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the treatment of chronic hepatitis c ( chc ) has evolved in the last 15 years from monotherapy with interferon alpha ( ifnalpha ) to the combination treatment with pegylated ifn ( peg - ifn ) plus ribavirin for 24 - 48 weeks . up to now , several viral , host and drug - related reactions in response to ifnalpha - based therapy have been identified .
recent studies suggest that liver inflammation in chc is controlled by several mechanisms , including host regulatory immune responses and viral polypeptides interacting with cells involved in innate and adaptive immunity .
it is well known that cutaneous side effects of treatment with ifnalpha alone or ifnalpha plus ribavirin in patients with chc have been widely reported , beyond the fact that the virus itself can cause skin lesions . however , the cutaneous side effects during therapy of chc are of inflammatory type with local erythema , edema and , much less frequently , necrosis at the injection skin sites . by contrast ,
skin side effects of such drugs have few data available in the literature , although the number of reports has been increasing in the last years , including cosmetic filler site injections .
roughly one half of these patients had cutaneous findings , either alone or with systemic involvement .
more recently there have been reported dermatological diseases in patients receiving the combination of ifn / ribavirin or ifn / ribavirin / amantadine for the treatment of chc .
a 44-year - old man with chc genotype 1a and a viral load of hcv > 800,000 iu / ml , a2-f1 on liver biopsy according to metavir criteria , was treated for the hepatic disease .
peg - ifnalpha-2b 1.5 g / kg weekly and 1,000 mg of ribavirin daily was introduced . in the 40th week
the patient reported sudden intense pain , pruritus , erythema and skin hypertrophy in the left deltoid area over the seahorse tattoo and in a scar on his face ( fig .
1 ) . a skin biopsy on the scar and tattoo showed granulomatous dermatitis ( fig .
chest x - ray was normal and the level of angiotensin enzyme converter was 50 iu / ml ( range 18 - 55 iu / ml ) .
we intend to call attention especially to dermatologists to the possibility of a granulomatous tattoo reaction as a side effect during chc treatment with peg - ifnalpha-2b plus ribavirin . until now
, eight cases of sarcoidosis as a complication of ifn therapy in chc have been reported in the english language literature .
the most relevant topic in this case is the occurrence of disease on a tattoo area .
six months later retreatment using peg - ifnalpha-2a 180 g once a week plus ribavirin 1,000 mg daily was tried . in the 6th week , cutaneous symptoms relapsed in an unbearable way .
the patient did not accept to continue therapy , since hydrocortisone cream was prescribed again but no satisfactory pruritus and pain improvement was noticed . a prompt regression of skin reaction occurred after stopping therapy .
sarcoidosis is a granulomatous disorder of unknown etiology and whose epidemiology suggest a genetic tendency face to infectious agents being supposed to result of immune system deregulation leading to non - caseating granulomas as an immune reaction to an unknown persistent antigenic stimulus .
ifn has been linked to pulmonary macrophage activation , a characteristic feature of sarcoidosis which has been assumed an exaggerated t helper 1 ( th-1 ) immune response to a variety of exogenous antigens .
it seems very likely that a potent immunoregulatory protein for th-1 response such as ifn may induce the disease . in january 2003 the eighth case of ifn - related sarcoidosis was reported with a review of the literature .
chest x - ray revealed hilar lymphadenopathy in three patients , with reticulonodular shadows in another three , while the patient with only cutaneous involvement had a normal chest x - ray .
the case we report presented with cutaneous sarcoidosis during chc therapy and spontaneous regression of the lesions was noted with treatment discontinuation .
we intend to emphasize , especially to dermatologists , the risks of a granulomatous tattoo and other cutaneous sites during chc treatment with peg - ifnalpha-2b plus ribavirin .
thalidomide has been also shown to have specific activity to the inflammatory mediators of sarcoidosis and to be an alternative beneficial therapy . | the treatment of chronic hepatitis c ( chc ) has evolved in the past 15 years and combination of pegylated interferon plus ribavirin is its current standard therapy . however , several side effects are commonly observed and frequently lead to transient or definitive interruption of treatment .
although sarcoidosis in its systemic or cutaneous form is a very rare side effect in such circumstances , some cases have been reported even with conventional interferon .
this brief review of the literature and description of a case of sarcoidosis occurring in a tattoo and a scar patient 's face , during treatment with pegylated interferon alpha-2b plus ribavirin , is an educative report directed in special to dermatologists .
the lesion improved after drug interruption and recurred after retreatment with pegylated interferon alpha-2a .
we conclude that this side effect must call the attention of doctors to seek for the diagnosis and therapy as soon as possible in such circumstances .
no differences were noticed neither with alpha-2a nor alpha-2b pegylated interferon employment . | [
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necrotising fasciitis ( nf ) is a serious soft tissue and life threatening infection , primarily involving the skin and superficial fascia , characterised by a rapid and extensive necrosis of the subcutaneous tissue .
most patients have associated comorbidities , such as diabetes , hiv infection or treatment with immunosuppressive drugs , but it can also affect previously healthy individuals .
infection is one of the common causes of morbidity and mortality in patients with systemic lupus erythematosus ( sle ) .
it is associated with immunosuppressive agents , renal failure , and increased disease activity . despite the increased propensity of sle patients to develop common and opportunistic infections , nf has rarely been reported . to our knowledge
, only 31 cases are described in the literature to date ( see table 1 ) .
nf has also been described in other rheumatic diseases including polymyositis , dermatomyositis , systemic sclerosis , rheumatoid arthritis and ankylosing spondylitis , but it appears that this association is more common in sle.1
2 clinical features of nf in systemic lupus erythematosus a , asian ; aa , african american ; aps , antiphospholipid syndrome ; art , arthritis ; avn , avascular necrosis ; aza , azathioprine ; cad , coronary artery disease ; cs , corticosteroid therapy ; cyc , cyclophosphamide ; d , died ; dm , diabetes mellitus ; f , female ; fg , fournier 's gangrene ; h , hispanic ; hcq , hydroxychloroquine ; lym , lymphocytes ; m , male ; mmf , mycophenolate ; mtx , methotrexate ; n , nephritis ; na , not available ; nf , necrotising fasciitis ; pe , pulmonary embolism ; rtx , rituximab ; s , survived ; sa , staphylococcus aureus ; ttp , thrombotic thrombocytopenic purpura ; wbc , white blood cells .
we describe the case of a nf caused by pseudomonas aeruginosa , complicated by recurrent sepsis and multiorgan failure in a young patient with a history of sle .
a 26-year - old asian woman with a complex history of sle , diagnosed at age 14 years when she presented with malar rash , arthralgia , mouth ulcers , pulmonary vasculitis , strongly positive anti - nuclear antibody and anti - dsdna antibodies .
subsequently , she had idiopathic detrusor overactivity with repeated urinary tract infection . linked to her corticosteroid treatment she developed avascular necrosis of her elbow ( at that time , on prednisolone 6 mg daily ) .
the patient was also treated with hydroxychloroquine ( hcq ) , azathioprine ( aza ) ( 100 mg from 1999 to 2009 ) and had 14 courses of cyclophosphamide ( cyc ) ( a cumulative dose of 14 g ) and two of rituximab ( rtx ) ( two 1 g intravenous infusions separated by 2 weeks ) , the last being given in march 2012 for a sle flare manifested by severe vasculitic rash . immediately prior to b - cell depletion , her c3 level was 0.33 g / l ( normal : 0.651.65 ) , cluster designation ( cd ) 19 count was 0.146/l ( 0.110.69 ) , and immunoglobulin g level was 23.4 g / l ( 716 ) . in may 2012
, she presented to her local hospital with painful swelling of her left lower limb , she denied a history of trauma , and her inflammatory markers and ultrasonography were normal , and she was discharged . within 12 h
she was found at home with a glasgow coma scale of 6 , and was admitted to the intensive care unit ( icu ) . on examination ,
the patient was in septic shock with hypotension ( systolic blood pressure 60 mm hg ) , tachycardia ( 135/min ) , and respiratory failure ( sao2 70% ) .
laboratory results showed erythrocyte sedimentation rate 96 mm / h ( normal : 17 ) , c - reactive protein 281 mg / dl ( 05 ) , white blood cells 15 400/l ( 300010 000 ) ( 97% neutrophils ; 1.9% lymphocytes ) , haemoglobin 9.6 g / dl ( 11.515.5 ) , platelet 50 000/l ( 150 000400 000 ) , na 138 mmol / l ( 135145 ) , creatine kinase 474 iu / l ( 26140 ) , urea 16.8 mmol / l ( 1.78.3 ) , alamine transaminase 384 iu / l ( 1035 ) , total bilirubin 11 mg / dl ( 0.31.9 ) , albumin 17 g / l ( 3450 ) , creatinine 290 mol / l ( 4992 ) and proteinuria 2.30 g / l ( 00,10 ) .
her c3 level was 1.0 g / l ( 0.651.65 ) , cd19 count was 0.001/l ( 0.110.69 ) , and immunoglobulin g level was 9.32 g /
cultures of muscle tissue only grew p aeruginosa , resistant to piperazillin - tazobactam ; therefore , she was treated with meropenem , teicoplanin and clindamycin . as her clinical condition
however , 1 week later , she deteriorated , with thick yellowish sputum , fever , respiratory distress and the chest x - ray showed right consolidation .
ciprofloxacin and teicoplanin were empirically started , and the patient 's clinical condition gradually improved . a week
after this treatment had finished , the patient became worse with a new left lower lobe consolidation , so the antibiotic therapy was changed to ceftazidime .
her lupus flared with a marked malar rash over both cheeks , so her steroids ( prednisolone ) were increased to 20 mg per day and hcq was restarted .
she was in the icu from may to august 2012 , but made a remarkable recovery and was discharged home with a steroid - tapering regimen and hcq .
later , she developed two more sle flares , which were treated again with cyc and rtx , achieving good response . at 18 months
follow - up , there is no evidence of new recurrent or severe ongoing infections .
most patients with nf ranged from 38years to 44 years , with a male to female ratio of 23 : 1 , and apparently an increased incidence in african and asian countries.3 the true incidence is not known ( it is estimated to be approximately 0.4 cases per 100 000 ) , with a reported mortality from 20% to as high as 80%.35 the causative agents of nf vary and include two main categories , polymicrobial ( type 1 ) and infection of group a streptococcal ( type 2 ) .
patients with sle have an increased risk of infections , due to immunological dysfunction and the use of steroids and immunosuppressive agents.6 other factors , such as the presence of a variant form of the fc receptor are also believed to contribute to the risk of pneumococcal infection.7 nf due to pseudomonas has very rarely been reported.8
9 the clinical presentation of patients with nf may be deceptively benign at onset , and it may not be possible to distinguish it clearly from minor soft tissue infections .
our patient was discharged from the original hospital before being admitted subsequently severely ill to university college hospital .
the lesion was rapidly progressive , probably due to her sle , and the prolonged steroid treatment and recent administration of cyc and rtx might have increased the risk of infection . during the initial period ,
exploration of the wound may be necessary even before the diagnosis is clear , particularly in a patient who is clearly toxic .
in addition to the present case , the other 31 cases of nf in sle patients that have been reported are summarised in table 1 .
the ages ranged from 12 years to 66 years , and 27 ( 84% ) were female .
most of the patients reported were asian ( 33% , 7 patients ) , 33% ( 7 patients ) hispanic and 19% ( 4 patients ) african american .
the sites of infection included the upper or lower limb ( 60% , 17 patients ) , the face , neck or tongue ( 18% , 5 patients ) , the genital area ( 11% , 3 patients ) and the abdomen ( 11% , 3 patients ) .
nf type 1 was identified in 22 patients ( 71% ) and type 2 in 7 patients ( 23% ) , and when reported , the most common isolation was streptococcus ( 50% , 16 patients ) .
most of the patients presented with intense pain , poorly demarcated erythematous , swollen lesions over the limbs and systemic inflammatory response ; 28 patients ( 88% ) were receiving corticosteroids and 7 hcq ( 22% ) . additionally , use of other immunosuppressive drugs has been reported ( 5 cyc , 5 aza , 2 rtx , 1 mycophenolate , 1 methotrexate , 1 plasmapheresis ) before or at the time of infection .
other associated conditions have been identified , including nephritis ( 54% ; 15 patients of 28 reported ) , either during the current presentation or in the past , 13 ( 42% ) had active disease ( presumably those taking > 20 mg prednisolone or equivalent per day ) , 76% ( 13 of 17 reported ) had lymphopenia , 100% ( 12 out of 12 reported ) low serum albumin levels and 12 ( 37% ) had anaemia . interestingly , before the admission , 9 ( 28% ) reported a previous episode of infection : urinary , bartholin abcess , diarrhoea , respiratory tract infection , carious tooth , colitis , hepatitis c , cutaneous ulcers ( 2 ) , breast abscess and diverticulitis .
most patients responded to therapy , but 8 of the 32 patients died ( 25% ) , probably due to the infection or because of complications ( such as sepsis or pulmonary embolism ) .
the case presented here and the literature reviewed suggests that active disease , nephropathy , lymphopenia , anaemia , low serum albumin levels , immunosuppressive therapy and significant infections in the past , may constitute risk factors for the development of nf in patients with sle .
a high index of suspicion for the diagnosis is required , and surgical exploration should not be delayed , especially during the early stages , to improve the prognosis of this devastating complication . | necrotising fasciitis ( nf ) is a rare infection of the subcutaneous tissue , known to be rapidly progressive and potentially fatal .
