hydantoin per day were administrated but had little effect .
when the acute pain attack began , it continued for about 1 hour with eating difficulty .
the pain was aggravated by touching the left mandibular area , brushing her teeth , and was associated with talking .
we initially blocked the left mental nerve under us with a 10 - 12 mhz linear transducer ( vivid e ; general electronics , fairfield , ct , usa ) using a mixture of 2 ml 2% mepivacaine and 20 mg triamcinolone .
her pain severity 1 month after this intervention was 1 - 2/10 on the vas , and 80 - 90% on a pain relief scale . because pain relief was maintained continuously throughout the 2 , 4 , 6 , and 12 months follow - ups after us - assisted mental nerve block , no additional treatment including medication was provided .
a 45-year - old man visited our pain clinic with the chief complaint of right mandibular area pain .
the patient had been diagnosed with facial herpes zoster in the right v3 area 9 months previously .
the right facial pain had continued for 9 months despite medical treatment . during this period
, 300 mg pregabalin , 10 mg amitriptyline , and three 37.5 mg tramadol/375 mg acetaminophen combination tablets were administrated per os daily but had little effect .
the pain was aggravated by touching the right mandibular area , and palpation over the right mental foramen reproduced the pain .
we initially blocked the right mental nerve under us with a 10 - 12 mhz linear transducer using a mixture of 2 ml 2% mepivacaine and 20 mg triamcinolone .
this procedure decreased the pain immediately , and pain severity was 0 - 1/10 on a vas .
one month after this intervention , his pain relief was > 90% . because relief was maintained continuously throughout the 2 , 4 , 6 , and 12 months follow - ups after us - assisted mental nerve block , no additional treatment including medication was provided
a 26-year - old women with phn in the left v3 area for 1 month visited our pain clinic for treatment by injection .
she had a cold , burning sensation on one her left teeth spreading to the left posterior ear .
her pain was aggravated by touching the left mandibular area , and palpation over the left mental foramen reproduced the pain .
we initially blocked the left mental nerve under us with a 10 - 12 mhz linear transducer using a mixture of 2 ml 2% mepivacaine and 20 mg triamcinolone .
when she returned to our pain clinic 1 month after the mental nerve block , the patient stated that the vas score had decreased from 9 to 4 for about 2 weeks following the mental nerve block but that her symptoms had then returned to their previous state .
therefore , we tried the same additional us - assisted left mental nerve treatment , but the outcome was the same with the earlier one .
the us - assisted mental nerve blocks were effective , but the effects were only maintained for 2 weeks , so we decided to perform prft . after explaining the procedure , efficacy , and possible side effects of prft , the patient was placed in a supine position .
the transducer was applied transversely at the level of the second premolar , midway between the upper and lower borders of the mandible .
we scanned her mandible in the cephalad direction from the inferior border of the mandible and easily identified the mental foramen with a hypoechoic cleft ( fig .
a radiofrequency needle ( 10 cm ) insulated with a 5-mm active tip ( 22 g , smk - c10 ; radionics inc , burlington , ma , usa ) was advanced slightly via the mental foramen under us assistance ( fig .
2 ) . confirmation of the needle position inside the mental foramen was achieved under fluoroscopy .
following negative aspiration , 0.5 ml of radio - contrast agent ( omnipaque ge healthcare , cork , ireland ) was injected to confirm no vascular uptake through the mental branch of the inferior alveolar artery ( fig .
2 ) . sensory stimulation using a 50 hz , 0.3 - 0.5 v electrical current showed paresthesia over the mandibular area . after confirming the needle position , we performed prft three times at 42 for 120 seconds .
her pain severity was 1 - 2/10 on the vas 1 month after prft , and her pain relief scale was 80 - 90% . because this pain relief was maintained continuously throughout the 2 , 4 , and 6 months follow - ups after prft , no additional treatment including medication was provided .
treatment for trigeminal phn is controversial . however , in our cases , precisely performed us - assisted mental nerve block and us - assisted prft resulted in excellent outcomes with no adverse effects .
trigeminal phn usually responds to pharmacotherapy , which should be employed before any intervention is attempted .
medical treatments result in good initial pain relief , but relief rates fall off dramatically over the long - term .
cases refractory to medical management can be treated with minimally invasive procedures such as a nerve block .
nerve blocks with local anesthetics and steroids for the painful area are a reasonable next step if pharmacological modalities fail to control the pain .
phn impairs all sensory fiber groups such as c , a , and a fibers that cause sharp pain , burning pain , allodynia and/or hypersensitivity .
the exact mechanism of pain relief from neural blockade during treatment of phn is unknown , but it may be related to modulating pain transmission .
reported that prft has repeatedly been demonstrated as a safe and effective procedure , even in instances where other treatment modalities have failed .
its use in a variety of conditions demonstrates the attractiveness of prft as a less invasive alternative to surgical intervention that may involve significant morbidity .
microstructure research has indicated that prf causes axonal changes more obviously in c fibers than that in a or a fibers , which is largely manifested within the mitochondria as micro - cytoskeletal edema , leading to an abnormality in atp metabolism and ion channel and pump function , thereby blocking the pain transfer in relevant nerves .
a neuroanatomic study suggested that the abnormal neuron conduction properties and synaptic activity in the hippocampus induced by prft can be quickly restored , which is unique and different from conventional radiofrequency lesioning .
therefore , the acute effects of prft are more reversible and less destructive in nature than the classic conventional rf mode .
the analgesic action of prft also involves enhancing noradrenergic and serotonergic descending pain inhibitory pathways .
although the precise mechanism is elusive , most studies suggest that the analgesia achieved by prft is through the pulse electric current and the biological effects induced thereby , including the effects on the drg , c - fos gene expression regulation in the cornu dorsale medullae spinalis , and nerve fiber edema .
long - term analgesia of prf is also closely connected with the gene expression alteration of neurons . according to koscielniak - nielsen , us - guided peripheral nerve block significantly shortens performance time and reduces the number of needle passages to the target .
the occurrence of paresthesia during block is also reduced but not the incidence of short - lasting post - operative neuropraxia .
however , limited information is available on the use of us for identifying bony structures .
reported complications of blind injections for the treatment of trigeminal neuralgia include soreness , infection , swelling , dysesthesia , and headache .
definitive identification of osseous landmarks may be important when the target nerve of the block is unidentifiable with us due to its small size or imaging artifacts .
when we perform us - assisted mental nerve block , bone appears as a hyperechoic linear structure . during transverse scanning over the inferior portion of the mandible at the level of the second premolar and scanning in the cephalad direction
after we advanced the needle slightly into the mental canal , we checked the needle position with fluoroscopy .
then , we reconfirmed that there was no vascular uptake through the mental branch of the inferior alveolar artery with radio - contrast agent ( fig .
2 ) . the mental foramen , which lies inferior to the outer lip at the level of the second premolar , midway between the upper and lower borders of the mandible , is localized using a transverse scanning in a cephalad direction from the inferior border of the mandible
. the most common position of the mental foramen in relation with the teeth appears to be below the second premolar regardless of race or age .
it lies at the level of root of second premolar in about 50% of cases , between the two premolars in about 20 - 25% and posterior to the second premolar in about 24% . in approximately 1 - 2% of cases
the mental foramen lies either anterior to the first premolar or posterior to the first molar . in summary
thus , we decided to perform us - assisted prft of the mental nerve and gained excellent results . here , we have presented our experience of us - assisted mental nerve block and us - assisted prft , which are safer and more effective techniques than that of the landmark method .
but , further randomized controlled studies will be needed to demonstrate the safety and efficacy of this treatment . | postherpetic neuralgia ( phn ) is one of the most difficult pain syndromes to treat .
invasive treatments may be considered when patients fail to obtain adequate pain relief from noninvasive treatment approaches . here , we present three cases of phn in the mandibular branch treated with ultrasound - assisted mental nerve block and pulsed radiofrequency treatment . none of the patients had adequate pain relief from the medical therapy , so we performed the mental nerve block on the affected side under ultrasound assistance .
two patients showed satisfactory pain relief continuously over 12 months without any further interventions , whereas one patient only had short - term pain relief .
for the patient had short - term pain relief we performed pulsed radiofrequency treatment ( prft ) on the left mental nerve under ultrasound assistance .
after prft , the patient had adequate pain relief for 6 months and there was no need for further management . | CASE REPORTS
1. Case 1
2. Case 2
3. Case 3
DISCUSSION |
atopic dermatitis ( ad ) is a most common chronic inflammatory skin disease , affecting about 10 million people in the world , leading to a significant reduction in quality of life , and its incidence is continuously increasing in westernized countries [ 1 , 2 ] .
the pathogenesis of ad is unknown , but the disease seems to be correlated with specific immune and inflammatory mechanisms .
the general characteristics of ad include excessive infiltration of inflammatory cells such as lymphocytes , macrophages , and granulated mast cells into the skin lesions , eosinophilia in peripheral blood , and a high level of serum immunoglobulin e ( ige ) .
mast cells are tissue - based inflammatory cells of bone marrow origin , which respond to signals of innate and adaptive immunity .
they play a major role in immediate hypersensitivity reaction and are activated through the high - affinity ige receptor , fcr .
in addition , it has been reported that a large number of mast cells can be found in ad skin lesion .
the majority of ad patients have elevated blood ige level and it is mediated of mast cell activation .
mast cell activation by ige is release inflammatory mediators , such as histamine , as well as cytokines , including th2 cytokines , such as il-4 , il-5 , and il-13 .
thus , cell - bound ige is cross - linked allergens and it is contributed to the development of ad through mediating activation of mast cells localization .
proinflammatory cytokines that are released by activated mast cells , including il-6 and il-8 , play an important role in allergic inflammation ; il-6 mediates allergic inflammation , while il-8 induces the migration of neutrophils into inflammatory regions as a potent chemotactic cytokine [ 9 , 10 ] .
cp001 is a mixture of four oriental herbal medicines composed of houttuynia cordata thunb , rehmannia glutinosa libosch , bark of betula platyphylla var .
houttuynia cordata thunb has long been used in traditional oriental medicine for the treatment of inflammatory diseases .
also , several studies demonstrated that houttuynia cordata thunb has been associated with a broad range of pharmacological activities , including anti - inflammatory , antiviral , and anticancer effects .
rehmannia glutinosa libosch has traditionally been used as an ingredient herb in east asian medicine for the effects of hemostasis , activation of blood circulation , and improvement of kidney function .
several studies indicated that rehmannia glutinosa libosch has antiallergic effects and anti - inflammatory function [ 1517 ] .
japonica is known to have antioxidant , anti - inflammatory , and anticancer effects and inhibits the development of ad in nc / nga mice [ 19 , 20 ] .
rubus coreanus miq . is a type of red raspberry that grows wild in korea , japan , and china .
the fruit , known as bokbunja in korean , has been used in traditional oriental medicine for reducing the risk of diseases such as asthma and allergy .
it is also known that rubus coreanus miq . has anti - inflammatory and antioxidative activities [ 2123 ] .
these collective observations indicate that cp001 may be good candidate for control of ad and beneficial in the treatment of human allergic disorders .
therefore , in our previous study , we already confirmed that topical application of km110329 ( cp001 modifying herbal mixture ) inhibits the atopic dermatitis in ovalbumin- and dncb - induced mouse model .
therefore , in this study , we investigated whether 30% ethanol extract of cp001 oral administration has anti - inflammatory activity in 2,4-dinitrochlorobenzene- ( dncb- ) induced ad mice model .
in addition , we also investigated whether 30% ethanol extract of cp001 has antiallergic effect inhibiting cytokine production in human mast cells , hmc-1 .
cp001 was prepared by hanpoong pharmaceutical ( jeon - ju , korea ) following good manufacturing practices ( gmp ) procedure .
cp001 is 30% ethanol extracted brown - colored powder , and it is composed of houttuynia cordata thunb , rehmannia glutinosa libosch , bark of betula platyphylla var .
the powder from the extract was dissolved in distilled water for in vivo and in vitro experiments .
six - week - old male balb / c mice were purchased from orient ( sung - nam , korea ) .
the mice were randomized into 6 groups ( normal , dncb , and 25 , 50 , 100 , and 200 mg / kg ( cp001 ) ) , each comprising five mice .
all mice were kept under pathogen - free environment and allowed free access to the diet and water .
all procedures performed on the mice were approved by the animal care center of kyung - hee university ( approval number khuasp ( se)-2012 - 004 ) .
induction of ad - like skin lesions procedure is described in figure 1 . for that purpose
, mice back skin was painted dermally with 200 l of a 1% dncb using 1 1 cm patches after shaving .
two weeks after sensitization , the back skin was challenged with 200 l of a 0.2% dncb solution twice per week .
this procedure was repeated for 2 weeks and cp001 was orally administrated together . at the end of the experiment ,
mice were sacrificed by co2 inhalation , and samples were collected . a portion of the skin biopsies were fixed in 4% paraformaldehyde ( pfa ) and embedded in tissue - tek optical cutting temperature ( o.c.t . ) compound ( tissue - tek , sakura , aa zoeterwoude , the netherlands ) on dry ice .
skin sections of 20 m were cut and stained with hematoxylin and eosin ( h & e ) for inflammatory cells or with toluidine blue for mast cells counts and examined under light microscopy ( olympus ) .
mast cells were counted in 10 parts of high - power fields ( hpf ) at 400x magnification .
after final cp001 administration , whole blood samples were collected by cardiac puncture for measurement of blood ige level .
the blood was placed in vacutainer tubes containing edta ( bd science , nj , usa ) and blood plasma was isolated .
total ige levels in plasma were determined by sandwich elisa using the bd pharmingen mouse ige elisa set .
briefly , plates were coated with capture antibody in elisa coating buffer ( sigma - aldrich ) and incubated overnight at 4c .
plates were washed with pbs - tween 20 ( 0.05% ) and subsequently blocked ( 10% fbs in pbs ) for 1 h at 20c .
serial dilutions of standard antigen or sample in dilution buffer ( 10% fbs in pbs ) were added to the plates and plates were incubated for 2 h at 20c .
after washing , biotin - conjugated anti - mouse ige and sav - hrp ( streptavidin - horseradish peroxidase conjugate ) were added to the plates and plates were incubated for 1 h at 20c . finally , tetramethylbenzidine ( tmb ) substrate solution was added to the plates and after 15 min incubation in the dark ,
optical densities were measured at 450 nm on an automated elisa reader ( versa max , molecular devices , ca , usa ) . il-6 and
il-8 levels were measured in hmc-1 supernatant by sandwich elisa using bd pharmingen human elisa set .
mice skin was immediately frozen in liquid nitrogen and kept at 70c until use . for real - time pcr assay
, mice skin was homogenized with ultra - turrax t10 ( ika labortechnik , seoul , korea ) and rna extraction was performed using trizol ( invitrogen life technologies , ny , usa ) .
rna content was measured using the nanodrop nd-1000 spectrophotometer ( nanodrop technologies inc . ) . 1 g of total cellular rna from each sample was reverse transcribed using cdna synthesis kit ( takara , japan ) .
quantitative pcr was performed using sybr green imaster and a lightcycler 480 ( roche , switzerland ) .
cells were harvested by centrifugation and the pellet was washed with ice - cold pbs .
rna was isolated from the pellet using easy - blue rna extraction kit ( intron biotech , republic of korea ) according to the manufacturer 's instructions .
isolated rna content was measured using the nanodrop nd-1000 spectrophotometer ( nanodrop technologies inc . ) .