patients with systemic lupus erythematosus ( sle ) may be predisposed to this condition , and early clinical recognition can be difficult .
we report a case of necrotising fasciitis in a 26-year - old woman with sle .
she presented with painful swelling of her left leg , then developed clinical features of septic shock .
emergency debridement was performed .
intraoperative findings revealed nf and cultures grew pseudomonas aeruginosa .
the patient survived after a lengthy hospital admission , following several further debridements complicated by recurrent chest sepsis and multiorgan failure .
we also review and discuss the published cases of nf in sle patients . | [
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spontaneous perforation of rectum is a rare event ; however evisceration of the small bowel through the perforated site without predisposing factors is extremely rare , complex and worth reporting .
the operative findings revealed it to be a case of spontaneous perforation of rectum with evisceration of the small bowel through the perforation .
sudden increase in the intra - abdominal pressure leads to the perforation in the chronically deranged rectal wall and pushes the small bowel loops into the pelvis and through the perforated rectum to appear transanally .
evisceration of small bowel through a spontaneous perforation in rectum is an extremely rare event .
although 55 cases of evisceration of small bowel through anus due to perforation of rectum have been documented in world literature since the first report in 1827 by benjamin brodie , the number of cases without any significant predisposing factor is very less .
the article reports a case of spontaneous perforation of rectum with evisceration of small bowel in a normal bowel without an apparent cause which is an extremely rare presentation .
a 14 years old male was admitted with the chief complaint of acute lower abdominal pain which started suddenly after he passed stools . the abdomen was tender on deep palpation in the left iliac fossa and bowel sounds were sluggish .
however , on per rectal examination gut loops were felt in the anal canal and finger was stained with blood thereby making intussusception the most probable diagnosis .
a lump was felt in the left iliac fossa once the patient was anaesthetized for surgery .
peroperative findings revealed a distended and a tubular sigmoid colon ( intussusception like appearance ) and twisting of ileal loops around the sigmoid colon simulating ileo - sigmoid knotting . on further mobilization and gentle traction on the ileal loops gangrenous segment of ileum became evident till it finally emerged out from a longitudinal perforation ( approximately15 mm ) in the anterior wall of rectum just above the peritoneal reflection ( fig .
rectal tear was closed primarily in single layer after taking the biopsy from the whole edge which revealed non specific inflammation . on repeated
questioning in postoperative period patient denied any history suggestive of constipation , rectal prolapse , trauma , weight lifting , rectal instrumentation or homosexual activity ; however intermittent straining at stools was present .
figure showing rectal perforation and the gangrenous segment of ileum which had eviscerated through the perforated rectum .
urinary bladder ( small square ) , the peritoneal reflection ( thin arrow ) and the rectal perforation ( thick arrow ) are clearly visible .
spontaneous rectal perforation usually occurs due to excessive straining on the anterior rectal wall with a pre - existing pathology like , diverticulosis , colitis , ulceration , malignancy , adhesions , irradiation , rectal and uterine prolapse and as a consequence of iatrogenic injuries and blunt trauma abdomen .
. chronic strain because of the underlying pathology causes progressive deepening of rectovesical and rectouterine pouches and the rectal wall becomes thin and weak as it is unsupported .
contraction of the abdominal muscles increases the intra - abdominal pressure and spontaneous perforation occurs through this thinned out area , mostly at the ant mesenteric border where the blood supply is poorest .
similarly , in a constipated patient the changed defecation pattern in some individuals per se causes a preliminary lesion in the intestinal wall which becomes friable due to chronic inflammation of its layers and may perforate .
nevertheless intestinal evisceration through the perforated gut without any contamination of the peritoneal cavity is enigmatic and little difficult to explain . in a recent study
presumed that two factors predisposed patients to this unusual complication : one was the sudden increase in intra - abdominal pressure and the other was the presence of rectal prolapse .
whatever the mechanics involved the sudden increase in the intra - abdominal pressure seems to be the main contributing factor .
it pushes the ileal loops into the rectovesical / rectouterine pouch which violently presses upon the anterior rectal wall . as the week rectal wall gives way the ileal loops sneak in .
this seems to be the only plausible explanation for the spontaneous act particularly when there are no signs of peritonitis as in our case . in a perforated simple ulcer of rectum
there is an area of induration around edges while a spontaneous perforation appears slit like , runs longitudinally ( rarely transversally ) with minimal signs of inflammation .
it can occur in all age groups , the youngest one reported was six years old and oldest being ninety six years old .
the treatment follows basic surgical principles . if reduced bowel is not viable it should be resected and followed by anastomosis or ileostomy depending upon the condition of the patient and contamination involved . in the follow up
period patients should be investigated to detect exact etiology and cause specific management will prevent recurrence and complications .
therefore , one must remember that per rectal palpation of bowel loops may be more than just rectal prolapse or intussusception . | context : spontaneous perforation of rectum is a rare event ; however evisceration of the small bowel through the perforated site without predisposing factors is extremely rare , complex and worth reporting.case report : a 14 years old presented to us apparently as a case of intussception .
the operative findings revealed it to be a case of spontaneous perforation of rectum with evisceration of the small bowel through the perforation.conclusion:sudden increase in the intra - abdominal pressure leads to the perforation in the chronically deranged rectal wall and pushes the small bowel loops into the pelvis and through the perforated rectum to appear transanally . | [
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the motor system is a dynamic network of cortical and subcortical areas interacting through excitatory and inhibitory circuits , modulated by somatosensory input .
modifications of cortical excitability enable recovery and reorganization of the remaining motor areas both in animal models [ 4 , 5 ] and in humans [ 1 , 6 ] .
transcranial magnetic stimulation ( tms ) and magnetoencephalography ( meg ) have both been applied in stroke patients to reveal cortical excitability changes .
motor threshold ( mt ) , that is , the minimal tms intensity eliciting motor evoked potentials ( meps ) , is related to membrane excitability as it is influenced by drugs affecting neuronal ion channels .
paired pulse tms ( pp - tms ) reveals short - interval intracortical inhibition ( sici ) , related to the activation of gaba - a receptors and intracortical facilitation ( icf ) attributed mainly to glutamatergic nmda receptor activation ( for references , see ) . in acute stroke
, the mt is increased in the lesioned hemisphere ( lh ) whereas in the nonlesioned ( nh ) hemisphere it is normal or decreased one month after stroke .
sici is decreased in both hemispheres early after stroke ; icf is stronger in nh in severe than mild strokes [ 1 , 6 ] .
finger movements and median nerve or finger stimulation modify spontaneous meg oscillations over the sensorimotor cortex in the beta band ( 1525 hz ) .
they are suppressed at 100300 ms after tactile stimulation ( event - related desynchronization ; erd ) , reflecting increased excitability , and increased at 5001000 ms ( event - related synchronization ; ers ) , reflecting removal of excitation [ 12 , 13 ] or reduced excitability .
inhibitory gaba - a agonist diazepam increases meg beta activity [ 15 , 16 ] .
combined meg and magnetic resonance spectroscopy showed a linear relationship between the beta ers strength and gaba concentration .
beta ers has been shown to be significantly weaker in the lh than nh ; stronger ers amplitude was correlated with a better affected hand function up to 3 months after stroke .
reduction of beta ers , however , correlated with clinical improvement after physiotherapy of patients with chronic stroke .
movement - related beta erd was significantly reduced in lh of stroke patients . the hand representation in the somatosensory cortex ( s1 ) , estimated by somatosensory evoked fields ( sefs ) , is the largest one month after stroke and its increase was correlated with the affected hand function . in tms mapping
, hand motor representation expands in the lh up to 1 month . in animal models ,
somatosensory reorganization is activated immediately after the lesion by diminished gaba - a - related inhibition and by glutamatergic activation after one month .
we hypothesized to see correlations between erd and mt related to the early cortical excitability and between ers and sici , both attributed to gaba - a - related processes .
moreover , we expected that icf , reflecting glutamatergic activity , would correlate with the somatosensory modifications in meg , as glutamate is associated with late somatosensory plasticity .
meg and ntms were recorded from thirteen patients ( age 67.3 11 years , 8 women ) , with first - ever ischemic stroke in the middle cerebral artery territory one ( t1 ) and three months after stroke ( t2 ) .
six patients had a subcortical and seven patients had a subcorticocortical or cortical stroke ( table 1 ) . at t1 ,
one patient used amitriptyline 10 mg / day and another used citalopram 10 mg / day .
one patient used zopiclone 7.5 mg for occasional sleeplessness . at t2 , citalopram 10 mg / day was used by one patient .
data from these patients has been presented previously [ 18 , 21 , 23 , 24 ] .
as the patients having both tms and meg recordings are a subset of the previous patient groups , we recalculated the group - level electrophysiological parameters to show that the present patient group is sufficiently similar to those reported previously ( see supplementary tables 1 and 2 in supplementary material available online at http://dx.doi.org/10.1155/2015/309546 ) .
an eximia navigated magnetic stimulator with a coplanar figure - of - eight coil of 70 mm wing radius ( nexstim ltd . , helsinki , finland ) was used for ntms .
patients ' mris , required for navigation , were scanned at t1 and used also at t2 .
the site was then stimulated by single tms pulses at 110% of mt and by the pp - tms at 90% for conditioning and 110% of mt for test pulses .
fifteen single pulses or pairs of conditioning and test pulses were delivered in each stimulation session .
the interval between stimuli was 3.3 s and the intersession interval varied between 1 and 3 min .
the isi selected for the paired - pulse stimuli was 2 ms for sici and 12 ms for icf .
the peak - to - peak amplitudes of meps elicited by pp - tms were normalized by dividing them by the corresponding single - pulse mep amplitude to simplify subject - to - subject comparisons .
meg during rest and tactile stimulation of the thumb ( d1 ) , index ( d2 ) , and little finger ( d5 ) were recorded with a 306-sensor neuromagnetometer ( elekta neuromag , helsinki , finland ) in biomag laboratory , right before the ntms measurement .
time - frequency representations ( tfr ) in the 1030 hz band were calculated to define the frequency range of beta modulation , which was quantified by the temporal spectral evolution method ( tse ) from signals of 2 to 4 meg sensors showing the strongest reactivity . only the contralateral beta modifications ( the affected hand stimulation for the lh and the unaffected hand for nh ) were analyzed .
onset and offset of the erd and ers were defined as a time point when the signal differed 2 sds from the baseline .
the absolute erd and ers strengths were calculated from the peak amplitudes and converted into relative values in relation to the 300 ms prestimulus baseline . for sefs , about 120 responses were averaged for stimulation of d2 ( isi 3005 ms ) , and d1 and d5 ( isi 1005 ms ) in separate sessions .
the size of the hand representation in the si was determined by calculating the euclidean distance in xyz - space between the equivalent current dipoles ( ecds ) of the earliest responses to d1 and d5 stimulation .
multiple comparison correction was carried out according to the number of tests ( n = 32 ) suggested by the four prior hypotheses ( t1 and t2 were tested separately ; lh and nh variations lead to four tests in each case ; n = 442 = 32 ) .
we also present significances of correlation values without multiple comparisons and supply all correlation coefficients and corresponding p values ( cf .
[ 1 , 9 , 28 ] for a similar approach and for its statistical aspects ) .
the differences between ntms and meg values obtained at t1 and t2 were tested with student 's t - test .
in the lh , meps were found in 11 patients both at t1 and at t2 and were present in the nh in all patients .
mt was higher for lh than nh in 9 patients at t1 ( p < 0.05 , binomial test ) and in 10 patients at t2 ( p < 0.05 , binomial test ) .
the mts in the lh and nh correlated strongly both at t1 ( r = .82 , p < .01 ) and at t2 ( r = .78 , p < .01 ) .
pp2ms stimulations of nh did not inhibit meps ( disinhibition ; diminished sici ) in 5 patients at t1 and in 3 patients at t2 .
pp12ms stimulation of lh facilitated meps ( icf ) in 10 out of 11 patients at t1 and in all patients at t2 . in nh
mep amplitudes elicited by pp12ms stimulations were correlated between the lh and nh at t1 ( r = .62 ; p < .05 ) but not at t2 ( supplementary table 2 ) .
erd started 140 10 ms after tactile stimulation and peaked at 250 10 ms .
the subsequent ers started at 520 40 ms and peaked at 900 70 ms . at t1 ,
erd was absent in one patient and ers in two patients in the lh ; ers was missing from the nh in one patient . at t2 ,
ers was smaller in the lh than nh at t1 ( 46 31% versus 63 32% ; p < .05 ) ; at t2 , the difference was nonsignificant .
sefs from both hemispheres were detected in 12 patients at t1 and in all patients at t2 .
they were smaller in the lh than nh at t1 ( 25 nam versus 32 nam ; p < .04 ) but not at t2 .
the si hand representation area was larger in the lh than nh at t1 ( 12 3 mm versus 10 3 mm p < .003 ) but not at t2 ( supplementary table 3 ) .
the plots of the most relevant correlations are depicted in figure 1 to show that they were not driven by outliers .
all correlations are displayed in table 2 to enable evaluation of significance of our hypotheses against general effects of the lesions .
the mt and erd were correlated in the lh at t1 ( r = .66 , p < .03 ) , indicating that small erd was associated with a high mt ( figure 1(a ) ) . at t2 , this correlation was weaker ( r = .58 , p < .06 ) . however , the mt of the lh correlated with the erd of nh ( r = .62 , p < .04 ) , and the mt of the nh correlated with erd of the lh ( r = .65 , p < .02 ) , suggesting that high mt was associated with a small erd in the opposite hemisphere at t2 ( table 2 ) .
sici and the ers did not correlate at t1 or in lh at t2 . in the nh ,
high ers was associated with a strong sici ( r = .59 , p < .04 ; figure 1(b ) ) .
in addition to hypothesized correlations , sici of the nh and erd of the lh at t2 were correlated ( r = .82 , p < .001 ) , indicating that strong erd in the lh was associated with a strong sici in the nh .
sici in the lh was correlated also with the si amplitude of the lh ( r = .64 , p < .04 ) , indicating that small si amplitude was associated with a weak sici ( table 2 ) .
icf and sef parameters did not correlate at t1 . at t2 , icf in the lh correlated with the s1 amplitude of lh ( r = .65 , p
in addition , icf in the nh correlated ( r = .82 , p < .001 ) with the si finger representation area of the lh ; this correlation remained significant also after bonferroni correction ( table 2 ) .
moreover , icf in the lh at t1 was correlated ( r = .83 ; p < .002 ) with the si finger representation area of lh at t2 ; high icf at t1 resulted in a small hand representation area at t2 ( figure 1(c ) ) .
our study is the first to compare meg and ntms excitability parameters during stroke recovery .
navigated tms , not applied previously in longitudinal studies of stroke patients , enabled the precise replication of the stimulus site between separate measurements , adding reliability to the follow - up .
we found correlations between cortical excitability estimates derived from ntms and meg . as expected , we found correlations between mt and erd , but in only one of the four possible intrahemispheric and two out of four interhemispheric conditions .
the sici and beta ers , both attributed to gabaergic mechanisms , were correlated in one of their four possible intrahemispheric conditions ( in the nh at t2 ) .
one reason for this may be that various gaba - a receptor subtypes contribute to sici .
nonselective gaba - a receptor activators modify sici whereas the gaba - a1 receptor specific zolpidem did not .
nonselective gaba - a agonist zopiclone increased meg beta activity whereas zolpidem suppressed beta activity in the vicinity of stroke lesion .
moreover , in healthy subjects , diazepam increased meg erd but did not affect ers when the increase of baseline beta activity was taken into account .
this nonspecificity could contribute to the correlations of sici with the erd and mt as well .
correlations between ntms parameters and meg erd / ers were stronger at t2 than at t1 .
analogously , most tms intracortical excitability measures did not correlate with the hand function acutely but did so 3 months after stroke .
recovery of sensorimotor fmri activation to digit stimulation from 1 to 3 months was correlated with final motor function , emphasizing the importance of this time period for stroke recovery .
is attributed mainly to glutaminergic mechanisms , glutamate may contribute to stroke - induced plasticity .
somewhat surprisingly , high icf at t1 correlated with small si hand area at t2 ( figure 1(c ) ) ; thus , the narrowing towards normal hand representation size may be supported by glutaminergic activity .
icf did not correlate with the meg erd / ers , and the si hand area and beta erd / ers were not correlated ; this suggests different mechanisms underlying sici and icf ( see for a detailed discussion ) .
for example , high mt was associated with a small erd in the opposite hemisphere , and strong erd in the lh was associated with a strong sici in the nh .
increased excitability within the unaffected motor cortex may cause imbalance between the homologous cortical motor areas and worsen also the ipsilesional hand coordination ( for references , see ) .
thus , some functional correlations may relate to the modified general excitability properties of the motor system , instead of effects in the immediate vicinity of the stroke .
tms results give direct information of the changes in the motor output and the immediate effects of tms are relatively local .
however , also subcortical and spinal processes affect the meps used to evaluate the tms effects .
meg reveals the activity of the whole cortical mantle and enables mapping of network effects generated by stroke .
meg source analysis suggests mainly motor cortex origin of beta ers [ 13 , 33 , 34 ] .
however , in electrocorticography , recorded directly from the cortex , beta erd and ers appear outside of pre- and postcentral gyri , in supplementary motor cortex , or broadly from pre- and postcentral gyri , frontolateral and medial cortex [ 37 , 38 ]
. the widespread cortical generation of the erd and ers may make them resilient to small cortical strokes .
multitude of generators may contribute to considerable variability of source locations of beta erd in stroke patients ( cf . ) .
multiple sources underlying meg signals may also explain resilience of auditory evoked fields after small strokes .
stronger correlations between icf and si parameters than between mt and sici and ers / erd may , in part , result from spatially more limited source areas of s1 responses than those of erd / ers .
it can be expected that meg and ntms produce complementary information about the effects of stroke on cortical networks . moreover ,
meg parameters in the affected hemisphere and ntms indices in the unaffected hemisphere were correlated with the motor performance of the affected hand ( cf . [ 18 , 21 , 24 ] ) .
erd in the 822 hz band may reflect downregulation of intracortical inhibition in the human motor cortex , as tms delivered during erd is associated with increased mep amplitudes and reduced sici .
however , 1 hz repetitive tms over the motor cortex reduces meps to subsequent single tms pulses , indicating inhibition , but decreases postmovement beta ers , and intermittent theta burst tms facilitates meps but increases postmovement beta ers .
beta ers is reduced in patients with myoclonus epilepsy , indicating increased cortical excitability . in line
, sici is decreased in myoclonus epilepsy patients ; however , mt is increased [ 44 , 45 ] , and the silent period after the tms pulse , reflecting motor cortical postsynaptic inhibition , is prolonged , indicating prevailing inhibitory cortical tonus .
thus , only some aspects of the cortical excitability may be shared in excitability estimates obtained by tms and meg .
our results suggest that some tms and meg excitability measures reflect the activity of the same transmitter systems .
however , high mt and absence of erd / ers may also correlate because of severely affected sensorimotor connections between the periphery and the cortex .
we detected sefs in 12/13 patients and meps in 11/13 patients already at t1 , indicating that both somatosensory and motor pathways were conveying signals .
motor function can be maintained despite significant damage to the corticospinal tract , as estimated from mt of stroke patients .
moreover , we observed fewer correlations at t1 , when the sensorimotor pathways probably were more affected , than at t2 .
however , such spurious correlations should remain stable or decrease during recovery from t1 to t2 but not increase , as in our data .
the limitations of our study include the small size of the patient group as the precise features of structural and functional changes may differ among the patients .
cortical excitability is modified differently in cortical and subcortical strokes [ 46 , 47 ] .
this , however , should not alter our correlations between the tms and meg , as both were recorded from the same patients .
possible effects of medication on excitability should go in parallel for meg and ntms , as the patients were tested sequentially during the same day .
the patients were not tested in the acute stage with tms , and meg recordings showed most dramatic erd and ers modifications between the acute phase and t1 . although mt in the lh is correlated with the paretic hand function in acute stroke , this correlation , however , weakens during recovery , and tms intracortical excitability parameters correlated with the clinical performance best at 3 months .
the 2 ms or 12 ms isis , selected for our paired - pulse stimuli , produce clear sici and icf in healthy subjects and in stroke patients , but we did not test other parameters , which could have produced stronger correlations between ntms and meg .
icf and si response amplitude and area size , mt and the erd of the hemisphere harboring the stroke lesion , and sici and ers of the nonlesioned hemisphere are correlated in stroke patients .
numerous correlations of the excitability parameters between the lh and nh emphasize the importance of the hemispheric balance of the excitatory - inhibitory properties of the sensorimotor system in analyzing the stroke - related dysfunction during stroke recovery . |
objective .
stroke alters cortical excitability both in the lesioned and in the nonlesioned hemisphere .
stroke recovery has been studied using transcranial magnetic stimulation ( tms ) .
spontaneous brain oscillations and somatosensory evoked fields ( sefs ) measured by magnetoencephalography ( meg ) are modified in stroke patients during recovery .
methods .
we recorded sefs and spontaneous meg activity and motor threshold ( mt ) short intracortical inhibition ( sici ) and intracortical facilitation ( icf ) with navigated tms ( ntms ) at one and three months after first - ever hemispheric ischemic strokes .
changes of meg and ntms parameters attributed to gamma - aminobutyrate and glutamate transmission were compared .
results .
icf correlated with the strength and extent of sef source areas depicted by meg at three months .
the ntms mt and event - related desynchronization ( erd ) of beta - band meg activity and sici and the beta - band meg event - related synchronization ( ers ) were correlated , but less strongly . conclusions .
this first report using sequential ntms and meg in stroke recovery found intra- and interhemispheric correlations of ntms and meg estimates of cortical excitability .
icf and sef parameters , mt and the erd of the lesioned hemisphere , and sici and ers of the nonlesioned hemisphere were correlated .
covarying excitability in the lesioned and nonlesioned hemispheres emphasizes the importance of the hemispheric balance of the excitability of the sensorimotor system . | [
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] |
the u.s . food and drug administration ( fda ) has approved around 30 antidepressant medications for the treatment of major depression . among them are selective serotonin reuptake inhibitors ( ssris ) .
these drugs change the balance of serotonin in the brain , such as fluoxetine ( prozac ) , paroxetine ( paxil ) , sertraline ( zoloft ) , escitalopram ( lexapro ) , and citalopram ( celexa ) .
another family of medications , selective serotonin and norepinephrine reuptake inhibitors ( snris ) , help increase serotonin and norepinephrine levels in the brain , such as venlafaxine ( effexor ) , duloxetine ( cymbalta ) , and levomilnacipran ( fetzima ) .
still others in this family include bupropion ( wellbutrin ) , vortioxetine ( brintellix ) , mirtazapine ( remeron ) vilazodone ( viibryd ) , nefazodone ( serzone ) , and trazodone ( desyrel ) .
in addition , tricyclics and monoamine oxidase inhibitors , which are two classes of older antidepressants that work by inhibiting the brain s reuptake of serotonin and norepinephrine , are also approved but tend to cause more side effects than the other classes of antidepressants .
but pharmacotherapy is nt the only option ; two other major classes of treatment are also available psychotherapy and somatic nonpharmacological treatments . in randomized
, controlled trials , cognitive - behavioral therapy ( cbt ) and interpersonal psychotherapy ( ipt ) repeatedly have been demonstrated to be effective in the treatment of major depressive disorder ( mdd ) .
whether other forms of psychotherapy , such as insight - oriented , psychodynamically based therapy , are effective in major depression remains controversial .
brain stimulation therapies involve activating or touching the brain directly with electricity , magnets , or implants , and the fda has approved three somatic nonpharmacological treatments for depression : electroconvulsive therapy ( ect ) , vagus nerve stimulation ( vns ) , and repetitive transcranial magnetic stimulation ( rtms ) .
ect is generally considered the most effective of all depression treatments , although no head - to - head , randomized , controlled trial has compared it with other interventions .
it generally requires inpatient hospitalization , at least initially , and general anesthesia with nine to twelve treatments over a three- to four - week period .
vns and rtms are both fda - approved for treatment - resistant depression ; the former requires an invasive surgical procedure .
researchers have conducted relatively few controlled studies of these devices compared with the vast number of pharmacotherapy and psychotherapy treatment trials .
with a plethora of drugs and psychotherapy approaches available , let us consider the problem psychiatrists encounter on a daily basis .
a 50-year - old academic physician suffers from a classic major depressive episode associated with severe work stress .
he has difficulty falling asleep , awakens several times during the night , and rises early with severe anxiety .
he has reduced appetite , difficulty concentrating , and trouble enjoying any leisure activities , and he feels pessimistic about the future .
he admits to passive contemplations about suicide , with recurring thoughts that if a car jumped the median and landed on his car , it would be an end to his suffering .
he has no underlying medical disorder that might be contributing to depression , such as hypothyroidism or drug or alcohol abuse
i want to recommend the treatment most likely to be successful in producing a complete remission of his depressive syndrome and relieving him of his considerable misery . what are the known and best - validated predictors of response ?
our group has previously reviewed the scientific findings in this area.36 the most reliable predictor is past response , but in this case the patient has never been treated for depression .
a positive response in first - degree family relatives is also predictive of a beneficial response to antidepressants , but again , this is not applicable to this patient .
some evidence suggests that certain subtypes of depression respond best to certain treatments monoamine oxidase inhibitors ( the first type of antidepressants developed ) are believed to be the most effective for patients with so - called atypical depression characterized by hypersomnia , overeating , extreme rejection sensitivity , and feeling better in the morning than later in the day .
combinations of antidepressants and antipsychotics or ect are best for patients with major depression with psychotic features .
surely patient choice is an important consideration , but it will likely be guided by my discussion with the patient . in 2013 ,
mayberg , holtzheimer , dunlop , and craighead ) were colleagues of mine for many years , and we continue to collaborate on various projects .
mayberg s study sought to identify a biomarker that could predict which type of treatment would benefit a patient based on the individual s brain activity . using regional brain glucose metabolism as measured by positron emission tomography ( pet ) as a proxy for neural activity , her group sought to determine whether baseline resting state activity predicted remission after twelve weeks of treatment with either the selective serotonin reuptake inhibitor escitalopram ( 10 to 20 mg per day ) or sixteen sessions of cognitive - behavioral therapy .
the study sample initially comprised eighty - two men and women who were randomized between the two treatments .
of these , sixty - five patients completed the study and thirty - eight had clear outcomes and acceptable pet data .
the thirty - eight patients who comprise the analyzable data set were distributed as follows : eleven who went into remission with escitalopram ( six non - responders ) and twelve who did so with cbt ( nine nonresponders ) .
the major finding were that hypometabolism of glucose in the insula , likely reflecting reduced activity of neurons in this brain region , was associated with remission using cbt , and with poor response to escitalopram .
contrariwise , insula hypermetabolism , reflecting increased activity of neurons in this brain region , was associated with remission using escitalopram and with poor response to cbt .