2 g of total cellular rna from each sample was reverse transcribed using cdna synthesis kit ( takara , japan ) .
pcr was conducted out in a 20 l reaction mixture consisting of dna template , 10 pm of each gene - specific primer , 10x taq buffer , 2.5 mm dntp mixture , and 1 unit of taq dna polymerase ( takara , japan ) .
pcr was performed using the specific primer and primer sequences for human il-6 , il-8 , and gapdh are shown in table 1 .
ellagic acid , quercitrin hydrate , and catalpol were purchased from sigma chemicals ( saint louis , mo ) .
hplc grade acetonitrile , methanol , and formic acid were purchased from j. t. baker ( phillipsburg , nj ) .
catalpol , ellagic acid , and quercitrin were chosen as marker compounds to standardize the extract sample .
cp001 was dissolved in distilled water at a concentration of 100 mg / ml and the solution was filtered through a 0.45 m membrane filter .
a 10 l aliquot of the sample solution was injected into a hplc system ( agilent technologies , palo alto , ca ) .
the sample was analyzed on a capcell pak ug120 c18 analytical column ( 250 4.6 mm , 5 m ; shiseido , japan ) .
statistical analyses presented as the mean standard error of the mean ( sem ) and were analyzed for statistical significance using the unpaired student 's t - test .
to determine whether cp001 decreases infiltration of inflammatory cells into ad - like skin lesions , we performed h & e staining on the skin after oral administration of cp001 .
we observed infiltration of inflammatory cells into the epidermis and dermis in dncb group , whereas cp001 decreased such infiltration of inflammatory cells into the skin ( figure 2 ) .
moreover , cp001 ( 25200 mg / kg ) abrogated skin thickening induced by dncb ( figure 2 ) .
next , we also performed toluidine blue staining for mast cell observation . repeated cutaneous application of dncb increased dermal mast cell number .
the th2 type cytokines are important in an acute phase of ad whereas mixed th2/th1 type inflammation is characteristic to a chronic phase of ad .
to determine whether cp001 decreases th2 type cytokines expression , we performed real - time pcr to measure il-4 and il-13 levels .
we found that oral administration of cp001 decreased il-4 mrna expression in ad - like skin lesions ( figure 4(a ) ) .
we also found that cp001 administration decreased il-13 mrna expression in ad - like skin lesions in a dose - dependent manner ( figure 4(b ) ) . in histology analysis , repeated cutaneous application of dncb increased dermal mast cell number and this feature was suppressed by cp001 oral administration .
activated mast cells secrete various chemokines and cytokines including il-6 and il-8 . to determine whether cp001 decreases il-6 and il-8 cytokines mrna levels
oral administration of cp001 did not affect the suppression of il-6 and il-8 mrna expression in ad - like skin lesions ( figures 4(c ) and 4(d ) ) .
hyperproduction of ige is a major characteristic of ad and patients with ad often exhibit elevated levels of total and allergen specific ige antibodies ( abs ) in their serum . to further test whether suppression of the progression of ad - like skin lesions by cp001 is associated with serum ige levels , we performed total ige elisa assay .
we found that total ige levels were dramatically elevated in dncb - treated group compared with normal group .
however , increased serum ige levels induced by dncb were significantly decreased by cp001 treatment ( figure 5 ) .
for that purpose , mast cells were pretreated with various concentrations of cp001 for 1 h and then treated with pma and a23187 for 24 h. the levels of il-6 and il-8 in culture supernatants were measured by elisa assay .
we found that il-6 secretion induced by pma and a23187 was significantly suppressed by cp001 ( figures 6(a ) and 6(c ) ) .
we also performed rt - pcr to measure il-6 and il-8 mrna expression in hmc-1 .
we observed that il-6 and il-8 mrna induced by pma and a23187 were decreased by cp001 ( figures 6(b ) and 6(d ) ) .
cp001 administration decreased il-4 and il-13 mrna expression in ad - like skin lesions ( figure 7(b ) ) .
therefore , we characterized the regulatory effect of cp001 on il-4 and il-13 mrna expression in hmc-1 using rt - pcr .
we found that il-13 expression induced by pma and a23187 was significantly suppressed by cp001 ( figure 7(a ) ) .
il-4 expression level was not increased by pma and a23187 , but it is suppressed by cp001 ( figure 7(a ) ) . to further evaluate the effective compounds of cp001 extract ,
hplc analysis was employed . in order to analyze catalpol , the mobile phase consisted of water ( w ) and methanol ( m ) and the flow rate was 1 ml / min .
the initial composition of the mobile phase was 97 : 3 ( w : m ) , which was linearly changed to 95 : 5 ( w : m ) over 1 min and changed to 91 : 9 ( w : m ) for 9 min . at 11 min ,
the composition of mobile phase returned to the initial condition , which was maintained for 9 min for column reequilibration .
chromatograms were acquired at 210 nm by uv detection ( figure 8) . for ellagic
acid and quercitrin , the mobile phase consisted of 0.1% formic acid ( f ) and acetonitrile ( a ) and the flow rate was 1 ml / min .
the initial composition of the mobile phase was 90 : 10 ( f : a ) , which was linearly changed to 85 : 15 ( f : a ) over 5 min and changed to 60 : 40 ( f : a ) for 35 min . at 41 min ,
the composition of mobile phase returned to the initial condition , which was maintained for 9 min for column reequilibration .
the retention times of catalpol , ellagic acid , and quercitrin were 6.2 , 14.4 , and 18.6 min , respectively ( figures 7(a ) and 7(b ) ) .
the concentrations of catalpol , ellagic acid , and quercitrin in the extract sample were determined using hplc analysis as described above .
the extract was standardized to contain 1.8% catalpol , 0.4% ellagic acid , and 0.3% quercitrin .
ad is a chronic inflammatory disease , which is accompanied by erythema , edema , and scaling in ad skin lesions .
recently , korean medicine has been the subject of increased interest for its potential in the treatment of inflammatory diseases , including atopic dermatitis and airway inflammation [ 16 , 17 , 25 , 26 ] .
the present study demonstrates that oral administration of korean herbal mixture extract , cp001 , inhibits dncb - induced ad .
we observed that cp001 decreases infiltration of inflammatory cells into ad - like skin lesions and dermal mast cell number .
generally , steroid therapy is used for ad treatment , but it can not be administrated over the long term because of the many side effects .
therefore , we find a new drug , which is effective in the treatment of ad without any side effects .
recently , we reported that topical application of km110329 ( cp001 modified drug ) reduced ovalbumin- and dncb - induced atopic dermatitis
. therefore , we were wondering whether cp001 oral administration may inhibit dncb - induced atopic dermatitis .
mast cells degranulation can be regulated by the recruitment , trafficking , and function of inflammatory response .
for example , il-4 and il-13 induce cell adhesion molecules on endothelium which can be recruitment of leukocytes [ 2729 ] . also , the production of il-4 cytokine in epidermal cells has been known to be the main factor for initiation of ad . in our data , we show that cytokine production and mrna levels of il-4 , il-13 , il-6 , and il-8 were suppressed by cp001 in hmc-1 .
also , quantitative real - time pcr of the skin lesions also showed that oral administration of cp001 diminished the mrna level of il-4 and il-13 in the ad - like skin lesions .
in addition , we found that cp001 reduces mast cell in dncb - induced ad - like skin lesion .
it seems that inhibition of infiltration of mast cell downregulates secretion of il-4 and il-13 cytokines and it may inhibit recruitment of leukocytes . thus , mast cell may be main factor for suppression of th2 cytokines in the ad - like skin lesions by oral administration of cp001 .
ige is mediator of mast cell activation and we observed that cp001 oral administration reduced elevated blood ige levels induced by repeated dncb sensitization .
cp001 also suppressed il-6 secretion and elevated il-6 and il-8 mrna expression induced by pma and a23187 in hmc-1 .
it seems that the reduction of infiltration of mast cells is related to decrease of degranulation of mast cells and maturation of eosinophils suppressing the release of various inflammatory cytokines .
our present study clearly demonstrates that cp001 suppresses the progression of ad induced by dnbc .
in addition , inflammatory related cytokine production and mrna levels of il-4 , il-13 , il-6 , and il-8 were suppressed by cp001 . | cp001 is four traditional herbal medicine mixtures with anti - inflammatory properties . in this study , we investigated the effect of oral administration of cp001 ethanol extract on the 2,4-dinitrochlorobenzene- ( dncb- ) induced ad mouse models . for that purpose
, we observed the effects of oral administration of cp001 on skin inflammatory cell infiltration , skin mast cells , production of serum ige , and expression of th2 cytokine mrna in the ad skin lesions of dncb treated balb / c mice .
histological analyses demonstrated that cp001 decreased dermis and epidermis thickening as well as dermal infiltration induced by inflammatory cells .
in addition , cp001 decreased mast cell infiltration in count as well as dermal infiltration induced by inflammatory cells . in the skin lesions , mrna expression of interleukin- ( il- ) 4 and il-13 was inhibited by cp001 .
cp001 also reduced the production of ige level in mouse plasma .
in addition , we investigated the effect of cp001 on the inflammatory allergic reaction using human mast cells ( hmc-1 ) . in hmc-1 , cytokine production and mrna levels of il-4 , il-13 , il-6 , and
il-8 were suppressed by cp001 . taken together , our results showed that oral administration of cp001 exerts beneficial effects in ad symptoms , suggesting that cp001 might be a useful candidate for the treatment of ad . | 1. Introduction
2. Material and Method
3. Results
4. Discussion
5. Conclusion |
nonsteroidal anti - inflammatory drugs ( nsaids ) are the most widely prescribed group of therapeutic drugs worldwide , and include many common analgesics and anti - inflammatory agents.1 selective cox-2-is have demonstrated improved gastrointestinal tract ( gi ) safety over nonselective nsaids.23 the clinical trials have evidenced that , compared to nonselective cox -is , cox-2-is associated with a reduced rate of serious gi events as well as a reduced requirement for concomitant gastro - protective therapies.4 this relative benefit may be due to a lack of cox-1-mediated inhibition of gastric mucous production and a lack of effect on platelet thromboxane production . however , the differential effects of cox-2-is on platelet aggregation , prostacyclin / thromboxane balance , and inflammatory mediators involved in the development of atherosclerosis have also led to concerns that cox-2-is increase the cv events.56 evidence from large prospective clinical trials on cv risks has been largely limited as those trials were designed to evaluate primarily gi events . the vioxx gastrointestinal outcomes research study ( vigor ) revealed an increased rate of thrombotic events in patients receiving cox-2 inhibitor ( rofecoxib).2 mukherjee and colleagues have compared rate of cv events in rofecoxib and celecoxib in vigor and celecoxib long - term arthritis safety study ( class ) trials with four aspirin studies viz .
, the us physicians health study , the uk doctors study , the thrombosis prevention trial , and the hypertension optimal treatment trial and revealed significantly higher cv event rate in cox-2-is treatment groups.37 in contrast to this , konstam and colleagues8 have analysed the results of multiple clinical studies involving rofecoxib and have not demonstrated any increased risk for cv events comparing cox-2 inhibitors and nonselective nsaids .
observational studies also have found mixed results when comparing select cox-2 is and nonselective nsaids.910 hence , we chose to research this question with an observational prospective cohort study of cv risk of selective and nonselective cox - is in adult arthritic patients .
many of the observational studies rely on case - control designs . in our cohort study
in this observational prospective cohort study adult arthritic patients of either sex those were freshly diagnosed or taking cox - is for < 3 months ; were included .
patients those with history of any other disease ( e.g. , diabetes , hypertension , stroke , ihd , etc . ) and medical treatment for the same were excluded .
initial ecg screening of every patient was done to rule out the cardiovascular disease . after getting the protocol approved by institutional ethics committee ( ref : sknmc no / ethics / corr/2011/103 )
patients were grouped into nonselective and selective cox-2-is groups with reference to treatment they received ( n = 34 ) .
their cv risk factors i.e. bmi , bp , bsl , lipid profile , etc . , were assessed at enrollment and recorded as baseline .
all arthritic patients were followed up and cv risk factors were assessed at 6 and 12 month of treatment [ see figure 1 ] .
demographics of cv risk factors ( i.e. , age , sex , smoking , alcohol , heredity ) were also studied .
bmi calculated by online bmi calculator while , 10-year cv risk was calculated using framingham 's calculator .
statistical test used was student 's t-test using openepi statistical software package version 2 .
in this prospective cohort study we have assessed the effects of nsaids on cv risk factors in arthritic patients over a period of time ( 1 yr )
. the demographic profiles of these arthritic patients were also studied and are presented in table 1 .
there were 22 males and 46 females with mean age 50.7 yrs enrolled in this study .
subset arthritic patients with history of smoking were 10 , alcohol consumption 6 and familial cvd 22 .
arthritic patients with family history of cvd were maximum ( 15 out of 22 ) in selective cox-2-is group .
arthritics in nonselective and selective cox-2-is group received eight different nsaids [ see figure 2 ] . among nonselective nsaids ,
diclofenac sodium was received by majority of the patients ( 23% ) followed by aceclofenac ( 10% ) and only 2% were treated by indomethacin . in selective cox-2-is
distribution of cyclooxygenase inhibitors use in arthritic patients ( n = 68 ) demographic profile of the arthritic patients from the results of this study it becomes evident that all nsaids reason potential cv risk when taken over a period of time as in arthritic patients .
bmi , bp , and lipid profile ; the potential cv risk factors , showed statistically significant impairment in selective cox-2-is - treated arthritic patients ; p < 0.01 , p < 0.001 ( sbp ) and p <
0.05 , respectively , compared to baseline and p < 0.05 for bmi compared to nonselective cox - is group at the end of 1yr treatment .
triglycerides ( tgs ) and cholesterol were apparently increased with apparent fall in hdl levels in cox-2-is group after 6 month of treatment but this increase was statistically insignificant .
no significant effect was observed on diastolic blood pressure and random bsl [ see tables 2 , 3 and figure 3 ] .