the authors conclude that baseline insula metabolism is the first objective marker to guide initial treatment selection in depression
first they eliminated from their primary analysis the responders to cbt or to escitalopram who did not go into remission . more specifically , partial responders to escitalopram or cbt were excluded from the analysis .
they did so in order to accentuate the differences between the extremes in the depressed population ; the results revealed clear differences in glucose metabolism in six regions : the right
anterior insula , right motor cortex , left premotor cortex , right inferior temporal cortex , left amygdala , and precuneus .
mean regional activity values for remitters and nonresponders segregated by treatment arm are plotted for the six regions showing a significant treatment outcome analysis of variance interaction effect .
regional metabolic activity values are displayed as region / whole - brain metabolism converted to z scores .
cbt indicates cognitive - behavioral therapy.7 when all six regions were compared , the right insula exhibited the greatest effect as a discriminator of treatment response , followed by the precuneus .
when the whole sample was studied , right insular activity was positively correlated with the depression symptom severity scale , and with the hamilton depression rating scale ( hrsd ) score in the cbt treatment group while right insular activity was negatively correlated with the hrsd in the escitalopram treatment group .
if additional research can replicates these results , it suggests that a simple brain imaging test could reliably predict whether a given patient should be treated with psychotherapy or antidepressant medication .
it also raises a plethora of additional questions :
a wealth of data , now summarized in a research meta - analysis , indicate that mdd patients with a history of child abuse and neglect exhibit a poorer response to pharmacotherapy and psychotherapy and exhibit unique brain imaging differences.8 mayberg s research does not address this critical clinical characteristic in this population.it is somewhat unclear how the six brain regions of interest were identified and why several regions repeatedly identified to be implicated in the pathophysiology of depression either were not selected or exhibited no significant effect , including the hippocampus , subgenual cingulate , and others.it is hard to know what to make of the findings that only the right anterior insula , right motor cortex , left premotor cortex , left amygdala , left precuneus , and right inferior temporal region show dramatic differences in the cbt versus escitalopram induced remission versus nonresponder groups whereas their counterparts , namely the left anterior insula , left motor cortex , right premotor cortex , right amygdala , right precuneus , and left inferior cortex did not . was a composite of the left and right sides of these structures informative?as the authors themselves point out , the study comprises a relatively small number of patients and our field is replete with pilot study findings that , unfortunately , have not been replicated in larger trials.this study utilized pet instead of the more often used functional magnetic resonance imaging ( fmri ) technology . as mayberg and
her colleagues appropriately point out in their paper , fmri studies have examined regional brain activity and , more recently , resting state connectivity to identify mdd or mdd subtypes , but neither type of imaging has been used to discriminate response either among antidepressants or between antidepressants and psychotherapy.9 a wealth of data , now summarized in a research meta - analysis , indicate that mdd patients with a history of child abuse and neglect exhibit a poorer response to pharmacotherapy and psychotherapy and exhibit unique brain imaging differences.8 mayberg s research does not address this critical clinical characteristic in this population .
it is somewhat unclear how the six brain regions of interest were identified and why several regions repeatedly identified to be implicated in the pathophysiology of depression either were not selected or exhibited no significant effect , including the hippocampus , subgenual cingulate , and others .
it is hard to know what to make of the findings that only the right anterior insula , right motor cortex , left premotor cortex , left amygdala , left precuneus , and right inferior temporal region show dramatic differences in the cbt versus escitalopram induced remission versus nonresponder groups whereas their counterparts , namely the left anterior insula , left motor cortex , right premotor cortex , right amygdala , right precuneus , and left inferior cortex did not . was a composite of the left and right sides of these structures informative ?
as the authors themselves point out , the study comprises a relatively small number of patients and our field is replete with pilot study findings that , unfortunately , have not been replicated in larger trials . this study utilized pet instead of the more often used functional magnetic resonance imaging ( fmri ) technology .
as mayberg and her colleagues appropriately point out in their paper , fmri studies have examined regional brain activity and , more recently , resting state connectivity to identify mdd or mdd subtypes , but neither type of imaging has been used to discriminate response either among antidepressants or between antidepressants and psychotherapy.9 the expanding area of genetics in general , and pharmacogenetics in particular , is also of vital importance .
a burgeoning database documents the role of certain genetic variations in vulnerability to mood disorders , and more recently how variations may affect treatment response to different antidepressants . whether genetic material was collected in mayberg s study is unclear , but this focus is crucial , particularly in view of recent findings in imaging genomics .
the lack of random assignment of the mdd patients as regards , for example , the vulnerability gene variants of the serotonin transporter or others , now shown to be associated with clear alterations in regional brain activity , could have confounded the results .
this research group has always been willing to take great leaps forward , and they should be applauded for it .
subsequent studies will reveal if the insula is truly the region that predicts response to cbt versus a selective serotonin reuptake inhibitor such as escitalopram or whether other regions or biomarkers also need to be a component of the ultimate formula .
this is part of the ongoing and exciting scientific process that is emblematic of the marriage of neuroscience and psychiatry .
ultimately , i believe this work will be judged as crucial in eventually attaining the goal all of us seek : a valid predictor of individual treatment response in depression , still the holy grail in psychiatry research . | editor s note:holy grail is a well - known metaphor for the eternal spiritual pursuit for truth and wisdom .
it suggests that in order for us to find what no one has found , we must search where few have looked . in 2013 ,
a group led by helen mayberg published a groundbreaking paper that sought an answer to one of the most discussed conundrums in psychiatry and neuroscience : can specific patterns of brain activity indicate how a depressed person will respond to treatment with medication or psychotherapy ? our author examines the findings and discusses their potential impact on treatment for a public health problem that affects millions of people worldwide . | [
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primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et al . , 2012 ) .
no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et al . , 2011 ) .
importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain .
additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et al . , 2000 ;
sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus .
sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et al . , 2012 ) .
the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein .
known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et al . , 2013 ) .
sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor .
for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et al .
recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice .
this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et al . , 2011 ) .
here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et al . , 2010 ) .
sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et al . , 2009 ) .
mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et al . , 1999 ) .
most of the patients carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele .
the second knockout allele in this animal model is functionally null ( yang et al . , 2010 ) .
while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by exercise intolerance and associated with ubiquitous cox deficiency .
to test the effects of persistent increase of the nad pool on the sco2 mouse , we first crossed it with a constitutive parp1 mouse ( de murcia et al . ,
1997 ) , which shows increased levels of nad in skeletal muscle ( bai et al . , 2011 ) .
the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure s1 available online ) .
the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4 weeks starting at 2 months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure 1a ) .
biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants ,
these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure 1b ; table s1 ) .
accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure 1c ) .
the activities of complex iii and iv ( figure 1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure 1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n = 4/group ) , as a food admix ( 400 mg / kg ) ( cant et al . , 2012 ) for four weeks .
metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in vivo .
nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure 2a ) , which showed no difference to each other .
these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et al . , 2012 ) .
accordingly , the nad / nadh ratio was significantly increased ( figure 2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure 2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals .
we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure 2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure 2d ) .
tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment .
as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 .
accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure 2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure 2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals .
the histochemical staining for cox reflected the biochemical results ( figure 2 g ) . in both c. elegans and mammalian cells
, nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et al .
we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure 2h ) .
no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) .
next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp < 1 nm ; pirinen et al . , 2014 , in this issue of cell metabolism ) at 50 mg /
kg as a food admix for 4 weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure s3 ) .
weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure 3a ) .
the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure 3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure 3c ) , indicating activation of sirt1 by mrlb-45696 .
similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure 3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure 3f ; table s3 ) .
histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure 3 g ) .
again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) .
in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure 4a ) , and respiratory chain activities in the brain ( figure 4b ; table s4 ) . the intensity of cox staining was increased as well ( figure 4c ) .
similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) .
the nad pool is set by the balance between de novo and salvage biosynthetic pathways and utilization by nad - consuming enzymes .
nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et al . , 2010 ) .
these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) .
nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle .
for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt .
parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) .
ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis .
these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et al . ,
we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr
was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals .
notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs .
increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6 months ) ( pirinen et al . , 2014 ) .
in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4 weeks ) compared to that of other studies ( 12 weeks ) .
this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much earlier than stimulation of mitochondrial proliferation and increase in mtdna content .
likewise , prolonged nr supplementation up to 6 months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et al . , 2013 ) .
while we observed hardly any effect of nr in our 4 week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice .
this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood .
our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease .
nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy .
our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et al . , 2013 ) or complex i deficiency ( karamanlidis et al . , 2013 )
initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et al . , 2013 ) .
several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et al . , 2013 ; tutt et al . ,
however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects .
mouse tissues were homogenized in 15 volumes of 10 mm potassium phosphate buffer ( ph 7.5 ) .
mitochondrial - enriched fractions were collected after centrifugation at 800 g for 10 min in the presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen .
aliquots , 70 g each , were run through a 12% sds - page and electroblotted onto a nitrocellulose membrane , which was then matched with different antibodies .
carlo besta neurological institute , in accordance with guidelines of the italian ministry of health .
mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12 hr light / dark cycle and free access to water and food .
a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et al .
series of 8 m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) .
muscle quadriceps samples stored in liquid nitrogen were homogenized in 10 mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et al . , 2004 ) .
note that in all panels the activity of cii has been multiplied by 10 for visualization clarity .
nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) .
tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) .
mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et al . , 2011 ) . | summarymitochondrial disorders
are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain . pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling , often fatal , disorders .
pathways related to mitochondrial biogenesis are targets of sirtuin1 , a nad+-dependent protein deacetylase . as nad+ boosts the activity of sirtuin1 and other sirtuins , intracellular levels of nad+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell .
we show here that supplementation with nicotinamide riboside , a natural nad+ precursor , or reduction of nad+ consumption by inhibiting the poly(adp - ribose ) polymerases , leads to marked improvement of the respiratory chain defect and exercise intolerance of the sco2 knockout / knockin mouse , a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis .
this strategy is potentially translatable into therapy of mitochondrial disorders in humans . | [
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noroviruses ( novs ) ,
a group of small , round - structured rna viruses
constituting the norovirus genus in
the family caliciviridae , infect both
humans and animals . human novs cause epidemic acute gastroenteritis ,
affecting millions of people and claiming over 200,000 lives annually
worldwide . at present
structurally , novs are nonenveloped ,
containing an outer protein capsid that encapsulates the single - stranded ,
positive sense rna genome of 7.7 kb .
the nov capsid is made
from a single major structural viral protein , vp1 .
crystallography
of recombinant nov - like particles ( vlps ) reveals a t = 3 icosahedral symmetry consisting of 180 copies of vp1 organized
into 90 dimers .
vp1 is divided into two
major domains , the shell ( s ) and the protruding ( p ) domains .
the s
domain forms the interior , icosahedral shell ; while the p domain forms
the dimeric protrusions extending outward from the shell . the p domain can be further divided into p1 and
p2 subdomains , corresponding to the legs and the head of the arch - like
protrusion , respectively .
the p2 subdomain forms the outermost surface
of the capsid with highly variable sequence , responsible for the virus
human novs are difficult to study due to the
lack of an efficient
cell culture system and a small animal model .
currently , research
into nov host interactions relies on various nov subviral particles .
expression of full - length vp1 results in vlps that are structurally
similar to an authentic virus . furthermore ,
expression of various subdomains results in smaller subviral particles
or complexes . for example , production of the s domain forms s particles , corresponding to the interior shell of the capsid , while expressions
of the p domains with or without modifications can form p dimers , 12-mer small p particles , or 24-mer
p particles .
in addition , various glutathione
s - transferase ( gst)-p domain fusion proteins have been shown to form
polyvalent complexes owing to the dimeric and oligomeric features
of the gst and the p domain .
these vlps , p particles
and p complexes retain the basic structures of the capsid or p dimer ,
recognize host ligands and , thus , have been used as tools or models
for the study of nov host interactions .
human novs recognize
histo - blood group antigens ( hbgas ) as attachment
factors or receptors , which play an important role in the host susceptibility
of nov infection , as shown by both human challenge studies and outbreak
investigations .
hbgas are oligosaccharides linked to membrane proteins
or lipids as glycoprotein or glycolipid that are distributed extensively
on the surfaces of red blood cells and mucosal epithelia .
they are also present as free oligosaccharides
in biological fluids , such as saliva or milk .
human novs interact with hbgas in a strain - specific manner , whereby
a number of nov - hbga binding patterns involved in all abo , lewis and
secretor / nonsecretor types have been identified .
the structural basis of these interactions have been elucidated
by x - ray crystallography of nov p dimers in complex with hbga oligosaccharides .
however , it has been observed
that some human novs , such as va115 ( gi.3 ) , desert shield virus ( gi.3 ) and noda485
( gii.1 ) , do not bind any hbgas .
a human
challenge study of snow mountain virus ( smv , gii.2 ) did not reveal
a dependence of host susceptibility on hbga type , despite the fact
that the smv vlp recognizes only the b antigen .
in addition , a recent study showed that nov vlps of ueno
7k ( gii.6 ) and noda485 binds caco-2 cells and human small intestinal
epithelium biopsy in a hbga - independent manner .
these data suggest that hbgas may not be the only receptors
for human novs .
for example , using thin - layer chromatography
and quartz crystal microbalance with dissipation monitoring , larson
et al . reported binding of gii.4 vlps to galactosylceramide and hbga
glycosphingolipids that were purified from human meconium samples .