effects of cyclooxygenase inhibitors on physical cardiovascular risk factors effects of cyclooxygenase inhibitors on biochemical cardiovascular risk factors cardiovascular risk of cyclooxygenase inhibitors when 10 year comparative cv risk was assessed using framingham 's calculator ; maximum over all cv risk percentage was reflected in selective cox-2-is treated arthritics [ see table 4 ] . while significantly higher risk of getting chd and mi ( p < 0.05 ) and apparently high risk of stroke , cvd , chd death and cvd death ( p > 0.05 ) over 10 years was also observed in arthritic patients treated with selective cox-2-is and same
. comparative percentage 10-year risk of chd , mi , stroke , cvd , chd death and cvd death in arthritic patients comparative 10-year cv risk in subset arthritic patients
arthritis is one of a 100 musculoskeletal conditions of varying etiologies and most prevalent disease involving middle age and elderly i.e. , 50 - 65yrs and go on increasing in prevalence with age i.e. > 65 yrs , the incident rate of arthritis is three times higher in females compared to males.1112 in this study also there were 22 men with 46 women with mean age 50.7 yrs , suggestive of high incidence rate in middle age women .
in present study arthritic patients received eight different nsaids ; among nonselective cox - is group 66% patients received diclofenac sodium and aceclofenac ( i.e. , phenylacetic acid derivatives ) remaining patients were treated with other nonselective nsaids . in selective cox-2-is group 94% patients received etoricoxib while only 6% treated with celecoxib .
older age ( 45 years of age for men and 55 years for women ) , smoking , hypertension , low hdl concentration , hyperlipidaemia , hyperglycaemia and a family history of heart disease are major cv risk factors.13 in present study we have evaluated cv risk of nsaids with reference to these risk factors . and the results have revealed that cox-2-is cause significant increase in bmi , sbp and also significant impairment in lipid profile ; the potential cv risk factors in arthritic patients .
nonselective cox - is also showed impairment in lipid profile of the arthritics but except hdl this impairment was statistically insignificant .
the effect of selective cox-2-is on these cv risk factors could be attributed to etoricoxib as 94% of patients received etoricoxib while in nonselective cox - is , these effects should be attributed to phenylacetic acid derivative with similar properties .
presently rofecoxib , valdecoxib and lumiracoxib have been withdrawn from the market worldwide on the basis of results of various trials viz .
vigor , approve and target.2141516 conflicting results of several clinical trials about cv safety of celecoxib created controversies.317 comparatively cardiovascular safety data about etoricoxib is scare although few clinical trials stated that etoricoxib exhibit less or comparable cv risk to nonselective nsaids and did not appear to significantly increase the risk for mi and stroke.31819 but in our study it has reasoned significant risk for chd and mi ( p < 0.05 ) whereas reflected apparent increase in risk of stroke , cvd and death due to chd and cvd which was statistically insignificant compared to nonselective cox - is .
hence though the results of our study confirm that selective cox-2-is exhibit comparable cv risk to nonselective nsaids ; our study results are in disagreement with the earlier studies that states selective cox-2-is exhibit less cv risk and did not significantly increase the risk for mi and stroke . increased cv risk of selective cox-2-is
is said to be due inhibition of formation of the vasodilator pgi2 , and leaving txa2 unopposed , which facilitates vasoconstriction , platelet activation and smooth muscle cell proliferation but , their exact role on lipid profile , atherosclerosis and plaque formation is still unclear.20 recently molecular studies have identified cytochrome p-450 ( cyp ) pathway in arachidonic acid ( aa ) metabolism along with cox and lipooxygenase pathway ( lox ) .
cyp epoxygenases are known to metabolise aa to four regioisomeric epoxyeicosatrienoic acids ( 5 , 6- , 8 , 9- , 11 , 12- , and 14 , 15-eet ) and by cyp w -hydroxylases to 20-hydroxyeicosatetraenoic acid ( 20-hete).21 it is potent vasoconstrictor and induces oxidative stress . in clinical studies ,
it is associated with increased bmi and the metabolic syndrome.22 cox pathway offer cardioprotection due to production of pgi2 by inducible cox 2 and blockade of this pathway may ultimately result into unopposed production of 20-hete.21 this could be the reason for increased bmi and impaired lipid profile in selective cox2-is treated arthritic patients .
cox2 inhibitors reduce renal medullary blood flow , decrease urine flow , and enhance the pressor effect of angiotensin ii .
in contrast , the pressor effect of angiotensin ii is blunted by cox1 inhibition.23 nonselective nsaids are said to increase blood pressure by nonselective blocking of cox1and 2 while selective nsaids by blocking of cox 2 ; hence nonselective nsaids are considered more renal toxic .
but in our study significant increase was observed in cox-2-is group and these findings are in agreement with various epidemiological studies that state hypertensive complications occur more commonly in patients treated with cox-2-is.24 lox pathway has predominant role in insulin release and cox inhibition does not affect this release.25 our study also revealed no significant difference in bsl in both cox - is groups compared to baseline . in this study
we have also explored the cv risk in subset arthritic patients i.e. with history of smoking , alcohol consumption and familial cvd which otherwise remained unexplored in majority of the earlier studies .
history of familial cvd has positive correlation with cv risk as stated in earlier few studies;226 our study results reflected higher cv risk in selective cox-2-is group ( 9.6% )
. this high cv risk could be due to maximum number of arthritics ( 15 out of 22 ) with familial cvd were there in cox-2-is group , which confirms this positive correlation .
smoking is one of the most important cv risk factors for the development and progression of atherosclerosis .
higher cv risk in smokers ( 23% ) observed in arthritics treated with cox2-is group could be explained on the basis of that all of these agents invariably cause an imbalance between pgi2 and txa2 ; as they block cox2-derived prostacyclin ( pgi2 ) keeping txa2 biosynthesis unopposed by cox 1 in smokers.27 several epidemiological investigations have shown that a low to moderate level ( i.e. , 20 g-70/day ) of alcohol intake has definitive protective role against chd and stroke .
mechanisms for cardioprotective effects include increased hdl , decreased ldl , prevention of clot formation , reduction in platelet aggregation , and lowering of plasma apolipoprotein ( a ) .
in contrast to this heavy intake ( i.e. , < 89 g-70/day ) increase the cv risk due to increased homocysteine levels.28 in this study cv risk was found to be high ( 20% ) in cox2-is group and alcohol consumption appears to compliment this .
several earlier studies have affirmed cv risk of selective cox-2-is like rofecoxib , valdecoxib , lumiracoxib and celecoxib .
our study mainly emphasizes the potential cv risk of etoricoxib ; cv safety data of which is scare and continued to be used in therapeutics .
the results of this study encourage the proclamation that cv risk of cox-2-is could be expressed as a class effect , derived from earlier studies in this regard .
although cox-2-is preferred over nonselective cox - is due to cardiovascular , gi and renal toxicity of these agents and are said to be safe , the use of selective cox-2-is must be constrained to the arthritic patients with absolute contraindications ( i.e. , peptic ulcer , asthma and renal disease ) for nonselective nsaids as arthritics need to take them over a long period of time and they impart substantial cv risk over a period of time .
thus this study could be a rewarding accumulation in cv safety data of selective cox-2-is . | background : cyclooxygenase-2 inhibitors ( cox-2-is ) have recently been concerned in the occurrence of adverse cardiovascular ( cv ) events .
rofecoxib and valdecoxib has been withdrawn from the market , but celecoxib , etoricoxib and parecoxib continues to be used .
other nonsteroidal anti - inflammatory drugs ( nsaids ) may also increase the risk of cv events .
however , clinical trial databases for cox-2-is had created lots of controversies regarding cardiovascular safety of selective and nonselective cyclooxygenase inhibitors ( cox - is ) .
this study was , conducted to assess and compare the cv risk of cox - is in arthritic patients over a period of time.materials and methods : in this prospective cohort study adult arthritics of either sex those were freshly diagnosed or taking cox - is for < 3 months ; were included .
patients were grouped into nonselective and selective cox-2-i groups with reference to treatment they received .
the cv risk factors like blood pressure ( bp ) , blood sugar level ( bsl ) , lipid profile , body mass index ( bmi ) were assessed and compared ; demography of cv risk factors was also studied .
data obtained was analysed using student 's
t-test of openepi statistical software.results:study clearly revealed that all nsaids exhibit variable cv risk ; however , selective cox-2-is found to exhibit more cv risk .
bmi , bp and lipid profile ; the potential cv risk factors , showed significant impairment in selective cox-2-is group ; p < 0.01 , p < 0.001 and p < 0.05 , respectively , compared to baseline and p < 0.05 vs. nonselective cox - is for bmi.conclusions:this study portrays the potential cv risk of selective cox-2-is ; confirms and re - evaluate the results of earlier studies in this regard . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
the italian physician ubaldo gasperini described in 1912 a patient with a lesion of the 5th , 6th and 7th cranial nerves of one side combined with a contralateral hypacusis and hemihypesthesia for pain and temperature , and only initially with a slight motor impairment .
the diagnosis was not confirmed by postmortem examination , but sudden onset of clinical symptoms was compatible with a vascular accident .
gasperini assumed a brainstem hemorrhage in the lateral caudal pontine tegmentum as the cause of the symptoms .
only a few cases of this crossed brainstem syndrome caused by pontine infarctions were published so far [ 2 - 4,6 ] . here
we report a patient with the classical gasperini syndrome due to demyelination in multiple sclerosis .
a 44-year - old man was referred with the left - sided facial weakness and numbness of his right arm and leg . on admission , he was alert and oriented .
he had an impaired abduction of the left eye with diplopia , a peripheral facial palsy with minimal lagophthalmus , a facial hypesthesia , and a hypacusis on the left side .
speech , soft palate movements , swallowing , gag reflex , and tongue movements were normal .
there was no disturbance of trapezius and sternocleidomastoid muscles , and no horner 's syndrome .
t2-weighted cranial mri revealed a high signal intensity area in the left lateral caudal pons ( figure 1 ) and several smaller demyelinating lesions in the periventricular white matter .
csf analysis yielded a slight lymphocytic pleocytosis ( 36 cells / mm ; normal < 5 cells / mm ) and oligoclonal bands . flair - weighted cranial mri showing an area of increased signal intensity in the left - sided lateral caudal pontine tegmentum .
all neurological signs and symptoms with the exception of the mild facial nerve paresis on the left side gradually disappeared within one week
. transverse section through the caudal pons showing : 1 - facial nerve and nucleus ; 2 - abducent nerve and nucleus ; 3 - lateral lemniscus ; 4 - spinal trigeminal tract ; 5 - spinothalamic tract ; 6 - fourth ventricle .
the mr imaging showed a demyelinating area in the left caudal pons ( figure 1 ) .
the anatomical basis of the gasperini syndrome can be considered as a lesion of the left - sided lateral caudal pontine tegmentum involving the ipsilateral facial nerve nucleus ( peripheral facial palsy ) , the abducens fibres ( abducens nerve palsy ) , the acoustic fibres ( hypacusis ) , the spinal trigeminal tract ( facial hypesthesia ) , and the spinothalamic tract ( contralateral hemihypesthesia for pain and temperature ) ( figure 2 ) . since the lemniscus lateralis carries predominant information from the contralateral cochlea , affection of the lateral lemniscus usually results in predominant hypacusis of the contralateral side .
however , affection of the lateral lemniscus did not cause a complete contralateral deafness or severe hypacusis described in the original case of gasperini and in another recent case because of the binaural representation of the ascending auditory tracts .
interestingly , gasperini predicted the possibility of deafness on the ipsilateral side which was lately described in the majority of published cases of the gasperini syndrome .
presumably , the ipsilateral deafness or hypacusis may be caused by the involvement of the more peripheral portion of the acoustic pathway . in our patient
since nearly 100 years only a very few cases of the gasperini syndrome were published [ 2 - 4,6 ] .
pontine ischemia or bleeding due to venous angioma , vertebral artery occlusion , basilar artery stenosis , and small vessel disease associated with diabetes mellitus , hyperlipidemia or hypertension were reported as causes of the gasperini syndrome so far .
sometimes the gasperini syndrome was even defined as the " aica syndrome with abducens nerve palsy " .
the gasperini syndrome with the typical combination of ipsilateral lesions of the cranial nerves v - viii and dissociated contralateral hemihypesthesia in a patient with pontine demyelination shows that classical crossed brainstem syndromes are topological terms not invariably associated with brainstem ischemia in particular vascular areas .
the present case may contribute to the differential diagnosis of peripheral facial nerve palsy , which also includes the gasperini syndrome . | the gasperini syndrome is a very rare brainstem disease characterized by the typical combination of ipsilateral lesions of the cranial nerves v - vii and dissociated contralateral hemihypesthesia , whereas both contralateral and ipsilateral hypacusis was described .
since the first description in 1912 , only a few cases of this crossed brainstem syndrome were published so far .
pontine infarction and bleedings were the reported causes of the syndrome . here
we report a 44-year - old man with the classical gasperini syndrome due to pontine demyelination in multiple sclerosis .
the clinical findings were correlated with changes on mri .
the present case shows that classical crossed brainstem syndromes are topological terms not invariably associated with brainstem ischemia in particular vascular areas and may contribute to the differential diagnosis of peripheral facial nerve palsy . | Introduction
Case report
Discussion |
despite the significant improvements that have been made in oral health , tooth loss remains a dental public health problem .
tooth loss is the basic indicator of failure or success in the dental community health programs .
the main purpose of these programs is to prevent tooth loss due to incurable caries or periodontal problems .
it is commonly known that having healthy teeth affects oral health . in developing countries , dental caries
tooth loss reduces one 's quality - of - life due to reduced aesthetics , speaking ability and chewing ability .
recently , some researchers have focused on the effect of early tooth loss on human life and found that tooth loss was associated with premature mortality in ancient times .