takeda and co - workers demonstrated that vlps of gii novs bound heparan
sulfate on the cell surface , while belliot
and co - workers showed that gii.4 vlps recognized sialic acid - containing
carbohydrates , such as sialyl lewis x ( le ) , sialyl - lacto - n - fucopentaose , sialyl - lacto - n - tetraose ,
and sialyl - lacto - n - neotetraose , with affinities comparable
to those of hbga ligands . using saturation
transfer difference nuclear magnetic resonance spectroscopy , peters
and co - workers detected the interactions between gii.4 vlps and the
sialic acid moiety of sialyl le and sialyl le .
however , they also found that carbohydrates
containing sialic acid , but not fucose , e.g. 3-sialyllactose
and 6-sialyllactose , do not exhibit detectable binding with
the vlp .
the results of these studies ,
taken together , imply that sialic acid - containing oligosaccharides
could also be ligands of human novs .
in fact , sialic acid - containing
oligosaccharides have been shown to be ligands or receptors for some
animal caliciviruses ( cvs ) , including murine nov ( mnv1 ) , feline calicivirus ( fcv ) and a porcine sapovirus ( psav , cowden strain ) . however , solid evidence to establish the ligand
status of sialic acid for human cvs ( human novs and human sapoviruses )
is lacking . here
, we report the first experimental evidence
that human novs
recognize sialic acid - containing glycosphingolipids ( gangliosides ) .
the catch - and - release electrospray ionization mass spectrometry ( car - esi - ms )
assay was used to screen a library of
gangliosides against the p particle of human nov va387 ( gii.4 ) .
the
affinities of 13 gangliosides for the p dimer of va387 and of a second
human nov strain , va115 ( gi.3 ) , were measured using the direct esi - ms
assay . using a competitive esi - ms assay ,
the proxy protein method , the highest
affinity ligand , gm3 ,
was subjected to additional binding measurements
and the affinities for both the va387 p particle and vlp were determined .
notably , the ganglioside affinities measured for nov va387 are comparable
to those of known hbga oligosaccharide receptors .
enzyme - linked immunosorbent assays ( elisa ) provided additional
evidence that both strains of novs exhibit binding to sialic acid - containing
oligosaccharides .
the vlps of va387 ( gii.4 ) were produced in
insect cells ( sf9 ) through a recombinant baculovirus containing the
gene encoding va387 vp1 ( genbank accession number ay038600 , molecular
weight ( mw ) of monomer 58,887 da ) as described previously .
the resulting vlps were purified by sucrose
gradient . va387 p particles ( 24-mer , mw 865,036 da ) , p dimers ( mw
69,312 da ) , and gst - p domain fusion proteins were produced based on
the p domain sequences ( residues 222539 ) of vp1 via e. coli as reported in our previous studies .
the gst gene fusion system ( ge healthcare life sciences ,
piscataway , nj ) with plasmid vector pgex-4t-1 was used for the p proteins
expression .
preparations of va115 ( gi.3 ) vlps and p particles were
attempted based on the vp1 sequences ( genbank accession number ay038598 ) and
the established procedure described above , but the yields for both
particles were found to be very low .
the p dimers ( mw 67,712 da ) and
the gst - p fusion proteins of va115 were produced in high yield ( > 20
mg l bacteria ) through the same procedure as used
for the production of the p proteins of va387 . formations of the 24-mer
p particles , p dimers , and the gst - p polymers were analyzed by gel - filtration
chromatography via a superdex 200 size exclusion column ( ge healthcare
life sciences ) controlled by an akta fast performance liquid chromatography
system ( fplc , model 920 , ge healthcare life sciences ) .
a single
chain fragment ( scfv , mw 26,539 da ) of the monoclonal antibody se1554
was produced using recombinant technology as described elsewhere .
a recombinant fragment of the c - terminus of
human galectin-3 ( gal-3c , mw 16,330 da ) was generously provided by
prof . c. cairo ( university of alberta ) .
bovine ubiquitin ( ubq , mw
8565 da ) was purchased from sigma - aldrich canada ( oakville , canada ) .
the proteins were concentrated and exchanged into an aqueous 200 mm
ammonium acetate ( ph 7 ) using vivaspin 0.5 ml centrifugal filters
( sartorius stedim biotech , gttingen , germany ) with a mw cutoff
of 10 kda and stored at 80 c until use .
the structures of the oligosaccharides and
glycoconjugates used in this study are shown in figure s1 ( supporting information ) .
the 17 ganglioside and
globoside oligosaccharides ( gm3 , gm2 , gm1a , gm1b , gd3 , gd2 , gd1a ,
gd1b , gt3 , gt2 , gt1a , gt1c , fucosyl - gm1 , asialo - gm2 , asialo - gm1 , gb3
and gb4 ) were purchased from elicityl sa ( crolles , france ) .
h type
3 trisaccharide , a type 3 tetrasaccharide , and b type 3 tetrasaccharide
were a gift from prof .
each solid compound was dissolved in ultrafiltered
milli - q water ( millipore , ma ) to give a 1 mm stock solution .
polyacrylamide
( paa)-conjugated neu5ac , 6-sialylacnac , and gm3 trisaccharide
were purchased from vector lab ( burlingame , ca ) .
all of the esi - ms assays were carried
out on a synapt g2s quadrupole - ion mobility separation - time - of - flight
( q - ims - tof ) mass spectrometer ( waters , manchester , u.k . )
the car - esi - ms and direct esi - ms
assays were performed in negative ion mode , whereas the proxy protein
esi - ms assay was implemented in positive ion mode .
, 0.68 mm i.d . )
pulled to 5 m using a p1000 micropipette puller
( sutter instruments , novato , ca ) .
a platinum wire was inserted into
the nanoesi tip , and a capillary voltage was applied to carry out
esi .
the source parameters for both negative and positive ion modes
were : capillary voltage 0.8 kv ( negative ion mode ) or 1.0
kv ( positive ion mode ) , source temperature 60 c , cone voltage
60 v ( negative ion mode ) or 35 v ( positive ion mode ) , trap voltage
5 v , and transfer voltage 2 v. data acquisition and processing were
performed using masslynx software ( version 4.1 ) .
the car - esi - ms assay was performed
to identify carbohydrate ligands of the nov va387 p particle .
ions
corresponding to ligand - bound p particle were isolated using the quadrupole
mass filter .
the quadrupole was set to transmit a broad mass - to - charge
ratio ( m / z ) window ( approximately
200 m / z units ) , which allows for
the simultaneous passage of free and ligand - bound p particle complexes
at a given charge state . protein
ligand complexes were subjected
to collision - induced dissociation ( cid ) in the trap region of the
synapt g2s by increasing the trap voltage from 5 to 200 v. argon ( 1.42
10 mbar ) was used to carry out cid in the
trap region . in most instances , the deprotonated ligands released
from the complexes could be identified from their mws . where required ,
ims was used to separate the released isomeric ligands . for ims separation
a wave height of 35
v was used , and the wave velocity was ramped from
2000 to 500 m s. in all cases a helium flow rate
of 150 ml min and a nitrogen flow rate of 40 ml
min were used .
the arrival time distributions
( atds ) for the released ligands were compared to reference atds , which
were measured for the deprotonated carbohydrates produced directly
from solution . the direct esi - ms
assay was used
to quantify the affinities of the carbohydrate ligands for the nov
p dimers of va387 and va115 .
at least four different initial ligand
concentrations were used for each oligosaccharide tested , and the
binding measurements were carried out in triplicate .
a complete description
of the data analysis method employed to calculate the intrinsic association
constants ( ka , int ) can be found elsewhere .
briefly , the abundance ratio ( ri ) of the ligand - bound protein ( pli ) , bound to i molecules of l , to free protein ( p )
measured by esi - ms ( after correction for nonspecific ligand - protein
binding ) is taken to be equal to the equilibrium concentration ratio
in solution , eq 1:1 assuming
the protein has h independent and identical binding
sites , ka , int can be expressed by eq 2:2where [ p]0 and [ l]0 are
the initial concentrations of the protein and ligand , respectively ,
and f is the fraction of occupied binding sites ,
eq 3:3 in the case of the p dimer , which has two
equivalent binding sites , ka , int can be
found using eq 4:4 the proxy protein esi - ms
assay was used to quantify the affinities of gm3 trisaccharide for
nov va387 p particle and vlp . a complete description of the data analysis
method employed to calculate ka , int can
be found elsewhere .
briefly , a proxy
protein ( pproxy ) , which binds to l with a known affinity ,
is used to monitor the extent of l binding to p. specifically , in
the presence of p , the abundance ratio rproxy (= [ pproxyl]/[pproxy ] ) will quantitatively
reflect the concentration of l bound to p and ka , int can be evaluated using eq 5:5where the initial concentrations
of target
protein ( [ p]0 ) , proxy protein ( [ pproxy]0 ) and ligand ( [ l]0 ) as well as the association
constant for binding of pproxy to the ligand ( ka , pproxy ) are known ; [ p]m,0 is the
initial concentration of binding sites in the target protein , i.e. ,
[ p]m,0 = h [ p]0 .
paa - conjugated
neu5ac , 6-sialylacnac and gm3 trisaccharide were dissolved
in 1x pbs ( ph 7.4 ) .
they were diluted and coated on a 96-well microtiter
plate at concentration of 2 g ml and stored
at 4 c overnight . after blocking with 5% nonfat dry milk , nov
vlp ,
p particle , or gst - p domain fusion proteins as well as gst ( negative
control ) at 50 ng l were added and incubated
for 2 h at 37 c .
the ligand - bound nov vlp and p proteins were
detected by homemade guinea pig hyperimmune serum against va387 vlp
and va115 p protein ( 1:3000 ) , respectively , followed by horseradish
peroxidase ( hrp)-conjugated goat antiguinea pig immunoglobulin g ( igg ,
1:3000 ; icn , aurora , oh ) .
the signals were displayed using a tmb kit ( thermo fisher
scientific , rockford , il ) .
evidence
of ganglioside binding to novs was initially revealed through the
screening of a small ( 20 components ) carbohydrate library against
the p particle ( 24-mer , mw 865,036 da ) of nov va387 ( gii.4 ) using
the car - esi - ms assay .
the library consisted
of the oligosaccharides of 17 glycosphingolipids , gm1a , gm1b , gm2 ,
gm3 , gd1a , gd1b , gd2 , gd3 , gt1a , gt1c , gt2 , gt3 , fucosyl - gm1 ( referred
to as fuc - gm1 ) , asialo gm1 , asialo gm2 , gb3 and gb4 as well as three
known hbga oligosaccharide ligands , h type 3 trisaccharide ( referred
to as h3 ) , a type 3 tetrasaccharide ( a3 ) , and b type 3 tetrasaccharide
( b3 ) .
the intrinsic affinities of the hbga ligands range from 700
to 1500 m. the car - esi - ms
assay was carried out by first incubating the p particle with the
carbohydrate library , followed by direct esi - ms analysis of the mixture .
because of the high mw of the p particle , the identity of the bound
ligands could not be established directly from the mass spectrum .
instead , using a quadrupole mass filter set to pass a range of mass - to - charge - ratio
( m / z ) ions , all of the ligand - bound
p particle ions at a given charge state were isolated and then activated
( heated ) using cid to release the ligands ( as ions ) from the complex .
given that carbohydrates have relatively low gas - phase acidities and
are able to effectively compete with proteins for negative charge ,
accurate mass analysis , alone or in combination
with ion mobility separation ( ims ) , which separates ions based on
size and shape , allowed for positive ligand identification . shown in figure 1a is a representative esi
mass spectrum acquired in negative ion mode for an aqueous ammonium
acetate ( 200 mm , ph 7 , 25 c ) solution of p particle ( 3 m )
and the carbohydrate library ( 10 m each ) . from the mass spectrum
it can be seen that the p particle exists predominately as a 24-mer ,
with a charge state distribution ranging from 60 to 65 .
signal corresponding to an 18-mer is also present , although at lower
abundance , with a charge state distribution of 51 to 54 .
due to the high mw of the p particle and the formation of adducts
during the esi process
, it was impossible to resolve the ions corresponding
to free p particle and its complexes with one or more oligosaccharide
ligands .
however , cid , performed using a 200 m / z wide isolation window centered at 14,350 to pass ions
corresponding to the 61 charge state of the p particle , led
to the appearance of singly deprotonated ions of the three hbga oligosaccharides
as well as gm3 ( m / z 632.2 ) , gm2
( m / z 835.3 ) , gd3 ( m / z 923.3 ) , gm1a and/or gm1b ( m / z 997.3 ) , and fuc - gm1 ( m / z 1143.4 ) ( figure 1b ) .
ions corresponding to
the singly deprotonated gd2 ( m / z 1126.4 ) and the doubly deprotonated ions of gd1a and/or gd1b ( m / z 644.1 ) were also detected , although
at low abundance ( figure 1b ) .
abundant multiply
charged protein monomer ions , pm at n = 1023 , were also evident
( figure 1b ) .
implementation of the car - esi - ms
assay using other charge states of the p particle complexes produced
similar results ( figure s2 , supporting information ) .
ion mobility separation of the released ligands revealed evidence
that both gm1a and gm1b are released from the p particle , with gm1a
being more abundant ( figure s3a , supporting information ) .
the doubly deprotonated ions of gd1a and gd1b could not be differentiated
using optimized ims conditions ( figure s3b , supporting
information ) , and therefore , it was not possible to establish
whether one or both oligosaccharides bind to the p particle directly
from these measurements . instead
, the car - esi - ms assay was applied
to solutions containing p particle ( 3 m ) and 10 m of
gd1a or gd1b .
these data revealed that only gd1a binds to the p particle
under these solution conditions ( figure s4 , supporting
information ) .
the car - esi - ms results provide compelling evidence
that the p particle of va387 exhibits a broad specificity for mono-
and disialylated gangliosides .
however , there is a clear preference
for gm3 , and the addition of saccharides to gal ( e.g. , gm1 or gm2 )
or sia ( e.g. , gd3 , gd2 or gd1b ) decreases binding , compared to gm3 .
these data , combined with affinities measured for ganglioside oligosaccharides , vide infra , suggest that the sia - gal - glc moiety represents
the dominant recognition epitope for this nov .