. however , the most common reasons for tooth extractions are dental caries , periodontal disease , impacted teeth , orthodontic treatment and prosthetic purposes .
different data relations that point to the most common reasons for tooth extraction are present in previous studies .
some of these studies suggested that periodontal diseases are the most common reason for tooth extraction , while others reported that deep carious lesions are highly linked with tooth extraction .
based on these studies , one can conclude that while teeth extractions generally occur in older adults due to advanced periodontal diseases , they generally occur in adolescents and children due to incurable caries .
therefore , early tooth loss in pediatric patients may be reduced by inhibiting these deep carious lesions .
similarly , the high availability of endodontically treated teeth ( ett ) and teeth requiring endodontic therapy ( tret ) as a result of deep caries is also a dental community problem .
the health - care system of a place indeed may influence the endodontic status of there .
several factors may affect the fate of the dental pulp : one of the most important factors is the caries disease that stems from microorganisms , which may result in pulp infection that necessitates endodontic treatment .
furthermore , endodontic treatment has some risk factors such as flare - ups , perforations and the fracture of an instrument in the root canal and anatomical failures .
a large number of epidemiological studies evaluate the frequency of ett and tooth loss in different populations .
eriksen and bjertness . have emphasized that the purposes of the few epidemiological studies were the adjustment of preventive and clinical procedures and the determination of endodontic treatment status .
furthermore , endodontic epidemiology is required to provide a general picture of endodontic disease , its treatment and the treatment outcome in different societies . in turkey ,
similarly , some investigators focused on tooth loss in recent decades , but did not provide detailed information about early loss of permanent first molars in children and adolescents .
the purpose of the present study was to evaluate the frequency and distribution of early missing as well as the endodontic status of permanent first molars based on age , gender , jaw and side factors in a turkish pediatric population .
this cross - sectional study was conducted in the cappadocia region of turkey and featured a total of 7,895 patients ( 3,752 boys and 4,143 girls ) ages 6 - 16 years .
panoramic radiographs taken routinely and anamnesis forms were obtained from the archive of erciyes university , faculty of dentistry , department of oral and maxillofacial radiology between august 2008 and december 2012 .
the scientific committee of the dental faculty approved the study and patient anonymity was strictly respected .
panoramic images were captured by a radiology assistant using a panoramic system ( orthopantomography op100 , tuusula , finland ) . two independent endodontists with more than 5 years of experience evaluated the digital radiographs . before this study ,
panoramic radiographs that were not included in the research were used to calibrate the two observations .
the teeth with deep carious lesions ( too close to the pulp ) , but no pain and mortality according to anamnesis forms were excluded from the study . in the present study ,
the following information was recorded on the prepared form for each individual : the number and location of missing teeth ( mt ) , teeth requiring extraction ( tre ) , tret and root - filled teeth .
the data collected were examined by jaw , gender , age and side . in the case of a disagreement
uncultivated data were analyzed using the excel 2007 ( microsoft corporation , redmond , wa , usa ) .
the chi - squire test was used to determine significant differences in terms of age , gender and location of the teeth for the following parameters : number of mt , tre , tret and root - filled teeth .
of the 19488 teeth examined in children group , 122 ( 0.63% ) were missing , 91 ( 0.47% ) required extraction , 780 ( 4% ) required endodontic therapy and 91 ( 0.47% ) ett were found . of the 12,092 teeth examined in the adolescent group , 501 ( 4.14% ) were missing ,
157 ( 1.30% ) required extraction , 736 ( 6.09% ) requiring endodontic therapy and 517 ( 4.28% ) ett were found . for all data , statistically significant differences existed between the child and adolescent groups ( p < 0.001 ) [ table 1 ] .
the results according to age , gender , jaw and side of the 15008 teeth examined in the boys group , 276 ( 1.84% ) were missing , 108 ( 0.72% ) required extraction , 724 ( 4.82% ) required endodontic therapy and 239 ( 1.59% ) ett were found . of the 16,572 teeth
examined in the girls group , 347 ( 2.09% ) were missing , 140 ( 0.84% ) required extraction , 792 ( 4.78% ) required endodontic therapy and 369 ( 2.23% ) ett were found . a statistically significant difference ( p <
0.001 ) was found for ett data between the child and adolescent groups , but no statistically significant difference for other data was found between these two groups ( p > 0.05 ) [ table 1 ] .
a total of 15,790 teeth were examined in the maxilla as well and 185 ( 1.17% ) were missing , 83 ( 0.53% ) required extraction , 551 ( 3.49% ) required endodontic therapy and 224 ( 1.42% ) ett were found . in the mandible , where the same number of teeth was examined
, 438 ( 2.77% ) were missing , 165 ( 1.04% ) required extraction , 755 ( 4.78% ) required endodontic therapy and 384 ( 2.43% ) ett were found .
all data were statistically higher in the mandible than in maxilla ( p < 0.001 ) [ table 1 ] .
when data were assessed in terms of the side factor ( right or left ) , 302 ( 1.91% ) teeth were missing , 116 ( 0.73% ) required extraction , 770 ( 4.88% ) required endodontic therapy and 305 ( 1.93% ) ett were found . in left side , where the same number of teeth that was on right side were examined , ( 2.037% ) were missing , 132 ( 0.73% ) required extraction , 746 ( 4.72% ) requiring endodontic therapy and 303 ( 1.92% ) ett were found .
no statistically significant difference between right and left were found ( p > 0.05 ) [ table 1 ] .
table 2 shows mt , tre , ett and tret data in each age separately .
for all data while 15 - 16-year - old patients showed the highest results , 12-year - old patients had the lowest results , except for the results dealing with tret and tre in adolescents .
tret was higher in 13 - 14 year - old patients and tre was too close to each other in groups 12 , 13 and 14 .
in addition , for all data while 10 - 11-year - old patients showed the highest results and 6 - 7-year - old patients had the lowest results in the child group .
each column were evaluated within itself separately as adolescent and child table 3 shows mt , tre , ett and tret data according to the side and jaw . while mt , tre and ett data were highest in the left mandible , tret was highest in the right mandible in adolescents .
in contrast while mt , tre and ett data were highest in the right mandible , tret was highest in the left mandible in children . the data according to side and jaw .
tooth loss and endodontic therapy need are major public health problems in many developing countries . consequently , many epidemiologic studies address these subjects in different countries .
the present study provides cross - sectional research on early loss of and endodontic status of permanent first molars in a turkish pediatric population .
this study was the first comprehensive study to assess both early tooth loss and the endodontic status of the turkish pediatric population and it included 31,580 teeth in 7,895 patients . in the present study ,
panoramic radiographs and patient anamnesis forms that were already in the faculty archive were evaluated because examining these items was far easier than performing clinic examinations of this high number of patients .
a periapical radiograph has a higher sensitivity than panoramic radiographs do for seeing pathologies and lesions , but panoramic radiographs and anamnesis forms were adequate for the present study to see mt and determine whether a tooth had endodontic therapy .
when a carious lesion is very close to the pulp , deciding on the presence of endodontic therapy is difficult looking only at the radiographs . in such situations ,
patient anamnesis forms were used for the final decision in this study . in the present study ,
only first permanent molar teeth were examined because these teeth erupted first and were the only permanent teeth in individuals 6 - 12 years of age until eruption of the second permanent molars . for the standardization ,
the same type of tooth was examined in the 13 - 16-year - old adolescent group , too .
the teeth were evaluated with regard to the number and percentage of mt , tre , tret and teeth treated endodontically . according to the results of present study , the adolescent group had higher tooth loss than those of the child group .
this is in agreement with a previous study that indicated that tooth loss increased with age .
similarly , the tre values were higher in the adolescent group than in the child group .
george et al . performed an epidemiologic study among children and adults in a suburban area of chennai .
it was observed that the percentage of tooth loss for groups of 6 , 12 and 15 years was 5.7% , 22% and 28% respectively . in the present study
; this data for permanent first molar was 0% , 2.22% and 6.53% respectively for the same age groups .
this condition may be caused by differences of the health - care system of the countries .
the percentage of permanent first molar loss among adolescents was 4.14% , which is relatively higher in comparison to study conducted in brazil , and saudi arabia .
gender may also influence tooth loss in a population . in a study , al shammary et al .
in contrast , the present study showed no statistically significant difference between males ( 1.84% ) and females ( 2.09% ) .
al shammary et al . found that mean tooth loss for males and females was 2.6 and 2.9 , respectively , which is lower than that of two studies conducted in saudi arabia and sri lanka , but higher compared with the present study .
these differences may be due to elderly people 's increasing the mean values of tooth loss because they had more mt than did children and adolescents . in the present study , only 6 - 16-year - old subjects who had few mt were evaluated . in the literature ,
we could not found considerable results evaluating early tooth missing according to jaws and side in pediatric patients .
hand selection when tooth brushing may affect brushing ability of both right and left side . a patient using the right hand
mechanical cleaning effects of foods when chewing can effect formation of carious lesion . in the present study
, we found that no significant difference existed between the right and left in terms of mt , tre , ett and tret ( p > 0.05 ) .
in contrast , su et al . reported that there were more fillings due to caries in mandible than maxilla that is in agreement with present study . in the present study
, we found that mandible had significantly higher values than maxilla ( p < 0.001 ) in terms of mt , tre , ett and tret .
this may be due to different saliva effects or anatomical structures of different teeth . in the present study ,
a statistically significant difference existed between adolescent and children in terms of both ett and tret .
this situation is normal because the number of carious tooth increases with age progress . in both adolescents and children , although the percentage of tret was low , the percentage of ett was high .
this may be caused that patient neglect their routine dentist control appointment or they go to the dentist only when they had pain .
this difference is higher in the 6 - 12 age child groups and this may be because they had fear of the dentist and needle .
the prevalence of a root - filled permanent first molar in a turkish population studied here is similar to those of previous studies , in which boys had significantly fewer ett than girls ( p < 0.001 ) .
this may be because the females have more esthetic concerns than males . however , no significant difference existed in terms of tret ( p > 0.05 ) .
regarding the limitation of the present study , adolescents have higher values of mt , tre , ett and tret than children .
furthermore , the mandible has higher values of mt , tre , ett and tret than does the maxilla . | objectives : the objective of this study is to evaluate the frequency and distribution of early tooth loss and endodontic treatment needs of permanent first molars in a turkish pediatric population.materials and methods : a total of 7,895 panoramic radiographs taken for routine dental examination at the department of oral maxillofacial radiology between 2008 and 2012 years were investigated .
two independent specialists evaluated early tooth loss and endodontic treatment needs of permanent first molars using panoramic radiography and patient anamnesis forms .
the teeth were classified according to the following data : ( a ) missing teeth , ( b ) teeth requiring extraction , ( c ) endodontically treated teeth ( ett ) , ( d ) teeth requiring endodontic therapy .
the data also classified according to four factors : age group ( 6 - 12 and 13 - 16 ) , gender ( boy and girl ) , jaw ( mandible and maxilla ) and side ( right and left ) . a chi - square test was used for statistical analyses.results:a total of 19,488 and 12,092 teeth were evaluated in the child group and adolescent group respectively .
all data were higher in adolescents than children ( p < 0.001 ) . for gender factor ,
only ett was higher in girls than it was in boys ( p < 0.001 ) .
for the jaw factor , all data were higher ( p < 0.001 ) in mandible than in the maxilla .
for the side factor , no statistical difference existed between right and left.conclusions:early tooth loss and endodontic treatment needs of permanent first molars showed variability according to age groups and jaws . when the results were compared according to the side and gender factors , no statistical difference was found ( p > 0.05 ) except with the data of ett in gender groups . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
bernardo alberto houssay in his lecture in 1947 the blood sugar and the production and consumption of glucose are kept within normal bounds , therefore there is an equilibrium between the glands of internal secretions which reduce the blood sugar ( pancreas ) and those which raise it ( anterohypophysis , adrenals , thyroid , etc . ) .
deficit or excess of thyroid hormones can break this equilibrium leading to alterations of carbohydrate metabolism .
overt hyperthyroidism has been related to glucose intolerance and even ketoacidosis . with regards to hypothyroidism ,
cases of hypoglycemia have been reported in the literature despite the fact that peripheral insulin resistance may be present . in the century that has elapsed , since the first observations of uncontrolled glucose metabolism in thyrotoxic diabetic patients , new pathways involved in the regulation of glucose homeostasis by thyroid hormones have been unveiled .
novel findings include the stimulation of hepatic glucose production by thyroid hormones acting via a sympathetic pathway from the hypothalamus and the discovery of transcriptional regulators of metabolic and mitochondrial genes that , influenced by intracellular t3 levels , may contribute to the development of insulin resistance .
the calorigenic - thermogenic activity of t3 long ascribed solely to uncoupling of mitochondrial oxidative phosphorylation has recently been related to t3-induced gating of mitochondrial permeability transition pore ( ptp ) of the inner mitochondrial membrane where the whole t3 transduction pathway integrates genomic and nongenomic activities of t3 in regulating mitochondrial energetics . in this paper
, we summarize the effects of thyroid hormones in glucose metabolism and its alterations when thyroid dysfunction is present .
thyroid receptor - mediated effects on gene transcription and translation are key in the regulation of glucose metabolism . according to the results of studies with complementary dna ( cdna ) microarray analysis in mouse liver ,
several genes involved in gluconeogenesis , glycogen metabolism , and insulin signaling that are regulated by thyroid hormones have been identified . in the study by feng
et al . , rna from hypothyroid mice treated with t3 was prepared , labeled with fluorescent dye , and hybridized with the cdna microarray .
this enzyme hydrolyzes glucose-6-phosphate and completes the final step in gluconeogenesis and glycogenolysis , therefore playing an important role in the homeostatic regulation of blood glucose levels .
another finding was a decrease in mrna expression of akt2 ( protein kinase b ) , a serine / threonine kinase that is an essential molecule in the insulin signaling pathway .
akt2 has been shown to promote glycogen synthesis in liver by inactivating glycogen synthase kinase 3 .
thus , a decrease in akt2 activity would decrease glycogen synthesis explaining the antagonistic insulin effect of thyroid hormones at the liver .
moreover , an induction of 2-adrenergic receptor mrna and repression of inhibitory g protein ( gi ) rna of the adenylate cyclase cascade by t3 were also reported .
all these results are in favour of a permissive influence of t3 in the glycogenolytic and gluconeogenic effects of epinephrine and glucagon .
other hepatic gluconeogenic enzymes that have been found to be positively regulated by thyroid hormones include phosphoenolpyruvate carboxykinase ( pepck ) , the enzyme that catalyzes the rate - controlling step of gluconeogenesis and pyruvate carboxylase , involved in the synthesis of oxaloacetate from pyruvate .
the catalytic activity of pyruvate carboxylase has been found increased approximately 2-fold in hyperthyroid rats compared with untreated or treated euthyroid controls .
another mechanism , whereby thyroid hormones are known to increase hepatic glucose output , is through increased hepatic expression of the glucose transporter glut2 as previously shown in a rat model where glut2 protein concentration in crude liver membranes was twice as high in chronically hyperthyroid versus hypothyroid animals .
it has been previously reported that , despite an expected resistance towards the insulin inhibitory effect on gluconeogenesis , the transcription of several enzymes involved in lipid synthesis or lipid metabolism is increased in hyperinsulinemic , insulin - resistant mice .
furthermore , t3 induction of lipogenesis through the transcriptional activation of malic enzyme , involved in fatty acid synthesis , has been previously reported .
therefore , it is possible that by the induction of lipogenic enzymes , t3 could be further aggravating the dysregulation of liver glucose and lipid metabolism characteristic of insulin resistance . as a result of the long time quest for thyroid analogs that possess the favourable actions on metabolism without the unwanted thyroid cardiac effects ,
the differential distribution of thyroid receptor ( tr ) isoforms in the tissues has been key for the development of these analogs . with regards to lipogenesis ,
carbohydrate - response element - binding protein ( chrebp ) is a major transcription factor controlling the activation of glucose - induced lipogenesis in liver and is a direct target of thyroid hormones in liver and white adipose tissue ( wat ) , the two main lipogenic tissues in mice .