( a ) esi mass spectrum
acquired in negative ion mode for an aqueous
ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va387
p particle ( 3 m ) and a 20-component ( 10 m each ) carbohydrate
library consisting of the oligosaccharides of gm1a , gm1b , gm2 , gm3 ,
gd1a , gd1b , gd2 , gd3 , gt1a , gt1c , gt2 , gt3 , fuc - gm1 , asialo gm1 , asialo
gm2 , gb3 , and gb4 as well as the h3 , b3 , and a3 oligosaccharides .
( b ) cid mass spectrum measured for the 61 charge state of
the free and ligand - bound p particle . based
on the relative abundances of the released oligosaccharide
ligands measured by car - esi - ms ( figure 1 )
it
would appear that the affinities of the ganglioside ligands are similar
to those of the highest affinity hbga oligosaccharides .
however , this conclusion is predicated on the
assumption that the release efficiency of the bound - ligands is essentially
independent of structure . because of the presence of the sialic acid ,
it is possible that gangliosides ( which are likely deprotonated in
the gaseous complexes ) are preferentially released from the p particle
due to a lower activation energy resulting from coulombic repulsion .
therefore , it was important to measure the affinities directly .
in order to do this ,
the affinities were measured using the direct esi - ms assay , which
has been shown to provide reliable ka values
for many protein carbohydrate interactions .
affinities were measured for the oligosaccharides of 13
gangliosides ( gm3 , gm2 , gm1a , gm1b , gd3 , gd2 , gd1a , gd1b , gt3 , gt2 ,
gt1a , gt1c , and fuc - gm1 ) for the va387 p dimer ( mw 69,312 da ) .
a reference
protein ( pref ) was used in all cases to correct the mass
spectra for the occurrence of nonspecific carbohydrate
a representative
esi mass spectrum acquired for an aqueous ammonium acetate solution
( 200 mm , ph 7 , 25 c ) of va387 p dimer ( 12 m ) and gm3
trisaccharide ( 80 m ) is shown in figure 2a as well as the distribution of ligand - bound p dimer after correction
for nonspecific binding . from the esi - ms data , ka , int values were calculated for each oligosaccharide ( table 1 ) .
affinities were also measured for a3 , b3 , and
h3 and shown to agree well with the reported values ( table s1 , supporting information ) .
inspection of the ka , int values reveals
that , of the tested gangliosides , gm3 exhibits the highest affinity
for the va387 p dimer , which is consistent with the results of the
car esi - ms measurements , vide supra .
moreover , the ka , int ( 1500 m ) is identical ,
within experimental error , to that of b3 ( 1500 150 m ) . of the 12 other gangliosides investigated ,
nine
bind weakly ( ka , int < 500 m ) and three ( gd1b , gt3 and gt1a ) do not show any detectable
binding .
notably , the quantitative binding data obtained for the p
dimer agree qualitatively with the relative affinities inferred from
the car - esi - ms measurements performed on the p particle . moreover ,
all ligands with affinities > 100 m were detected
in the car - esi - ms measurements ( table s2 , supporting
information ) .
( a ) esi mass spectrum acquired in negative ion mode for
aqueous
ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va387
p dimer ( p2 , 12 m ) , gm3 trisaccharide ( 80 m )
and pref ( 4 m ) . another minor form of p dimer ( p2 , mw 74,080 da ) was also detected with lower abundance .
inset ,
normalized distribution of gm3 bound to p2 after correction
for nonspecific ligand binding .
( b ) esi mass spectrum acquired in
negative ion mode for aqueous ammonium acetate solution ( 200 mm , ph
7 and 25 c ) of nov va115 p dimer ( p2 , 12 m ,
mw 67,712 da ) , gm3 trisaccharide ( 80 m ) , and pref ( 4 m ) .
inset , normalized distribution of gm3 bound to p2 after correction for nonspecific ligand binding .
nb = no binding
detected . to demonstrate
the relevance of the affinity data acquired for
the p dimer
, affinity measurements were also carried out for gm3 trisaccharide
binding to the va387 p particle and vlp ( 180-mer , mw 10.5
mda ) .
an adaptation of the proxy protein esi - ms method , which combines
direct esi - ms binding measurements and competitive protein binding ,
was used to evaluate the affinities . a
recombinant fragment of the c - terminus of human galectin-3 ( gal-3c ,
mw 16,330 da ) , which contains a carbohydrate recognition domain and
interacts with a -galactoside moiety , served as the proxy protein ( pproxy ) .
importantly , gal-3c
binds to gm3 trisaccharide with an affinity of ( 1.20 0.02 )
10 m. the extent of binding
of gm3 trisaccharide to gal-3c , as determined by esi - ms , in the presence
of known concentrations of the target protein ( p particle or vlp )
allowed for a quantitative measure of gm3 binding to the target .
esi - ms measurements were performed on aqueous ammonium acetate
solutions ( 160 mm , ph 7 and 25 c ) of pproxy ( 3.0
m ) , pref ( 1.0 m ) , gm3 trisaccharide ( 40 m ) ,
and either p particle , at concentrations ranging from 0 to 7.2 m
( corresponds to monomer concentration of 0172.8 m ) ,
or vlp , at concentrations ranging from 0 to 570 nm ( monomer concentration
of 0102.6 m ) .
representative esi mass spectra acquired
in positive ion mode in the absence and presence of nov vlp ( 570 nm )
are shown in figure 3a and 3b , respectively .
the distributions of ligand - bound pproxy , following correction for nonspecific ligand binding , are also given .
inspection of the distributions reveals a measurable decrease in the
extent of gm3 trisaccharide binding to gal-3c upon addition of vlp .
the
dependence of the extent of gm3 trisaccharide binding to pproxy on vlp concentration is shown in figure 3c .
binding measurements performed on solutions containing p particle
yielded qualitatively similar results ( figure s5 , supporting information ) .
analysis of the pproxy binding data acquired in the presence of vlp or p particle using
the procedure outlined in experimental section yields gm3 affinities of 2600 200 and 5500 600 m for the p particle and vlp , respectively .
the slight
differences in the magnitude of the affinities measured for the binding
of a common carbohydrate ligand to the p dimer , p particle , and vlp
of a nov ( the first such data set to be reported ) , likely reflect
subtle differences in the structure of the carbohydrate binding site
presented by these related protein complexes .
these differences notwhithstanding , the present results suggest
that the p dimer can serve as a surrogate of the vlp for carbohydrate
binding studies .
representative esi mass spectra measured in positive ion
mode for
aqueous ammonium acetate solutions ( 160 mm , ph 7 and 25 c ) of
pproxy ( gal-3c , 3.0 m ) , pref ( ubq , 1.0
m ) , and gm3 trisaccharide ( 40 m ) without ( a ) or with
( b ) nov va387 vlp ( 570 nm , 180-mer ) .
insets show the fraction of free
and gm3-bound pproxy , after correction for nonspecific
ligand binding .
( c ) plot of the abundance ratio of gm3-bound pproxy to free pproxy ( rproxy ) versus vlp concentration .
the solution conditions for each measurement
were the same as in ( a ) , but with the addition of vlp .
it has been proposed that nov
va387 has a binding interface that
recognizes hbgas through the -l - fuc epitope as the major binding
interaction and either the -d - galnac or -d - gal epitope
as a minor binding interaction .
however , these core recognition elements are missing in the ganglioside
ligands identified in the present study .
therefore , it is of interest
to establish whether the ganglioside ligands interact with the nov
through the hbga binding site or through a distinct ganglioside binding
site .
it is not possible to answer this question through competitive
binding measurements carried out using a ganglioside oligosaccharide
( e.g. , gm3 trisaccharide ) and va387 p dimer in the presence of varying
concentrations of a hbga oligosaccharide ligand due to the low affinities
of these ligands . instead
, future efforts will rely on x - ray crystallography
to establish whether va387 nov has distinct binding sites for hbga
and ganglioside ligands .
the aforementioned
binding data reveal that nov va387 binds to mono- and disialylated
gangliosides , with affinities comparable to those of the highest affinity
hbga oligosaccharide ligands . to demonstrate that this is not an isolated
example of a human nov that recognizes gangliosides , the affinities
of the 13 ganglioside oligosaccharides for the p dimer of nov va115
( gi.3 genotype ) , which does not bind to human hbgas , were also measured ( table 1 ) .
a
representative esi mass spectrum acquired for an aqueous ammonium
acetate solution ( 200 mm , ph 7 , 25 c ) of nov va115 p dimer ( 12
m ) and gm3 trisaccharide ( 80 m ) is shown in figure 2b as well as the distribution of ligand - bound p
dimer after correction for nonspecific binding .
notably , the va115
p dimer binds to all 13 oligosaccharides tested , and overall , the
affinities are slightly higher than those for va387 .
additional evidence for the recognition of sialic
acid by human novs comes from elisa measurements carried out on the
va387 vlp , p particle , and gst - p fusion protein as well as va115 gst - p fusion protein , with paa - conjugated neu5ac ,
6-sialylacnac , and gm3 trisaccharide . as shown in figure 4 ,
it is curious that the va387 vlp
exhibited weaker binding than that of the p particle to the three
glycoconjugates and the cause of the weaker binding is , at this time ,
unknown .
nevertheless , the fact that all three assemblies of nov capsid
protein exhibit a similar binding pattern to the three glycoconjugates
( gm3 > 6-sialylacnac > neu5ac ) validate their applications
as models for nov ligand interaction . moreover , comparing the
binding of gst - p fusion protein of va387 to that of va115 indicates
that the sialic acid - containing glycoconjugates have slightly higher
affinities for va115 , consistent with the esi - ms data .
these results ,
together with those from esi - ms , suggest both -(2,3)- and -(2,6)-
linked sialic acids as critical motifs in va387 and va115 binding ,
similar to what has been reported for mnv1 and psav .
it is important to point out that , although
sialic acid - containing oligosaccharides have been identified as receptors
for an animal nov ( mnv1 ) and two other animal
cvs ( fcv and psav ) , human novs generally recognize
gangliosides in addition to hbgas . furthermore , human novs differ
greatly from mnvs in many other important aspects , including host
tropism ( human vs mouse ) , clinical manifestation ( with vs without
diarrhea / vomiting ) , and pathogenesis . binding
of nov vlp , p particles , and gst - p fusion protein of va387
as well as gst - p fusion protein of va115 to paa - conjugated gm3 trisaccharide ,
6-sialylacnac , and neu5ac in 1x pbs ( ph 7.4 ) .
gst , which does
not show binding to any of the three glycoconjugates , served as a
negative control .
taken
together , the results of esi - ms and elisa measurements performed
on two human novs representing two different genogroups ( gi and gii )
provide the first experimental evidence of interactions between human
novs and gangliosides and sialic acid - containing glycoconjugates .
notably , the affinities measured for the oligosaccharides of the ganglioside
ligands by esi - ms are comparable in magnitude to those reported for
the oligosaccharides of known hbga receptors .
these experimental data
demonstrate sialic acid - containing oligosaccharides as alternative
( to hbgas ) ligands for human novs and suggest a new mechanism of human
nov
host interaction , one that involves hbga and sialic acid - containing
oligosaccharide receptors and co - receptors for attachment and penetration
into host cells and opens a new direction in human nov research .
further
studies to characterize the role of cell surface sialic acids / gangliosides
in the early stage of viral infection and its potential coordination
with hbgas for viral attachment and/or entry are needed . | human
noroviruses ( novs ) are known to recognize histo - blood group
antigens ( hbgas ) as attachment factors .
we report the first experimental
evidence that sialic acid - containing glycosphingolipids ( gangliosides )
are also ligands for human novs .
electrospray ionization mass spectrometry - based
carbohydrate binding measurements performed on assemblies ( p dimer ,
p particle , and virus - like particle ) of recombinant viral capsid proteins
of two nov strains , va387 ( gii.4 ) and va115 ( gi.3 ) , identified binding
to the oligosaccharides of mono- , di- , and trisialylated gangliosides .
the intrinsic ( per binding site ) affinities measured for these ligands
are similar in magnitude ( 102103 m1 ) to those of human hbgas . binding of nov vlps , p
particles , and glutathione s - transferase ( gst)-p domain fusion proteins
to sialic acid - containing glycoconjugates , observed in enzyme - linked
immunosorbent assays , provided additional confirmation of the nov ganglioside
interactions . | [
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hypertension is one of the most common if not the commonest medical complication in pregnancy.19 hyper - tensive disorders in pregnancy are found to be the greatest single cause of maternal mortality.911 it also causes a lot of prenatal mortality.12 most of the complications caused by this problem could be reduced by early detection and proper management.1314 studies on the incidence of hypertensive disease in pregnancy in most developing countries , including saudi arabia , are scarce.15 few of those studies , if any , have looked at the extent of knowledge , attitude or practice of doctors or nurses dealing with hypertensive pregnant mothers .
hence , the objective of this study was to determine the status of knowledge , attitude and practice ( kap ) of doctors and nurses in phc centers with regard to hypertension in pregnancy and to analyze factors affecting kap in the al - khobar area of the eastern province of the kingdom of saudi arabia .
terms used to describe hypertension and its complications in pregnancy differ according to its presentation , gestational age of discovery and the presence of previous history of hypertension before pregnancy .
the one defined by the 1972 committee on terminology of the american obstetricians and gynecologists ( acog)131519 was adopted for the purpose of this kap study .
doctors and female nurses working at all 8 phc centers in al - khobar area were enrolled for the study .
two self - administered questionnaires structured to examine the essential knowledge , attitude , and practice of doctors and nurses in dealing with hypertensive disorders during pregnancy .
the first was intended to gather demographic characteristics and information on the respondent that might affect the kap .
these variables included age , sex , nationality , year of graduation , certification , training and experience in obstetrics , duration of work in phc in the country and any in - service training received .
the second part of the questionnaire consisted of 49 questions divided into three sections dealing with practice , attitude and knowledge .
the practice sections included questions designed to test competence of doctors and nurses in the skills of measuring blood pressure , management of the hypertensive pregnant mother and practices of health education .
the knowledge section was composed of 28 questions to test basic medical knowledge regarding blood pressure reading , management of the problem and its complications .
both questionnaires were given to three consultant obstetricians and two nurse educators for consent validity and elimination of non - essential questions .
both knowledge and practice areas were scored on a zero and one additive scoring system in which each correct answer or practice was given one score and no mark given for a wrong response .
the attitude part included questions that measured attitudes of participants towards the seriousness and commonness of hypertension in pregnancy and their motivation to improve their knowledge on the subject .
attitude questions were scored using the five - point likert scaling system . in this scale ,
a high attitude was assigned to the answer if the respondent 's answer was to agree or
strongly agree to the question in the scale and a low attitude level for not sure or
final scoring of the knowledge and practice section was satisfactory if the participant scored at least 60% of the total marks in these two sections .
this was an arbitrary cut - off point based on the pilot study and judgment of the experts . on attitude
, the questionnaire was handed over to five postgraduate doctors in general practice and their responses to attitude questions were used as a reference standard . the questionnaire for nurses
both versions of the questionnaire were pilot tested on a pilot group of 10 doctors and 22 nurses , selected randomly from phc centers in a nearby city , al - dammam to assess level of difficulty , clarity , suitability and time required for their completion .
data were fed into a personal computer , cleaned and analyzed using epi info and spss statistical packages .
frequency distribution tables were constructed and appropriate tests including the multiple regression analysis to identify significant independent factors were applied .
the total number of phc doctors in al - khobar area was 44 . among those 36 ( 81.4% ) who were present at time of data collection
their ages ranged between 25 - 47 with mean of 37.1 years ( sd 6.4 ) and 56% were in the 35 - 44 years group .
arab nationals other than saudis including egyptians , palestinian , and jordanian formed the majority of doctors ( 41.7% ) , while saudis constituted 22.2% of the group and the rest were from the indian subcontinent . the mean number of years in practice after graduation was 12.6 years ( sd 7.38 ) .
distribution of phc doctors according to years of practice after graduation the mean number of years working in phc was 4.7 years ( sd 3.4 ) and 17 doctors ( 47.2% ) had worked for over 5 years in phc centers . only , 4 doctors ( 11.1% ) had postgraduate qualifications ; out of these only one held a diploma in obstetrics .
five doctors ( 13.9% ) had had 3 - 6 months post - internship training in obstetrics but no certification .
of the entire group , only 2 doctors ( 5.6% ) had had courses in hypertensive disorders in pregnancy in the course of their work . in describing their general attitude towards managing hypertension in pregnancy ,
a majority of doctors ( 80% ) stated clearly that they did not feel confident in managing hypertensive pregnant mothers and would prefer to see children or adult male patients in their clinic . nevertheless , the attitude of 34 doctors ( 94.4% ) toward learning more about hypertension in pregnancy was positive .
out of the maximum attitude score of 45 marks , the mean score for the attitude questions for the group reached a reasonably high figure of 30.69 marks .
doctors attitudes towards hypertension in pregnancy doctors management of hypertension in pregnancy was quite deficient as 16 doctors ( 44.4% ) reported that they had not actually seen any hypertensive pregnant mother during last year .
around 53% of the doctors depended on bp measurement taken by a competent staff and did not check it .
the doctors scores for practice was generally low , as only 60% of them got the correct answers to the skill of blood pressure measurement , a basic skill necessary for any medical graduate .
in addition , almost all doctors wrongly reported that they had advised their pregnant hypertensive patients to lose weight and reduce their salt intake .
another major mistake noted among 25% of the doctors was that they said they would start treatment of hypertensive cases immediately after diagnosis in their clinic without considering referral .
the scores on practice of 75% of the doctors were below the satisfactory cut - of point .
again the doctors score of 58.3% on all areas of knowledge was unsatisfactory since this directly affected the diagnosis and quality of care for hypertensive mothers .
some of the responses dealt with normal and abnormal readings of blood pressure and the presenting symptoms of preeclampsia .
doctors mean score 12.1 marks out of a total score of 21 on knowledge was moderate .
the total number of phc nurses in al - khobar area was 120 . among these ,
the 91 ( 75.8% ) who were present at the time of data collection answered the questionnaire .
their mean age was 29.8 years ( sd 6.3 ) ranging between 20 - 44 years , 74% of them fell within the 20 - 34 age group .
indians formed the second largest group ( 37.4% ) followed by filipinos ( 11% ) . according to their qualification , 14 nurses ( 15.4% ) held bachelor degrees and 77 ( 84.6% ) had diplomas .
mean years of practice after registration was 8.7 ( sd 5.4 ) and their distribution is shown in table 3 .
mean duration of work in phc was 4.1 years ( sd 2.8 ) and most nurses ( 69.2% ) had spent < 5 years working in phc in the country .
distribution of phc nurses according to years of practice after registration thirty - seven nurses ( 40.7% ) had spent < 6 months in obstetrics training and 10 nurses ( 11% ) had spent > 1 year .
most of the group ( 48.3% ) , mainly the saudis , had minimal training of 3 weeks in obstetrics ward during their internship period as required by the saudi female health institution .
a majority of the nurses 73 ( 80.2% ) had not worked in a phc setting before their current positions . out of those who had had previous phc experience ,
nine of them had had courses in hypertensive disorders in pregnancy during their work in phc . in describing their general attitude towards hypertension in pregnancy
, 86% of them reported that it was a common health problem and they were all positive about learning more .
they were all keen to talk to pregnant women about their problem and advice them to take bed rest .
out of the maximum attitude score of 35 marks , the highest mean score was 30.02 marks . table 4 shows some of the attitude questions and their responses . nurses attitude towards hypertension in pregnancy
the nurses were quite good on practice in the area of hypertension in pregnancy , as 95.6% of them scored satisfactory .
the mean score of this section on practice was quite high for nurses being 8.3 marks out of a total score of 12 marks .
the nurses level of knowledge was very low since only five of them ( 5.5% ) scored satisfactory on the questions in this section .
the deficiencies were in areas dealing with diagnosis and quality of care for hypertensive mothers .
most of them ( 96.7% ) wrongly considered salt restriction and weight reduction as important in the health education of hypertensive pregnant women .
mean scores on knowledge was 9.02 marks out of a total score of 20 marks which was quite low . in the regression analysis of independent factors associated with kap of doctors and nurses , all variables in the first part of the questionnaire such as age , nationality and certification
were included in a series of multiple regression equations against each of the dependent variables , namely , scores on knowledge , attitude , and practice for doctors and nurses .
there was a positive significant association of the doctors attitude with their sex i.e. , male , nationality , being older and previous enrollment in a course on hypertension ( r square = 0.26 p=0.02 ) .
their knowledge was only associated with previous training in obstetrics ( r square = 0.14 , p=0.02 ) , while practices of both doctors and nurses were not associated with any of the factors under study .
the attitude of nurses , on the other hand , showed different associations as non - saudi nationals and training in obstetrics had a positive significant effect on their attitude ( r square = 0.08 , p=0.02 ) .
their knowledge was only associated significantly with being non - saudi ( r square = 0.19 , p=0.001 ) .
hypertension in pregnancy is one of the major causes of prenatal morality and morbidity.20 it is responsible for about 18% of maternal mortality,102122 maternal mortality in hypertensive disorders of pregnancy is primarily due to low standard of care and delay in referral.14 antenatal care ( anc ) is a major part of maternal and child services in phc .
one of the most important functions of anc is to detect high - risk pregnancies and to give them the necessary care .
findings from this study will help to identify the status kap of workers in phc on hypertensive disorders in pregnancy .
it was discovered in this study that the definition of raised blood pressure and its management was not standardized among doctors and nurses in phc .
this is not surprising as previous study by bisson in bristol23 in which a large group of general practitioners , hospital doctors , hospital midwives , community midwives and student midwives who were questioned , gave variety of action plans according to their understanding of diagnostic criteria .
they considered the reading of 90 mmhg the model value of diastolic bp at which further action would be taken , whether proteinuria was present or absent .
edema was considered a useful indicator by 93% of the respondents and 49% would use ankle edema in their assessment .
another questionnaire - based survey by hutton24 on the management of hypertension in pregnancy completed by 65 new zealand obstetricians found that 40 ( 61.5% ) doctors considered the diastolic of 80 - 85 mmhg , the lowest abnormal reading , at 28 weeks , and by 18 ( 27.7% ) doctors at 36-week gestation . however , 20 ( 30.8% ) doctors considered the diastolic of 90 mmhg , at 28 weeks and 42 ( 64.6% ) doctors at 36 weeks gestation , the lowest abnormal reading . around 47% of phc doctors
had spent more than 5 years in phc service in ksa and only 14% of them had had post - internship training in obstetrics but being males had not been involved in anc activity in phc . only 2 had had courses in hypertensive disorders of pregnancy .
about 44% of the doctors had not seen any cases of hypertensive pregnant mothers during the last year .
questions asked were concerned with four aspects namely , techniques of blood pressure measurements , history and physical examination , health education practice and action management to be taken by the phc doctors on discovered cases .
it is obvious that having such little contact with hypertensive cases and lacking the basic training or refresher courses , their management skills will be inadequate .
since hypertension is one of the commonest medical complications in pregnancy , it is important to educate the pregnant mother about it .
almost all doctors report that they advise hypertensive mothers on the need for bed rest .
this is an important non - pharmacological measure in the management of the problem.25 on the other hand , almost all doctors report that they advise their patients to restrict their salt intake and to go on a weight - reducing diet .
salt restriction in hypertensive non - pregnant women can be effective but in pregnancy may aggravate the condition .
moreover , diet restriction in pregnancy can lead to delivery of small - for - date fetus.25 similar findings of wrong advice were reported in other studies by trudinger26 and bisson.23 around 85% of phc doctors reported that they would not refer hypertensive cases after diagnosis and 25% of them would start medication immediately after diagnosis .
in fact , the cornerstone of management of hypertensive cases starts in the phc center with accurate diagnosis and undelayed referral of these cases from the phc to the specialist or hospital.1621 phc doctors should be aware of their limitation in the management of these cases and not to jeopardize the health of the mother and her fetus .
around 31% did not recognize this is a common health problem in pregnancy , while 97% of them stressed the importance of taking blood pressure reading at each anc visit .
that 53% of phc doctors depended on blood pressure measurement taken by clinical staff without confirming it , the reading themselves is an alarming negative attitude as well as wrong practice .
the diagnosis of hypertension in general or in pregnancy in any patient is not an easy task and proper management and modifications depend on it .
it is therefore , important that the person responsible for the management , namely , the doctor , should confirm the reading again . in looking for factors affecting the attitude of doctors
, it was found that gender i.e. , male , arab , older and previous training in a course on hypertension were factors associated with higher scores . longer experience in phc work , being male and older and the absence of a language barrier for arabic speakers , including saudis , might explain to some extent their better attitude scores as compared to those of the females and younger doctors .
the knowledge of about 58% of doctors on questions that bare on the diagnosis and quality of care of hypertensive mothers as well as
nurses provide most of the vital anc services in the phc . around 53% of the phc nurses
were non - saudis and non - arabic speakers and the language barrier adversely affects the health education in hypertensive cases .
it is obvious from the results that there is a shortage of specialized courses for nurses .
none of the saudi nurses had more than a diploma in nursing and there did nt seem to be any arrangements for their further movement at universities .
a period of 3 weeks in an obstetrics ward for new graduate nurses as part of an internship period is not enough to give clear , pragmatic information on anc services in a phc setting .
about 31% of the nurses have spent > 5 years in the phc centers in the ksa , but only 10% had taken courses in hypertension .
this clearly , demonstrates the necessity of a better arrangement for on - job training classes for them .
their responses to the question of history and physical examinations were 87% correct and about 90% of them responded correctly to the questions on health education .
their erroneous responses were on the items on restriction on salt intake and weight reduction .
these good scores indicate that except for their wrong ideas on salt and diet restriction , which should be corrected at refresher courses , the correct procedure have been learnt . although the phc nurses were very good with practice in general , around 95% of them had poor knowledge .
knowledge and facts , on the other hand , need to be updated by continuous education .
around 86% of them felt that it was a common health problem and reported that they needed to know more about it .
the importance of a health education as a vital task for nurses can not be ignored .
the presence of these cases in the clinic , therefore , provides a good opportunity for the performance of this task .