chrebp is shown to be specifically regulated by trbeta but not by tralpha in vivo , in liver where trbeta represents 80% of the thyroid hormone bound tr , but also in wat where both tr isoforms are expressed .
although the area of thyroid analogs is beyond the scope of this paper , it is to be mentioned that some thyromimetic analogs , such as 3,5-l - diiodothyronine ( t(2 ) ) , exert their beneficial action on metabolism , without inducing a thyrotoxic state , through a mechanism that does not involve binding to thyroid hormone receptors . in rats
fed a high - fat diet , the addition of t(2 ) to lipid - overloaded cells resulted in reduction in lipid content ; downregulation of peroxisome proliferator - activated receptors ( ppar) , ppar , and alternative oxidase ( aox ) expression ; increase in ppar expression ; and stimulation of mitochondrial uncoupling thus preventing and reversing hepatic steatosis in this animal model .
surprisingly , in this study , these lipid - lowering actions not mediated by tr were also observed with t3 . to summarize ,
all these findings have helped to understand that thyroid hormones have insulin antagonistic effects at the liver that lead to an increased glucose hepatic output , via an enhanced rate of gluconeogenesis and glycogenolysis . with regards to lipid metabolism ,
however , in the context of insulin resistance , the conversion of glucose into fatty acids together with nonsuppressed gluconeogenesis is simply perpetuating the hyperinsulinemic state . furthermore , nutritional influences , such as those of high - fat diets , should also be taken into consideration as modifiers of the effects of thyroid hormones on insulin sensitivity .
opposite to what occurs at the liver level , at peripheral tissues , thyroid hormones have been shown to exert some of their actions synergically with insulin .
the upregulation of the expression of genes such as glut-4 or phosphoglycerate kinase ( pgk ) , involved in glucose transport and glycolysis respectively , is a good proof of concept . in skeletal muscle , the main site of insulin - mediated glucose disposal , glucose transporter glut4 , is induced by t3 , revealing that it can increase basal and insulin - stimulated glucose transport in this tissue .
another t3 target in skeletal muscle is mitochondrial uncoupling protein 3 ( ucp3 ) . unveiling
this association may be important since progressive reduction of ucp3 levels results in insulin resistance accompanied by decreased fatty acid oxidation and a less intense akt / pkb and 5 adenosine monophosphate - activated protein kinase ( ampk ) signaling .
although discrepancies between the regulation by t3 of ucp3 expression in rats , humans , and mice have been observed , the rat model has shed some light into t3 actions in this tissue .
t3 intravenous ( i.v . ) administration in hypothyroid rats showed a rise in serum fatty acid levels concomitant with a rapid increase in ucp3 expression in gastrocnemius muscle .
these findings point to ucp as a possible molecular determinant of the action of t3 on energy metabolism .
, t2 actions have been also explored in skeletal muscle . in a model of high - fat diet - induced insulin resistance in rat
, the administration of t2 , on the gastrocnemius muscle , induced remarkable changes on the metabolic / structural phenotype and insulin signaling .
t2 increased insulin - stimulated akt phosphorylation levels , the muscle contents of fast / glycolytic fibers and sarcolemmal glut4 . moreover ,
the result of cdna microarray analysis on skeletal muscle of a group of healthy men receiving 75 g / d of t3 for 14 days has shown that not only genes with agonistic insulin effects but several others with antagonistic insulin effects are upregulated by treatment with t3 , underlying the pleiotropic effect of thyroid hormone in energy metabolism .
cdna array data have also provided a molecular basis to the effect of t3 on adipose tissue . in human adipocytes ,
t3 increases the mrna levels of the lipolytic 2-ar , favouring catecolamine - induced lipolysis and it also downregulates sterol regulatory element binding protein ( srebp1c ) , involved in lipogenesis , which may constitute a link between hyperthyroidism and insulin resistance .
skin fibroblasts have been also used to study thyroid hormone - responsive genes involved in metabolism in human cells .
although they are not as metabolically active as hepatic cells , they are easily obtained and also , thyroid hormone - responsive . in cultured human fibroblasts , moeller et al . observed that , opposite to a posttranscriptional regulation as reported for other growth factors and hormones , the mrna of the transcription factor hif-1 ( hypoxia - inducible factor 1 ) , a key mediator of glycolysis , increased in response to t3 .
as the glucose transporter glut1 , several enzymes of glycolysis , and the lactate exporter slc16a3 were all also found induced by t3 and are target genes of the transcription factor hif-1 , the authors postulated that the effect of thyroid hormones on the induction of these genes most probably was indirect and hif-1 mediated .
furthermore , a new mechanism of thyroid action was unraveled by this group of researchers .
it was shown that t3 bound to trbeta , in lieu of initiating gene transcription in the nucleus , activates the phosphatidylinositol 3-kinase ( pi3k ) signaling pathway in the cytosol in order to activate hif-1 gene expression . at the cellular level
, thyroid hormones can also increase mitochondrial biogenesis , fatty acid oxidation , and tca cycle activity .
these findings are quite relevant since the role of mitochondrial dysfunction , leading to cellular lipid excess and impaired oxidative metabolism , has been clearly demonstrated in the pathogenesis of type 2 diabetes [ 2325 ] .
furthermore , it has been described that in skeletal muscle , the lack of thyroid hormones might dysregulate mitochondrial gene expression .
ppar gamma coactivator-1 alpha ( pgc-1 alpha ) , a key transcriptional regulator of mitochondrial content and function , fatty acid oxidation , and gluconeogenesis , has been involved in the process whereby thyroid hormones regulate mitochondrial function .
it has been shown that pgc-1 alpha gene expression is increased by t3 , as much as 13-fold 6 hours after t3 treatment .
the regulation pattern of t3 on pgc-1 alpha is complex and may occur through nongenomic activation of kinases to induce the expression of pgc-1 alpha or through transcriptional upregulation via the presence of a thyroid responsive element ( tre ) in the pgc-1 alpha promoter or by genomic upregulation of a transcription factor ( via a tre ) , which then binds to the pgc-1 alpha promoter and increases pgc-1 alpha transcription .
it is hypothesized that pgc-1 alpha can be dysregulated by reduced t3 levels , thus contributing to insulin resistance .
not only low circulating but also , intracellular t3 levels , could count for this matter . a lower expression and activity of type 2 iodothyronine - deiodinase ( d2 ) , the enzyme that is key for the conversion of t4 into t3 in muscle and thus , amplifies
thyroid hormone signaling in individual cells , has been found linked to insulin resistance [ 29 , 30 ] .
if pgc-1 alpha effects on mitochondrial gene expression may indeed be regulated by thyroid hormone , normal activity of deiodinase type 2 is very relevant .
bile acids are potent stimulators of the enzyme and may play an important role in the relationship between thyroid action and glucose metabolism . on the other hand , the natural occurrence of polymorphisms of deiodinase type 2 such as thr92ala , with a lower activity , has also been implicated with increased risk for diabetes type 2 .
it has been shown that the hypothalamus can modulate endogenous glucose production by using functionally reciprocal sympathetic and parasympathetic autonomic outputs to the liver . moreover ,
a sympathetic pathway from the hypothalamic paraventricular nucleus to the liver has been proposed as a central pathway for modulation of hepatic glucose metabolism by thyroid hormone .
klieveric et al . demonstrated that upon selective administration to the paraventricular nucleus ( pvn ) , t3 increases endogenous glucose production and plasma glucose , and that these hypothalamic t3 effects are mediated via sympathetic projections to the liver . in order to arrive to such remarkable findings
first they administered an intracerebroventricular ( i.c.v . ) t3 or vehicle ( veh ) infusion , and there was a significant increase in plasma glucose compared with veh - treated rats .
this meant that central t3 infusion could reproduce the characteristic increase in hepatic glucose output of thyrotoxicosis .
to further identify the neuroanatomic region responsible for these changes , the authors infused t3 within the hypothalamus at the pvn .
a similar response was obtained , that was independent of plasma t3 , insulin , and corticosterone concentrations .
they repeated the experiment in surgically hepatic sympathectomized animals ( hsx ) and sham - denervated animals .
the principal finding of this study is the description of a neural ( autonomic ) modulation of hepatic glucose metabolism by t3 at the hypothalamus that takes place independently of plasma glucoregulatory hormone concentrations .
thyrotoxic subjects frequently show impaired glucose tolerance . this is a result of increased glucose turnover with increased glucose absorption through the gastrointestinal tract , postabsorptive hyperglycemia , elevated hepatic glucose output , with elevated fasting and/or postprandial insulin and proinsulin levels , elevated free fatty acid concentrations and elevated peripheral glucose transport and utilization .
the literature about this topic is vast and has been previously comprehensively reviewed by dimitriadis and raptis .
although ketoacidosis may result per se from the insulin resistance present in thyrotoxicosis , the stimulatory action of thyroid hormones in excess on lipolysis and free fatty acids availability can also contribute to increased ketogenesis .
thyrotoxicosis has been reported to increase endogenous glucose production in the liver in the basal state and to decrease hepatic insulin sensitivity in humans .
the different mechanisms to explain this phenomenon include increased rates of gluconeogenesis and glycogenolysis mainly explained by the above - mentioned effects on the liver by thyroid hormones . to summarize , these effects include thyroid receptor - mediated effects on liver gene transcription , increased sympathetic action in the liver mediated by hypothalamus , and increased concentrations of the glut2 glucose transporters in the liver plasma membrane that allows for glucose efflux [ 8 , 39 ] together with increased concentration of free fatty acids in plasma .
the interpretation of the effects of hyperthyroidism on glucose utilization by peripheral tissues is by far the most complex issue on this topic .
on one hand , the rates of glucose uptake in peripheral tissues have been found increased by thyroid hormones , suggesting that glucose utilization is highly increased , specially in skeletal muscle [ 34 , 37 , 4144 ] .
this increased utilization , as shown by indirect calorimetry during euglycemic hyperinsulinemic clamps , is mainly due to an increase in insulin - stimulated glucose oxidation rates [ 43 , 4548 ] .
however , a decrease in insulin - stimulated nonoxidative glucose disposal , through reduced glycogenogenesis [ 43 , 44 , 49 ] , takes place , with intracellular glucose being redirected towards glycolysis and lactate formation .
the release of lactate from peripheral tissues back to the liver is a major contributor to the cori cycle where more hepatic glucose is being produced [ 43 , 4951 ] .
although glucose intolerance in hyperthyroidism can be easily explained by hepatic insulin resistance without involvement of peripheral tissues , impaired insulin - stimulated peripheral glucose uptake has also been proven in some studies . by means of the arteriovenous difference technique in the forearm muscles of hyperthyroid subjects after the consumption of a mixed meal , it has been clearly demonstrated that muscle blood flow is increased , masking a defect in insulin - stimulated glucose uptake .
moreover , in disagreement with previous reports [ 41 , 45 ] , shen et al . also described decreased peripheral insulin sensitivity in hyperthyroidism .
alternative explanations for peripheral insulin resistance in hyperthyroidism include an increased secretion of bioactive mediators ( adipokines ) such as interleukin 6 ( il6 ) and tumour necrosis factor a ( tnf ) from adipose tissue in hyperthyroidism .
these adipokines , that exert both proinflammatory and insulin resistant effects , have been found elevated in hyperthyroid women . in hyperthyroidism , decreased , normal , or even increased levels of plasma insulin have been reported .
however , a rather consistent finding has been the increased degradation of insulin in hyperthyroid subjects [ 43 , 55 ] .
it has been postulated that , in the long run , severe thyrotoxicosis can lead to irreversible pancreatic damage [ 56 , 57 ] . with regards to glucagon , its secretion and metabolic clearance rates have been reported increased , explaining the normal fasting plasma levels described in hyperthyroidism .
subclinical hyperthyroidism has also been associated with insulin resistance [ 5961 ] in some but not all studies .
endogenous subclinical hyperthyroidism may have a larger impact on glucose metabolism due to its chronicity and higher t3 levels when compared to exogenous administration of t4 .
this phenomenon can be interpreted in the light of reduced levels of gluconeogenesis leading to decreased liver glucose output [ 63 , 64 ] . on the other hand ,
insulin resistance has been shown to be present in peripheral tissues in hypothyroid animal models [ 65 , 66 ] .
hence , a poor utilization of glucose in hypothyroidism may be offset by a reduced release to circulation maintaining a balance of the glucose metabolism .
studies performed in adipocytes and skeletal muscle of rats made hypothyroid have shown that these tissues are less responsive to insulin with regards to glucose metabolism [ 63 , 6569 ] .
czech et al . studied insulin responsiveness in adipocytes and skeletal muscle of mature rats rendered hypothyroid by a low iodine diet and propylthiouracil .
it was observed that glucose conversion to glycogen was partially inhibited while the glycolytic flux stimulation by insulin was totally frustrated .
other authors [ 66 , 67 ] showed decreased insulin - stimulated glucose transport and/or phosphorylation , as well as a lower rate of glycolysis in the isolated , incubated soleus muscle of the hypothyroid rat and also suggested that the effects of hypothyroidism in muscle were not mediated through an interference of insulin binding to its receptor .
it was postulated that they rather occurred through a postreceptor mechanism that may include abnormal phosphorylation of insulin signaling proteins .
insulin resistance was confirmed in another study of rats with mild hypothyroidism . in this study
, insulin responsiveness was measured by an insulin tolerance test and an euglycemic - hyperinsulinemic clamp followed by measurements of tissue - specific glucose utilization indices with the labelled 2-deoxy - d-[1 - 3h]glucose ( 2-dg ) technique in muscles ( red quadriceps ) and white adipose tissue ( epididymal fat ) .
several other parameters were also determined such as muscle triglyceride content , plasma leptin and nonesterified fatty acids ( nefa ) levels , and mrna expression of resistin in adipose tissue as well as adipose tissue and liver carnitine palmitoyl transferase 1 ( cpt-1 ) , and muscle carnitine palmitoyl transferase 1 ( cpt-1 ) mrna levels , explored by real - time quantitative pcr .
plasma leptin levels were lower and adipose tissue mrna expression of resistin higher , in the hypothyroid state .
looking for a potential role of leptin in the metabolic consequences of hypothyroidism , leptin was infused , and it was found that glucose disposal was recovered . increased expression of muscle and adipose tissue carnitine palmitoyl transferases , decreased plasma nefa levels , and reduced muscle triglyceride content after leptin infusion were interpreted as the mediators of the recovery of the insulin resistant state .
therefore , one possible explanation to decreased insulin responsiveness in hypothyroidism , according to the authors of this study , includes a dysregulation of leptin action at the hypothalamus .
adipocyte - myocyte crosstalk by adipokines has been reported to play a significant role in skeletal muscle insulin resistance and may partially explain insulin resistance present in hypothyroidism .
however , other factors associated with insulin resistance in hypothyroidism , such as altered blood flow , impaired glut4 translocation , decreased glycogen synthesis , and decreased muscle oxidative capacity have to be also considered .
compared to the number of reports about insulin resistance in hyperthyroid patients , there are relatively fewer studies in humans dealing with the effects of hypothyroidism on glucose metabolism .
measured whole - body sensitivity of glucose disposal to insulin in hypothyroid patients using the euglycemic - hyperinsulinemic clamp technique .
they demonstrated that hypothyroidism induced a decrease in the insulin - mediated glucose disposal that reverted upon treatment .
the same clamp technique and also measuring glucose tolerance and beta - cell activity with an oral glucose tolerance tests ( ogtt ) , handisurya et al
. confirmed these findings and added the knowledge that glucose - induced insulin secretion is diminished by thyroid replacement corresponding well with the observed improvement of insulin sensitivity .
dimitriadis et al . explored glucose uptake in muscle and adipose tissue of hypothyroid and control subjects by means of the arteriovenous difference technique in the anterior abdominal subcutaneous adipose tissue and the forearm muscles after the consumption of a mixed meal .
a decreased net extraction of glucose and blood flow after the meal in hypothyroid muscle and adipose tissue was reported .
this impairment in the ability of insulin to increase blood flow rate to the hypothyroid tissues is an alternative explanation to the mechanism whereby hypothyroidism can induce lower glucose disposal .
insulin tolerance test has been used to explore insulin sensitivity in acute overt hypothyroid patients .
a previous study in overt hypothyroid patients based on the homeostasis model assessment ( homa - ir ) showed no association between hypothyroidism and insulin sensitivity .
found unimpaired insulin - stimulated glucose disposal in the forearm of hypothyroid patients after treatment with levothyroxine . with regards to subclinical hypothyroidism , insulin resistance has been demonstrated in some [ 60 , 74 , 79 ] but not all studies , where homa levels were found comparable to a control group [ 8082 ] .
however , in some of these negative studies [ 80 , 81 ] , hyperinsulinemia was reported in subclinical hypothyroid subjects and interpreted as an early sign of impairment of glucose metabolism .
thyroid hormones have a large impact on glucose metabolism . a direct regulation on thyroid responsive genes at the target organ has been described and more recently an indirect effect involving hypothalamic pathways that regulate glucose metabolism via control of the sympathetic nervous system has been reported . furthermore , thyroid hormone effects can be insulin agonistic , such as demonstrated in muscle or antagonistic such as observed in the liver . in hyperthyroidism ,
dysregulation of this balance may end in glucose intolerance mainly due to hepatic insulin resistance . in hypothyroidism
the results are less evident . however , the available data suggest that insulin resistance is present mainly at the peripheral tissues .
possible explanations hypothesized to explain this phenomenon span from the dysregulation of mitochondrial oxidative metabolism to the reduction of blood flow in muscle and adipose tissue under hypothyroid conditions . | evidence for a relationship between t4 and t3 and glucose metabolism appeared over 100 years ago when the influence of thyroid hormone excess in the deterioration of glucose metabolism was first noticed .
since then , it has been known that hyperthyroidism is associated with insulin resistance .
more recently , hypothyroidism has also been linked to decreased insulin sensitivity .
the explanation to this apparent paradox may lie in the differential effects of thyroid hormones at the liver and peripheral tissues level .
the purpose of this paper is to explore the effects of thyroid hormones in glucose metabolism and analyze the mechanisms whereby alterations of thyroid hormones lead to insulin resistance .
| 1. Introduction
2. Effects of Thyroid Hormones on Glucose Metabolism (
3. Insulin Resistance as a Consequence of Hyperthyroidism
4. Hypothyroidism Can Also Lead to Insulin Resistance
5. Conclusions |
systemic lupus erythematosus ( sle ) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear antigens and by dysregulated activation of t and b cells .
polyclonal activation of b cells leads to the production of large quantities of autoreactive antibodies and the formation of immune complexes , which causes tissue damage . in some sle patients
, it has been shown that bone marrow mesenchymal stem cells exhibit impaired capacities for proliferation , differentiation , migration , and immune modulation .
genetic defects , drug exposure , infectious agents , and environmental factors can also contribute to the pathogenesis of this disease [ 3 , 4 ] .
sle has an incidence in europe and north america of approximately 10 cases per 100,000 population per year , and it is estimated that 10% of these cases are drug - induced .
drug - induced lupus erythematosus ( dile ) is a lupus - like syndrome that resolves upon drug discontinuation .
the drugs more frequently associated with the induction of this lupus - like syndrome are procainamide ( antiarrhythmic ) , hydralazine ( antihypertensive ) , and chlorpromazine ( antipsychotic ) [ 5 , 6 ]
. animal models of sle include lupus - prone mice , which spontaneously develop lupus , and normal mice that develop lupus after injection of lymphocytes from lupus - prone mice , immunization with prototypical lupus antigens ( dna- and rna - protein complexes ) , or injection of pristane ( 2,6,10,14-tetramethylpentadecane ) [ 3 , 7 ] .
the most commonly used lupus - prone mice are the f1 hybrids of new zealand black ( nzb ) and nz white ( nzb / nzw f1 ) mice , the murphy - roths large / lymphoproliferative locus ( mlr / lpr ) mice , and the recombinant c57bl/6 female and sb / le male strain / y - linked autoimmune accelerator ( bxsb / yaa ) mice [ 3 , 8 , 9 ] . our group has also developed a mouse model of autoimmune disease resembling human lupus that can be induced in normal mice . in this model ,
liposomes are model membranes made of cylindrical phospholipids , such as phosphatidylcholine , and hii - preferring ( conical shaped ) phospholipids , such as phosphatidic acid , phosphatidylserine , or cardiolipin .
conical phospholipids can form molecular associations distinct to lipid bilayers , known as nonbilayer phospholipid arrangements , in the presence of inducers such as mn [ 12 , 13 ] or the drugs chlorpromazine and procainamide , which can trigger dile in humans .
nonbilayer phospholipid arrangements are formed by an inverted micelle ( made of conical phospholipids with their polar heads towards the center of the micelle , where the inducer is also located ) inserted into and distorting the shape of the phospholipid bilayer ( figure 1(a ) ) . we demonstrated that liposomes with nonbilayer phospholipid arrangements induced by mn , chlorpromazine , or procainamide cause an autoimmune disease resembling human lupus in mice .
a similar disease is produced by treating mice directly with mn , chlorpromazine , or procainamide ( which induce nonbilayer phospholipid arrangements on mouse cells ) or by injecting the monoclonal antibody h308 ( which binds specifically to nonbilayer phospholipid arrangements and stabilizes these arrangements on mouse cells ) [ 10 , 14 ] .
igm and igg antibodies against nonbilayer phospholipid arrangements are found in the sera of mice with the autoimmune disease resembling human lupus , and also in the sera of patients with lupus [ 10 , 15 ] .
usually , the efficient production of igg antibodies requires an activation of the innate immune response .
therefore we hypothesized that nonbilayer phospholipid arrangements could be toll - like receptor- ( tlr- ) 4/md-2 agonists , as their molecular structure is similar to that of the lipid a from bacterial lipopolysaccharide ( lps ) .
lipid a is formed by a -1,6-d - glucosamine disaccharide with two ( negatively charged ) phosphates and six saturated acyl chains in an asymmetric distribution ( four chains are bound to the nonreducing and two to the reducing glucosamine ) . hexaacylated asymmetric lipid a molecules have a conical molecular shape , because the cross section of the hydrophobic region is larger than that of the hydrophilic region ( figure 1(b ) ) . hexaacylated symmetric lipid a ( with three acyl chains bound to the nonreducing and three to the reducing glucosamine ) and penta- and tetraacylated lipid a molecules have a cylindrical molecular shape , and they do not have biological activity [ 16 , 17 ] .
the intrinsic conformation of lipid a is not altered when saccharide groups are added , as in lps .
the lps molecules form multimeric aggregates in water : if the lipid a is cylindrical , they form a smooth bilayer arrangement , but conical lipid a molecules form a nonbilayer or hexagonal ( hii ) arrangement .
lps - binding protein ( lbp ) is a plasma protein that facilitates the transfer of lps molecules from these hexagonal ( hii ) arrangements to cd14 , and membrane - bound cd14 delivers lps to tlr-4/md2 . since the conical molecular shape of lipid a is a requirement for tlr-4/md-2 triggering [ 1619 ] , we hypothesized that liposomes with nonbilayer phospholipid arrangements , but not smooth liposomes ( with phospholipids in a bilayer arrangement ) , could trigger tlr-4/md-2 signaling . in this study , we investigated whether liposomes with nonbilayer phospholipid arrangements are tlr-4/md-2 agonists , because the activation of this innate immune receptor leads to the production of proinflammatory cytokines
. we also looked for proinflammatory cytokines in the sera of mice with the autoimmune disease triggered by liposomes with nonbilayer phospholipid arrangements , and we determined the gene expression profile in the spleens of these mice , focusing on the expression of proinflammatory genes .
in addition , we determined the relative percentage and activation of t , nkt , dendritic , and b cells in the spleen of mice with the disease .
this study contributes to the understanding of the pathological and genetic features of a novel mouse model of human lupus .
egg - yolk l--phosphatidic acid , bovine brain l--phosphatidylserine , egg - yolk l--phosphatidylcholine , chlorpromazine , procainamide , and chloroquine were purchased from sigma ( st . louis , mo , usa ) .
liposomes contained the cylindrical shaped phospholipid phosphatidylcholine and a conical phospholipid ( phosphatidic acid or phosphatidylserine ) . the molar ratios ( phosphatidylcholine / phosphatidic acid 2 : 1 , phosphatidylcholine / phosphatidylserine 4 : 1 ) were optimized for the induction of nonbilayer phospholipid arrangements .
nine micromoles of phospholipid mixture was dissolved in 1 ml diethyl ether and 330 l of ts buffer ( 10 mm tris - hcl , 1 mm nacl , ph 7 ) , mixed and sonicated three times in a g112spi sonicator ( laboratory supplies , hicksville , ny , usa ) .
the diethyl ether was then removed under a stream of oxygen - free dry nitrogen at reduced pressure , using a rotary evaporator at 37c .
the liposomes were filtered through 0.45 m mf - millipore membranes ( billerica , ma , usa ) to homogenize their size . to induce the formation of nonbilayer phospholipid arrangements , liposomes in ts buffer
were incubated for 30 min at 37c in the presence of 0.54 mm mncl2 , 0.53 mm chlorpromazine , and 432 mm procainamide .
all of the final preparations of liposomes were negative for lps contamination , as assessed by the gel clot lal method ( charles river endosafe , charleston , sc , usa ) .
the detection of nonbilayer phospholipid arrangements by flow cytometry was previously validated by freeze - fracture electron microscopy and p - nmr spectroscopy [ 10 , 14 , 15 ] .
therefore , in this study we only used flow cytometry to demonstrate the formation of these arrangements on liposomes .
liposomes and liposomes with nonbilayer phospholipid arrangements in ts buffer were analyzed with a facscalibur flow cytometer ( becton dickinson , san jose , ca , usa ) with cellquest software .
human embryonic kidney ( hek ) 293 cells , nontransfected or stably transfected with human tlr-4/md2/cd14 , tlr-2/tlr-6 , tlr-5 , or tlr-8 , were purchased from invivogen ( san diego , ca , usa ) .
the hek - tlr transfectants were maintained at 37c in 5% co2 in dulbecco 's modified eagle 's medium ( invitrogen , carlsbad , ca , usa ) containing 4.5 g / l glucose , 10% heat - inactivated fetal bovine serum ( gibco , grand island , ny , usa ) , 10 g / ml blasticidin ( invivogen ) , and 100 g / ml normocin ( invivogen ) .
hygrogold ( 25 g / ml ; invivogen ) was also added to the media of the hek - tlr-4/md2/cd14 cell line .
the viability of these cell lines in the presence of mn , chlorpromazine , procainamide , or chloroquine , and in the presence of liposomes or liposomes with nonbilayer phospholipid arrangements , was evaluated with the alamar blue method . to assess tlr activation , the cell lines were incubated in the presence of liposomes made of phosphatidylcholine / phosphatidic acid ( 2 : 1 ) , alone or with nonbilayer phospholipid arrangements induced by mn ( 24 mm ) . as a negative control
, the liposomes with nonbilayer arrangements were previously incubated with 0.1 mm chloroquine . for the positive controls ,
the cell lines were incubated in the presence of their known tlr agonists : 100 ng / ml escherichia coli 0111:b4 lps for hek - tlr-4/md2/cd14 , 1 g / ml fsl-1 ( a synthetic lipoprotein derived from mycoplasma salivarium ) for hek - tlr-2/tlr-6 , 1 g / ml salmonella typhimurium flagellin for hek - tlr-5 , and 2.5 g / ml ssrna40 ( a 20 mer phosphorothioate - protected single - stranded rna oligonucleotide containing a gu - rich sequence ) for hek - tlr-8 . all tlr agonists were sourced from invivogen .
after 24 h , the cell culture supernatants were harvested and assayed for il-8 production ( bd opteia set human il-8 , bd biosciences , san diego , ca , usa ) .
nf-b activation was assayed in cell culture extracts using the reporter plasmid pnifty - luc ( promega corporation , madison , wi , usa ) . in order to determine
if chloroquine affects the viability of hek293 cells , the live / dead fixable violet dead cell stain kit ( invitrogen ) was used .
hek293 cells were incubated with 0.05 , 0.1 , and 0.5 mm of chloroquine for 24 h at 37c and 5% co2 .
the cells were then transferred to a tube and stained with 50 l of live / dead diluted 1 : 100 in distilled water and incubated for 15 min at room temperature in the dark .
facs lysis buffer ( 1 ml ; becton dickinson ) was added for erythrocyte lysis , and the cells were incubated for 10 min at room temperature in the dark .
the cells were washed with 2 ml of phosphate - buffered saline ( pbs ) and resuspended in 300 l of pbs and analyzed by flow cytometry .
forty thousand events were acquired for each sample with a lsr fortessa cytometer ( becton - dickinson ) . to evaluate
whether chloroquine can induce apoptosis of hek293 cells , the annexin v - propidium iodide staining method was used .
hek293 cells were incubated with 0.05 , 0.1 , and 0.5 mm of chloroquine for 24 h at 37c and 5% co2 .
the cells were then transferred to a tube and washed with 1 ml of annexin v - binding buffer ( ebioscience , san diego , ca , usa )
. 100 l of 2 g / ml annexin v - apc ( ebioscience ) in annexin v - binding buffer was added , and the cells were incubated for 15 min at room temperature in the dark .
the cells were washed with 1 ml of annexin v - binding buffer , resuspended in 100 l of the same buffer containing 1 g of propidium iodide ( biolegend , san diego ca , usa ) and incubated for 15 min at room temperature in the dark .
the cells were washed and resuspended in the annexin v - binding buffer and analyzed immediately by flow cytometry .
forty thousand events were acquired for each sample in a lsr fortessa cytometer ( becton - dickinson ) .
tlr stimulation was also analyzed in bone marrow - derived macrophages ( bmdm ) from balb / c mice .
bmdm were obtained from the femur and shinbone of female 2-month - old balb / c mice and they were cultured in rpmi media with 10% heat - inactivated fetal bovine serum , 50 u / ml penicillin ( gibco ) , 50 g / ml streptomycin ( gibco ) , and 10 ng / ml recombinant m - csf ( biolegend ) for 7 days . for tlr stimulation , the bmdm were cultured in 96-well plates and stimulated with 10 ng / ml lps , 1 g / ml peptidoglycan ( pgn ; invivogen ) , or 10 or 20 l of smooth liposomes or liposomes bearing nonbilayer phospholipids arrangements , respectively .
after incubation for 24 h at 37c , the supernatants were collected and analyzed by enzyme - linked immunoabsorbent assay ( elisa ; biolegend ) for tumor necrosis factor- ( tnf- ) production . for the blocking experiments , 10 g / ml of anti - tlr-2 ( clone t2.5 , biolegend ) or 20 g / ml of anti - tlr-4 ( clone mts510 , biolegend ) was added 2 h before the liposomes .
the cells were incubated for 24 h at 37c and the supernatants were collected and analyzed for tnf-. igg1, ( clone mopc-21 , biolegend ) and igg2a, ( clone rtk2758 , biolegend ) isotype controls were used for the blocking antibodies .
forty female 2-month - old specific - pathogen - free balb / c mice were divided into four groups .
the first and the second groups were injected intrasplenically , on days 1 and 15 , with phosphatidylcholine / phosphatidic acid ( 2 : 1 ) liposomes that had been incubated with 5 mm mncl2 ( mn group ) or 3 mm chlorpromazine ( cpz group ) .
mice received the same amount of liposomes by intraperitoneal injection on day 30 and then every week for 6 months .
the negative control groups consisted of 10 mice treated in the same way but using ts buffer alone ( control group i ) , or liposomes made of phosphatidylcholine / phosphatidic acid ( 2 : 1 ) alone ( control group ii ) .
blood was taken from mice before liposome injection and each month after the first intraperitoneal injection , for a total of 6 months .
sera were heated at 56c for 30 min to inactivate complement and frozen in aliquots at 70c . to confirm
that these mice developed the disease resembling human lupus , we measured anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies in their sera .
anti - nonbilayer phospholipid arrangements antibodies were measured by elisa where the wells were coated with liposomes with or without nonbilayer phospholipid arrangements .
results are reported as arbitrary units ( au ) calculated as ( asp asw)/(ash asw ) , where asp is the absorbance obtained with the sera of mice injected with the liposomes , ash is the absorbance obtained with the sera of mice before the injection of liposomes , and asw is absorbance of controls without sera .
a modification of the kaolin - activated thromboplastin time test was used to determine the anti - coagulant antibodies ; results are reported as the coagulation time in seconds .
three mice from each of the four groups indicated above were euthanized 4 months after the first injection of nonbilayer phospholipid arrangements , when they had the highest titers of anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies , and their spleens were used for gene and protein expression studies .
the experimental protocols for animal care and use were reviewed and approved by the bioethics committee of our institution according to the guide for the care and use of laboratory animals , which was published by the us national institute of health .
the serum concentrations of interleukin-6 ( il-6 ) , il-10 , il-12p70 , interferon- ( ifn- ) , tnf- , and monocyte chemoattractant protein- ( mcp- ) 1 were measured with a bead - based multiplex immunoassay ( bd cba mouse inflammation kit ) .
mouse spleens were sectioned and placed in two cryotubes , one with rnalater ( invitrogen ) for rna expression studies and one with tissue - tek ( sakura finetek , torrance , ca , usa ) for protein analysis .
the cryotubes were stored at 70c until use . to isolate rna , the tissue stored in rnalater was thawed and disaggregated at 15,000 rpm with a tissueruptor ( qiagen , valencia , ca , usa ) , and total rna was extracted from the tissue homogenates using an rneasy mini kit ( qiagen ) . the quality and quantity of the rna samples
were assessed in an agilent bioanalyzer 2100 ( agilent , palo alto , ca , usa ) and a nanodrop 2000 ( thermo fisher scientific , auburn , al , usa ) , respectively ; only rna samples with a rna integrity number ( rin ) 7 were used for the gene expression analysis .
total rna ( 400 ng ) was amplified and labeled using the quick amp labeling kit ( agilent ) , and the cyanine-3- or cyanine-5-labeled crna was purified with an rneasy mini kit ( qiagen ) .
the crna were hybridized to 4 44 k whole mouse genome microarray chips ( agilent , g4122f ) ; the microarrays were scanned with an agilent microarray scanner ( g2565ba ) and the data were extracted with agilent feature extraction software ( v.9.5.3.1 ) .
the cutoff for over- and underexpressed genes was set at a mean fold change log2 ratio greater than + 2 or lower than 2 , as assessed by two - way analysis of variance ( anova ; partek pro software , partek inc . ,
st . charles , mo , usa ) with p < 0.01 . to evaluate protein expression ,
the spleen samples stored in tissue - tek were thawed , rinsed with pbs , and disaggregated at 15,000 rpm with a tissueruptor ( qiagen ) .
the homogenates were centrifuged at 5,000 g for 5 min at 4c , and the supernatants were used to measure c3 ( elisa kit mbs700250 , mybiosource , san diego , ca , usa ) , c5 ( elisa kit mbs704792 , mybiosource ) , c3a ( elisa kit mbs70381 , mybiosource ) , c5a ( elisa kit mbs700538 , mybiosource ) , and ifn- ( elisa kit 439407 , biolegend ) .
tlr-4 was measured by flow cytometry in cells obtained from fresh spleens , which were disaggregated and passed through a 70 m nylon mesh .
the cells were labeled with a fluorescein isothiocyanate- ( fitc- ) conjugated anti - f4/80 antibody ( biolegend ) , a pe - conjugated rat anti - mouse tlr-4 antibody ( biolegend ) , and fixable viability dye 450 ( ebiosciences ) and acquired in a facscalibur flow cytometer .
single viable cells were analyzed , and the percentage of f4/80 tlr-4 cells of the total live cells was determined .
the spleens of three mice from the groups injected intrasplenically with liposomes without nonbilayer phospholipid arrangements or with liposomes bearing nonbilayer phospholipid arrangements were placed in fluorescence - activated cell sorting ( facs ) buffer containing 0.1% bsa and 0.01% sodium azide ( sigma aldrich ) .
red blood cells were lysed and spleen cells were resuspended in facs buffer . before staining
, cells were incubated with universal blocking reagent ( block biogenex , san ramn , ca , usa ) in pbs for 10 min at 4c and then washed .
splenocyte suspensions were labeled with anti - cd19-apc , anti - cd5-af488 , anti - cd69-percp , and anti - tlr4-pe ( ebioscience ) to evaluate b cells ; with anti - cd3-fitc , anti - cd4-pe ( ebioscience ) , anti - cd8-apc , and anti - cd69-percp to evaluate t cells ; with anti - gr-1-percp , anti - cd11c - apc , anti - mhc - ii - pe ( ebioscience ) , anti - cd80-fitc , and anti - cd86-pe / cy7 ( ebioscience ) to evaluate dendritic cells ; and with anti - cd3-fitc and anti - nk 1.1-apc to evaluate nkt cells .
the cells were incubated for 30 min at 4c , then washed with facs buffer , and fixed with 1% paraformaldehyde ( sigma aldrich ) .
labeled cells were acquired in a lsr fortessa flow cytometer ( becton dickinson ) ; single , viable cells were analyzed with flowjo 10.0.6 ( tree star , inc . ,
we had previously shown that the presence of nonbilayer phospholipid arrangements can be detected by flow cytometry as an increase in side scatter ( ssc ) value [ 10 , 14 , 15 ] .
thus , the increase in ssc signal after the addition of mn , chlorpromazine , or procainamide to liposomes made of phosphatidylcholine / phosphatidic acid or phosphatidylcholine / phosphatidylserine indicated the presence of nonbilayer phospholipid arrangements ( figures 1(c)-1(d ) and 1(f)-1(g ) ) . as a negative control , we added 5 mm of mg to liposomes ( figures 1(c ) , 1(d ) , and 1(e ) ) ; mg does not induce the formation of nonbilayer phospholipid arrangements , as was previously shown for phosphatidylcholine / phosphatidic acid liposomes .
liposomes made of the cylindrical lipid phosphatidylcholine , without any conical lipid , did not increase in complexity in the presence of mn or mg ( figure 1(e ) ) , chlorpromazine , or procainamide ( data not shown ) .
the addition of lps caused an increase in ssc signal when it was used alone ( figure 1(h ) ) or in combination with mn ( figure 1(i ) ) , which suggests that lps modifies the lipid bilayer .
the addition of 0.1 mm chloroquine , a drug that blocks or reverses the formation of nonbilayer phospholipid arrangements , decreased the liposome complexity induced by procainamide or mn ( figures 1(g)-1(j ) ) or chlorpromazine ( data not shown ) .
we evaluated the effects of phosphatidylcholine / phosphatidic acid liposomes and phosphatidylcholine / phosphatidylserine liposomes , alone or in the presence of mn , chlorpromazine , or procainamide , on the viability of hek , hek - tlr-4/md2/cd14 , hek - tlr-2/tlr-6 , hek - tlr-5 , and hek - tlr-8 cell lines .
we found that liposomes made of phosphatidylcholine / phosphatidic acid with mn - induced nonbilayer phospholipid arrangements had no effect on the cell viability ( 90% or more of cells were viable ) .
liposomes with mn - induced nonbilayer phospholipid arrangements stimulated il-8 production by hek - tlr-4/md2/cd14 cells and , to a lesser degree , by hek - tlr-2/tlr-6 cells , but not by hek - tlr-5 or hek - tlr-8 cells ( figure 2(a ) ) .
liposomes without nonbilayer phospholipid arrangements or liposomes in which mn - induced nonbilayer phospholipid arrangements had been reversed by chloroquine did not induce il-8 production ( figure 2(a ) ) .
similar results were obtained when nf-b activation was measured through the reporter plasmid pnifty - luc ( figure 2(a ) ) . nontransfected hek cells did not produce il-8 in the presence of liposomes with mn - induced nonbilayer phospholipid arrangements ( data not shown ) .
the production of il-8 by hek - tlr-4/md2/cd14 or hek - tlr-2/tlr-6 cells in response to liposomes with mn - induced nonbilayer phospholipid arrangements was dose - dependent , and the effect was inhibited by chloroquine ( figure 2(b ) ) .
cell viability in the presence of chloroquine was 90% or higher , and chloroquine did not induce apoptosis of these cells at the tested concentrations ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/369462 ) .
thus , the effects observed in the presence of chloroquine can be attributed to a reversion of mn - induced nonbilayer phospholipid arrangements by this drug .
additionally , we found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by bmdm from balb / c mice .
furthermore , anti - tlr-2 and anti - tlr-4 antibodies blocked the production of tnf- by bmdm in response to nonbilayer phospholipid arrangements ( figures 2(c)-2(d ) ) .
liposomes with nonbilayer phospholipid arrangements induced by mn or chlorpromazine were used to produce an autoimmune disease resembling human lupus in mice .
antibodies against nonbilayer phospholipid arrangements were detected 1 month after the first injection of liposomes with nonbilayer phospholipid arrangements , and the titers in mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements were higher than in those injected with mn - induced nonbilayer phospholipid arrangements ( p < 0.001 ) .
these antibodies appeared 1 month before the anti - cardiolipin , anti - histone , and anti - coagulant antibodies ( figures 3(a ) , 3(b ) , 3(c ) , and 3(d ) ) .
the presence of the four autoantibodies confirmed that the disease had been developed in the mice .
control mice injected with ts buffer or with liposomes without nonbilayer phospholipid arrangements did not generate any of the four autoantibodies .
il-6 , il-10 , il-12p70 , ifn- , tnf- , and mcp-1 were found in the sera of mice injected with liposomes with mn - induced nonbilayer phospholipid arrangements ; il-6 , ifn- , and tnf- appeared 1 month after treatment , while il-10 , il-12p70 , and mcp-1 were found after 2 months .
these cytokines were also found in the sera of mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements ; il-6 , tnf- , and mcp-1 appeared 1 month after treatment , while ifn- and il-12p70 were found 4 months after treatment .
none of the tested cytokines were found in the sera of mice treated with smooth liposomes ( figure 4 ) .
we evaluated gene expression in the spleens of mice from the four treatment groups : group 1 , mice injected with ts buffer alone ( control i ) ; group 2 , mice injected with smooth liposomes ( liposomes without nonbilayer phospholipid arrangements , control ii ) ; group 3 , mice that received liposomes with mn - induced nonbilayer phospholipid arrangements ( mn group ) ; and group 4 , mice that received liposomes with chlorpromazine - induced nonbilayer phospholipid arrangements ( cpz group ) .
spleens were collected 4 months after treatment , and the crna derived from the spleens of three mice from each group were pooled and hybridized to a whole mouse genome microarray chip .
no significant differences were found between control i and control ii groups ; 426 genes were overexpressed and 62 genes were underexpressed in the mn group , compared with the control ii group ; 542 genes were overexpressed and 73 genes were underexpressed in the cpz group , compared with the control ii group ; and 383 genes were overexpressed and 44 genes were underexpressed in the cpz group , compared with the mn group .
table 1 shows a list of genes that were overexpressed in both the mn and cpz groups , compared with the control ii group .
this includes genes for complement components ( c3 and c5 ) , molecules involved in the presentation of exogenous antigens , in the production of antibodies , and in tlr-4 and nod-2 signaling .
table 1 also shows a list of genes that were underexpressed in both the mn and cpz groups , compared with the control ii group .
these are genes for molecules that are involved in apoptosis and in nk cell recognition .
the c3 and c5 complement proteins were increased in the control i and control ii groups , compared with the mn and cpz groups .
however , c3a and c5a , two active fragments that are produced by c3 and c5 cleavage , were increased in the mn and the cpz groups , compared with the control i and control ii groups ( figures 5(a)-5(b ) ) .
ifn- was also increased in the spleens of mice with the autoimmune disease , compared with healthy mice ( figure 5(c ) ) .
the number of cells expressing tlr-4 increased in the mn and the cpz groups , compared with the control i and control ii groups ( figure 5(d ) ) . activated cd4 and cd8 t cells ( figures 6(a ) , 6(b ) , 6(c ) , and 6(d ) ) , nkt cells ( figures 6(e)-6(f ) ) , activated dendritic cells ( figures 6(g)-6(h ) ) , and activated and tlr4 expressing b1 and b2 cells ( figures 6(i ) , 6(j ) , and 6(k ) ) were identified by flow cytometry in the spleens of mice .
fifteen days after the mice were injected intrasplenically with liposomes bearing nonbilayer phospholipids arrangements induced by mn or chlorpromazine , the percentage and activation of cd4 and cd8 t cells were not increased , compared with the control mice that received ts buffer or liposomes alone ( figures 6(l)-6(m ) ) .
in contrast , the percentage of nkt , dendritic , and b2 cells was increased ( figures 6(n ) , 6(o ) , and 6(q ) ) , and the activation of dendritic and b2 cells was also increased ( figures 6(o)-6(q ) ) .
an increase in tlr4 expression was also observed in b2 cells ( figure 6(q ) ) .
b1 cells did not increase in percentage , but the number of activated and tlr4 expressing b1 cells did increase ( figure 6(p ) ) .
sle is a systemic autoimmune disease of unknown etiology characterized by b and t cell hyperactivity , by defects in the clearance of apoptotic cells and immune complexes , and by production of a complex mixture of various cytokines , chemokines , signaling molecules , and pattern - recognition receptors involved in immunity [ 4 , 24 ] .
we have previously demonstrated that liposomes with nonbilayer phospholipid arrangements trigger a disease that resembles human lupus in mice and that igm and igg specific to nonbilayer phospholipid arrangements are produced in these mice .
the activation of this innate immune receptor leads to the production of proinflammatory cytokines ; a proinflammatory environment is needed for efficient activation of the adaptive immune response and the production of igg antibodies .
these findings were supported by the increase in the percentage of nkt cells and by the increase in the percentage and activation of dendritic and b2 cells .
in addition , the activation of tlr-4/md2/cd14 by liposomes with mn - induced nonbilayer phospholipid arrangements supports our hypothesis on the similarity of the structure of conical phospholipids , which form an inverted micelle inside the nonbilayer arrangement , with the conical association of the acyl chains of the lipid a moiety of lps .
the importance of the lipid a moiety of lps was taken into account in the design of glucopyranosyl lipid a ( gla ) , a synthetic lipid a with six acyl chains and a single phosphate group . gla as a stable oil - in - water - emulsion ( gla - se ) is a tlr-4 agonist , which signals through myd88 and trif and drives a polyclonal th1 response in vivo , characterized by ifn- , tnf- , and il-2 producing cells and igg2c isotype switching [ 25 , 26 ] .
interestingly , we also found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by balb / c mouse bmdm .
furthermore , anti - tlr-2 and anti - tlr-4 antibodies blocked the production of tnf- by these macrophages in response to nonbilayer phospholipid arrangements .
these findings confirmed our observations with the hek cells transfected with human tlrs , which also showed that nonbilayer phospholipid arrangements are agonists for tlr-4/md-2 and tlr-2/tlr-6 .
we observed that liposomes with nonbilayer phospholipid arrangements were agonists for tlr-2/tlr-6 , but the activation was 3-fold lower than for tlr-4/md2/cd14 .
bacterial macroamphiphilic molecules , such as lipoproteins ( including the synthetic lipoprotein fsl-1 ) , lipoteichoic acids , lipoglycans , glycolipids , and lipoarabinomannans , are anchored on bacterial envelopes through a lipidic structure , which is usually a diacylglyceryl moiety .
these amphiphilic molecules are mainly recognized via their lipid anchor through tlr-2 , alone or as a heterodimer with tlr-1 or tlr-6 [ 27 , 28 ] . because the liposomes bearing nonbilayer phospholipid arrangements are made of phosphatidylcholine and phosphatidate , which also have the diacylglyceryl moiety , it is possible that this lipid moiety activated the tlr-2/tlr-6 heterodimer .
tlrs not only recognize pathogen - associated molecular patterns , such as lps , but also recognize damage - associated molecular patterns , which are released by cells that are either under stress or undergoing apoptosis or necrosis .
examples of damage - associated molecular patterns that are tlr-4 agonists include heat - shock protein 60 , fibronectin , fibrinogen , -defensins , and hyaluronan .
the molecular structure of these agonists is different from that of lps , but they all have hydrophobic regions , which are probably recognized by tlr-4 .
the modification of the lipid bilayer of cell membranes could be a signal of cell stress : nonbilayer phospholipid arrangements are normally transitory , but if they are stabilized by mn or by the drugs chlorpromazine or procainamide , they could activate the innate immune response via tlrs and then induce the production of antibodies , with the subsequent development of an autoimmune disease .
tlr-4 signaling leads to the activation of nf-b and the production of proinflammatory cytokines , including tnf- , il-12 , and ifn- , and chemokines , such as mcp-1 .
we found these cytokines and chemokines in the sera of mice treated with liposomes with mn- or chlorpromazine - induced nonbilayer phospholipid arrangements .
the increase in the concentration of the proinflammatory cytokines il-6 and tnf- correlated with the appearance of anti - nonbilayer phospholipid arrangement antibodies 1 month after the first injection of mice with nonbilayer phospholipid arrangements , and this also corresponds to the period of disease onset .
the chemokine mcp-1 and the proinflammatory cytokines inf- and il-12p70 increased between months 2 and 4 and correlated with the development and establishment of the disease , given by an increase in the titers of anti - nonbilayer phospholipid arrangement antibodies and the presence of anti - cardiolipin , anti - histone , and anti - coagulant antibodies .
the proinflammatory cytokines il-1 , il-6 , ifn- , and tnf- and the immunomodulatory cytokines il-10 and tumor growth factor- ( tgf- ) have been identified as important players in the development of sle [ 31 , 32 ] .
the cytokine pattern that we report indicates another similarity of this mouse model with the human disease .
tlr-4 was increased at the mrna level and the number of cells that express tlr-4 increased in the spleens of mice that received liposomes with nonbilayer phospholipid arrangements .
other genes associated with tlr-4 signaling , such as tram , trif , tbk1 , and irf3 , were also increased at the mrna level in these mice .
these genes are associated with trif - dependent , but not myd88-dependent , tlr-4 signaling .
trif - dependent tlr-4 signaling leads to the production of ifn- and . ifn- was increased in the spleens of mice that had received liposomes with nonbilayer phospholipid arrangements compared with healthy mice , and increased levels of ifn- and are reported in patients with sle .
the expression of genes associated with the classical pathway of complement activation ( c1ra , c1s , c1q , c3 , c5 , and c7 ) was increased in mice that had received liposomes with nonbilayer phospholipid arrangements , and this mrna increase correlated with the detection of c3a and c5a proteins in the spleens of mice with the autoimmune disease .
complement has an important role in the immune response , but it also has the potential to cause tissue damage , as has been reported in sle and other autoimmune diseases [ 35 , 36 ]
. it will be interesting to evaluate the role of complement in the tissue damage that is observed in this mouse model of autoimmune disease .
in contrast , the expression of genes associated with nk cell activation ( klrb1a , klrb1c , klra23 , klra7 , gzmb , and klra22 ) was decreased in mice that received liposomes with nonbilayer phospholipid arrangements .
this decrease could reflect a reduction in the absolute number of nk cells or a lower activation of the existing nk cells .
the expression of genes associated with apoptosis ( casp8 , cycs , apaf1 , and aaifm1 ) was also decreased in mice that received liposomes with nonbilayer phospholipid arrangements .
this could be relevant for disease development , since deficient apoptosis could favor the survival of autoreactive t cells .
an important additional support for our hypothesis on the effect of nonbilayer phospholipid arrangements on the innate immune response is our finding that mice with the autoimmune disease resembling human lupus have an increase in nkt and dendritic cell percentages , together with increased dendritic cell activation .
these cells could recruit and activate b1 and b2 cells , which are the precursors of plasma cells that produce antibodies against nonbilayer phospholipid arrangements .
the findings reported in this paper are consistent with a mouse model in which nonbilayer phospholipid arrangements directly activate tlr-4 and tlr-2/tlr-6 and lead to the production of proinflammatory cytokines .
the proinflammatory environment leads to the efficient activation of the adaptive immune response to the production of igg antibodies specific for nonbilayer phospholipid arrangements .
these antibodies bind to the nonbilayer phospholipid arrangements that are transitorily formed on the surface of many cells and cause cell lysis ; the exposure of intracellular antigens could then lead to the formation of anti - cardiolipin , anti - histone , and anti - coagulant antibodies .
furthermore , the inflammatory environment can cause complement - mediated tissue damage and ifn- production . | systemic lupus erythematosus is characterized by dysregulated activation of t and b cells and autoantibodies to nuclear antigens and , in some cases , lipid antigens .
liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice , including igm and igg antibodies against nonbilayer phospholipid arrangements . as the effect of these liposomes on
the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation , we evaluated the effect of these liposomes on toll - like receptor ( tlr ) signaling , cytokine production , proinflammatory gene expression , and t , nkt , dendritic , and b cells .
liposomes induce tlr-4- and , to a lesser extent , tlr-2/tlr-6-dependent signaling in tlr - expressing human embryonic kidney ( hek ) cells and bone marrow - derived macrophages .
mice with the lupus - like disease had increased serum concentrations of proinflammatory cytokines , c3a and c5a ; they also had more tlr-4-expressing splenocytes , a higher expression of genes associated with trif - dependent tlr-4-signaling and complement activation , and a lower expression of apoptosis - related genes , compared to healthy mice .
the percentage of nkt and the percentage and activation of dendritic and b2 cells were also increased .
thus , tlr-4 and tlr-2/tlr-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus - like disease in mice . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
a 72-year - old male patient with interstitial lung disease presented to a local clinic because of difficulty breathing and a subjective fever .
he had a fever of 39. the fever went down to 37 with the administration of antibiotics ; the patient was then given oral corticosteroids ( predinisolone 60 mg / day ) .
nine days after admission to a local clinic he developed blurred vision in both eyes ; however , ophthalmic examination was not performed initially .
three days after this decrease in visual acuity he was referred to our hospital . on initial examination ,
anterior segment examination revealed severe conjunctival injection , severe anterior chamber reaction without hypopyon , and mild complicated cataracts in both eyes .
fundus examination revealed a vitreal reaction and multiple whitish - yellow exudates in both eyes .
a single exudate was seen in the right macula and multiple exudates were observed in the left macula ( fig .
1 ) . we initially suspected a fungal infection , such as candida , given his history and fundus findings .
cytological examination and cultures of the blood , aqueous humor , and vitreous were ordered .
intravitreal vancomycin ( 1 mg ) , amikacin ( 200 g ) , and amphotericin b ( 5 g ) were then injected into both eyes .
oral ketoconazole ( 400 mg / day ) was given for the suspected ocular fungal infection . despite this treatment ,
five days after the initial intravitreal injection , visual acuity of the right eye had decreased to hand motion .
nine days after admission the patient underwent a pars plana vitrectomy with cataract surgery in the right eye .
intravitreal gentamicin ( 20 g / ml ) , cefazolin ( 50 g / ml ) , amphotericin b ( 1.0 g / ml ) , vancomycin ( 1 mg ) , and ceftazidime ( 2 mg ) were administered at the end of the surgery .
five days after the initial ocular presentation , p. agglomerans was identified in the vitreous culture , but the aqueous humor and blood cultures were negative . based on sensitivities , intravenous antibiotics ( ciprofloxacin 200 mg every twelve hours and ampicillin sodium 500 mg every eight hours ) were administered .
the inflammation of the right eye gradually decreased in response to this therapy , but no response was observed in the left eye . on hospital day 24
six months after surgery the patient 's final visual acuity improved to 25/20 in the right eye and 20/200 in the left eye .
an exudative scar of the paramacular area in his left eye was noted , but his symptoms were markedly improved .
endogenous bacterial endophthalmitis is a rare disease that occurs when microorganisms spread to the eye from a source elsewhere in the body , usually through the bloodstream .
it accounts for only 2 to 6% of all cases of endophthalmitis . in this case
we initially believed that the infection was fungal in origin because of the clinical features , no history of ocular trauma , the presence of interstitial lung disease with a fever , and the prior administration of immunosuppressive treatment .
early diagnosis and treatment were delayed and the inflammatory reaction worsened because this species of enterobacteriace is refractory to antifungal agents .
after aspiration of the vitreous , p. agglomerans was confirmed as the causative agent of the intraocular infection .
early detection of visual disturbances is important in patients who have received corticosteroids or immunosuppressive therapy .
p. agglomerans ( formerly named enterobacter agglomerans ) is a rare cause of endogenous endophthalmitis .
all species of the genus p. agglomerans can be isolated from feculent materials , plants , and soil ; they can be either pathogens or commensals . within the genus ,
p. agglomerans is the most commonly isolated species in humans , resulting in soft tissue or bone / joint infections following penetrating trauma by vegetation .
the organism also colonizes cotton and cotton plants and has been shown to cause a benign self - limited fever in intravenous drug abusers who filter drugs through cotton before injecting .
tissue toxicity associated with gram - negative rods in endophthalmitis is known to generally have a poor outcome . however , in our case the ocular infection subsided after vitrectomy , intravitreal injection , and tailored antibiotic treatment .
agglomerans produces yellow pigments that are typically only noticeable after delayed incubation at room temperature .
yellow pigment - producing colonies that were 2 mm in size , nonhemolytic , and convex were detected on a blood agar plate ( fig .
we believe that our case is the first report of bilateral endogenous endophthalmitis caused by p. agglomerans in korea and the first case reported outside of the united states . | we here in report a case of bilateral endogenous endophthalmitis caused by pantoea agglomerans ( p. agglomerans ) in a patient who had interstitial lung disease and was treated with oral corticosteroids . a 72-year - old man presented with decreased visual acuity in both eyes nine days after he received oral corticosteroids .
he had marked uveitis , cataracts , and vitreous opacities .
cultures were taken of blood , aqueous humor , and vitreous .
we initially suspected a fungal etiology and treated him with antifungal drugs ; however , the intraocular disease progressed without improvement .
vitreous culture was positive for p. agglomerans .
the patient underwent pars plana vitrectomy with cataract surgery bilaterally , followed by a 2-week course of antibiotics .
the final visual acuity was 20/25 in the right eye and 20/200 in the left eye .
this is the first report of bilateral endogenous endophthalmitis caused by p. agglomerans in korea ; it is also the first case reported outside of the united states . | Case Report
Discussion |