there was a strong positive association of attitude of nurses to non - saudi nationality and training in obstetrics .
while all saudi nurses hold diplomas , about 30% of the non - saudi nurses have bachelors degrees .
there is a masked difference in the type , duration and content of curriculum at the pre - graduate level for both groups and non - saudi nurses had higher knowledge scores than saudis .
the implication of these findings clearly points out the need to improve the knowledge and attitude of phc nurses through refresher courses .
good in attitude questions but had low scores on the practice and knowledge component of the questionnaire .
the nurses scored high on practice and attitude but had low scores on the knowledge component .
it is recommended that appropriate regular refresher courses on common and serious problems like hypertension be organized for doctors and nurses in the phcs .
there should be opportunities for effective training of reasonable duration with clearly defined objectives under proper supervision in good hospitals to improve their knowledge and practice
. it would be also appropriate to offer saudi doctors extra incentives for postgraduate study in family medicine to deal with these common problems , and to institute a suitable program of continuing medical education within the health centers for both doctors and nurses .
it is also vital to review the curriculum of the female nursing institutions to update both its theoretical and practical content , and extend the duration of training in such common problems as hypertension in obstetrics . | objective : to assess the status of knowledge , attitude and practice ( kap ) of doctors and nurses in primary health care ( phc ) centers with regard to hypertension in pregnancy and to identify factors associated with kap in al - khobar , saudi arabia.methodology:using a self - administered comprehensive questionnaire , all available doctors and nurses in phc centers of the al - khobar area were approached to determine their knowledge , attitude and practice in hypertension during pregnancy .
questionnaires were validated and pilot tested .
each section of the questionnaire was scored and the mean scores calculated .
factors affecting each section were identified by means of multiple regression analysis.results:a total of 36 doctors and 91 nurses were enrolled in the study .
saudis formed 22.2% of the doctors and 47.3% of nurses .
mean years of practice after graduation were 12.6 and 8.7 years for doctors and nurses , respectively .
saudi nurses spend only 3 weeks in the obstetrics ward during the whole period of their internship .
all saudi nurses hold only diplomas and not many courses on the hypertensive disorder are offered to both doctors and nurses after graduation .
the practice of doctors particularly in the management of patients after reaching a diagnosis and educating them on diet and salt intake was poor .
furthermore , their knowledge was also poor . though their level of knowledge was poor , the nurses practice was satisfactory , particularly in taking history and physical examination
.
the attitude of both doctors and nurses towards hypertensive disorders was in general , positive and satisfactory towards health education .
nurses nationality and duration of post - internship training were the factors that influenced their attitude and scores on knowledge.conclusion and recommendation : the study revealed that both doctors and nurses working in the phc lacked training and knowledge in this area of their work .
it is therefore necessary to give phc doctors and nurses refresher courses on common and serious problems like hypertension . a longer period of training in action management is needed to improve the knowledge and practice of doctors and nurses working in antenatal clinics in this area . | [
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renal cell carcinoma ( rcc ) is the most common primary malignant neoplasm of the kidney and accounts for about 3% of all adult neoplasms .
the incidence of rcc has been increasing during the past two decades in all age groups .
the greatest increase has been in patients with localized tumors as a result of incidental detection , which is thought to be the result of more widespread use of abdominal ultrasound and computed tomography . however , about 30% of patients with rcc already have metastatic disease at the time of diagnosis , and 10% to 14% of patients experience local recurrence or distant metastasis after nephrectomy despite a finding of pathologically confined disease at the time of nephrectomy [ 3 - 5 ] .
well - known pathological and clinical variables have been identified and sometimes integrated into nomograms to better predict disease recurrence , but with only partial success . with a better understanding of the biology of rcc
, laboratory values and molecular markers are increasingly being investigated as potential predictive factors for rcc [ 6 - 8 ] .
disease progression appears to depend on a complex interaction between the tumor and host inflammatory responses .
it is estimated that underlying infection and inflammatory responses are linked to 15% to 20% of all deaths from cancer worldwide .
the serum level of c - reactive protein ( crp ) , a nonspecific inflammatory acute - phase protein , is frequently increased in patients with metastatic rcc and is a predictor of prognosis .
however , secondary thrombocytosis has been reported with many malignant diseases ; the survival of such patients has been reported to be significantly shorter . the prognostic value of the platelet count has been studied in patients with localized and metastatic rcc ; patients with thrombocytosis had a poorer survival than did patients with a normal platelet count . the purpose of this study was to investigate the association of preoperative crp elevation and thrombocytosis with the prognosis of patients with non - metastatic rcc .
the medical records of 177 patients with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet levels were available for analysis were retrospectively reviewed .
there were 130 men and 43 women , with a mean age of 53.5 years ( range , 28 - 83 years ) .
preoperatively , all patients were evaluated with a physical examination , routine hematology and biochemical analysis , and radiology studies , including abdominal computed tomography and chest x - ray .
the tumors were staged by using the 2002 tnm classification of the american joint committee on cancer ( ajcc ) and were graded according to fuhrman 's nuclear grading system .
all patients were evaluated postoperatively every 3 months for the first 2 years , every 6 months for the next 2 years , and yearly thereafter .
crp elevation was defined as a crp level 0.8 mg / dl , and thrombocytosis was defined as a platelet count 380,000/l in men and 370,000/l in women according to the normal reference range in our hospital .
disease recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis .
the chi - square test or fisher 's exact test was used to analyze the correlation between preoperative crp elevation or thrombocytosis and the clinical and pathological variables , including age , gender , anemia , histology subtype , tumor size , t stage , nuclear grade , and metastasis during follow - up .
univariate and multivariate analyses were performed by using the log - rank test or the cox proportional hazards regression model . for all tests ,
the clinical and pathological characteristics of the 177 patients with non - metastatic rcc are summarized in table 1 .
the mean tumor size was 5.12 cm ( median , 4.50 ; range , 1 - 22 cm ) .
regional lymph node dissection was performed in 24 patients who showed suspicious lymph node metastasis on computed tomography . however , none had a pathological diagnosis of lymph node metastasis .
there were 38 patients ( 21.5% ) with crp elevations and 11 patients ( 6.2% ) with thrombocytosis . among patients with crp elevation ,
the mean follow - up period was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) .
twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period .
preoperative crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) , but not with age , gender , tumor histology subtype , or tumor size ( table 2 ) .
preoperative thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) , but not with age , gender , tumor histology subtype , tumor size , or grade ( table 3 ) .
no significant correlation was found between preoperative crp levels and platelet counts when analyzed by using pearson 's correlation coefficient ( r=0.132 , p=0.079 ) .
kaplan - meier recurrence - free survival curves according to crp level showed that the survival rate of patients with crp elevation ( crp0.8 mg / dl ) was significantly lower than that of patients with normal crp levels ( p<0.001 ) ( fig .
the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors for recurrence - free survival , whereas the multivariate analysis showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors ( table 4 ) .
crp is an acute phase reactant that is produced exclusively by hepatocytes in response to cytokines such as interleukin ( il)-6 , which are known mediators of ongoing inflammatory processes .
an elevation of the serum concentration of this protein indicates the presence of acute inflammation , which is observed 8 to 12 hours after the onset of infection . the relationship between cancer and crp elevation
hepatic production of crp is induced by cytokines such as il-1 , tumor necrosis factor ( tnf ) , and primarily il-6 , which is frequently overproduced by the tumor cells themselves .
experimental studies on rcc cell lines and expression studies of renal surgical specimens have shown that at least some renal tumors produce il-6 , which functions as an autocrine growth factor of the rcc .
therefore , the presence of a systemic inflammatory response might be associated with aggressive rcc behavior .
the prognostic value of crp levels has been reported in many recent studies of cancers , including those of the esophagus , ovaries , and colon [ 21 - 23 ] .
however , the mechanism by which a systemic inflammatory response affects cancer - specific survival in patients with rcc is unclear .
hwang et al studied the relationship between crp and survival in patients with rcc who underwent a radical nephrectomy .
they reported a statistically insignificant difference in the survival rates of patients with and without crp elevation and concluded that crp was not a significant prognostic factor for rcc when compared with tumor stage , grade , tumor size , and cell type .
however , komai et al demonstrated that the 5-year and 10-year disease - specific survival rates ( 75% and 30% , respectively ) in patients with high crp levels were significantly worse than the rates in patients with normal crp levels ( both 93% , p<0.001 ) and suggested that the preoperative crp level is associated with poor survival in patients with localized rcc .
in addition , they reported that the crp level is a useful preoperative screening tool in patients with localized rcc .
blay et al reported that pretreatment il-6 and crp were significantly associated with the effects of cytokine therapy and high il-6 and that the crp level indicated a poor response to cytokine therapy . in the present study ,
preoperative crp elevation was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc .
this result confirms the importance of the preoperative crp level as an independent prognostic factor for tumor recurrence and is consistent with the findings of earlier reports showing that a high crp level is associated with poor survival in localized rcc .
secondary or reactive thrombocytosis has been associated with tumor metastasis and poor survival in cancer patients .
the pathophysiologic mechanism of reactive thrombocytosis may be tumor - associated elevation of circulating platelets by tumor - derived humoral factors such as il-6 and macrophage colony - stimulating factor ( m - csf ) that may be responsible for the development of cancer - related thrombocytosis .
il-6 is a potent stimulator of megakaryocytopoiesis , and tumor cells have been shown to release il-6 both in vitro and in vivo .
these observations suggest that il-6 plays a role in the development of tumor - associated thrombocytosis .
karakiewicz et al analyzed 1828 patients with rcc by univariable , multivariable , and predictive accuracy analyses with regard to rcc - specific mortality .
in that study , they showed that the addition of thrombocytosis to the base model ( age , tumor size , tnm stage , ecog - performance status , fuhrman grade , and histology subtype ) increased the predictive accuracy by only 0.3% ( from 85.3% to 85.6% ) ; these changes in predictive accuracy were not statistically significant .
consequently , they concluded that patients who presented with thrombocytosis did not have poorer prognosis than did their counterparts who did not exhibit these apparently unfavorable characteristics , as long as the effects of the tnm stage , histology , tumor grade , and ecog - performance status were considered .
however , many investigators have reported that thrombocytosis is related to a poor prognosis in patients with rcc .
, they showed that patients with thrombocytosis had a mean survival of 45.2 months compared with 76.6 months in patients without thrombocytosis ( p=0.0002 ) and concluded that preoperative thrombocytosis is associated with a poorer survival than is a normal platelet count . in the present study
, we showed that preoperative thrombocytosis was significantly correlated with anemia , t stage , and metastasis .
in addition , thrombocytosis was a significant prognostic factor associated with recurrence - free survival in the univariate analysis .
therefore , thrombocytosis was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc ; these findings are consistent with those of previous studies .
ito et al showed that preoperative crp levels and platelet counts had a significant correlation and suggested that this was because the reactive thrombocytosis and crp elevation in rcc were both caused by the production of inflammatory cytokines such as il-6 .
as in the study by ito et al , we analyzed the relationship between preoperative crp and platelet count .
however , we did not find a statistically significant association between preoperative crp and platelet count ( r=0.132 , p=0.079 ) .
therefore , the exact relationship between preoperative crp and platelet count remains to be determined .
a limitation of this study is that several variables could not be considered in the current analysis .
nonetheless , all available variables were included , and our findings indicate that several known prognostic factors were valuable as prognostic factors
the reason for this result was , to some extent , too many patients in stage t1 rather than other t stages and a small number of patients with grade 4 compared with other grades according to the early detection of rcc nowadays .
preoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . moreover ,
however , preoperative thrombocytosis was not an independent prognostic factor for tumor recurrence and prognosis . | purposethe aim of this study was to investigate the association of preoperative c - reactive protein ( crp ) elevation and thrombocytosis with the prognosis of patients with non - metastatic renal cell carcinoma ( rcc).materials and methodsthis was a retrospective review of the medical records of 177 patients ( 130 men and 47 women ) with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet data were available for analysis .
preoperative crp elevation and thrombocytosis were compared with clinical and pathological variables.resultsthere were 38 patients with crp elevation and 11 patients with thrombocytosis .
the mean follow - up time was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) .
twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period .
crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) .
thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) .
the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors associated with recurrence - free survival , whereas the multivariate analyses showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors.conclusionspreoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc .
moreover , preoperative crp elevation appeared to be an independent predictor of tumor recurrence and prognosis .
preoperative thrombocytosis , however , was not an independent prognostic factor for tumor recurrence and prognosis . | [
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angiogenesis is a constant hallmark of multiple myeloma ( mm ) progression and has prognostic potential .
we have previously isolated endothelial cells ( ec ) from bone marrow of patients with mm ( mmec ) .
they show intrinsic angiogenic ability , because they rapidly form a capillary network in vitro , and extrinsic ability , because they generate numerous new vessels in vivo in the chick embryo chorioallantoic membrane ( cam ) .
moreover , mmec secrete angiogenic cytokines , such as fibroblast growth factor-2 ( fgf-2 ) and vascular endothelial growth factor ( vegf ) , as well as matrix metalloproteinase-2 and 9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
studies by rajkumar et al . , dominici et al . , and laroche et al .
the bone marrow microenviromnent plays a crucial role in inducing the angiogenic response in mm [ 6 , 7 ] .
reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells , namely haematopoietic stem cells , fibroblasts , ostoblasts / osteoclasts , ec , ec progenitor cell , t lymphocytes , macrophages and mast cells are mediated by an array of cytokines , receptors and adhesion molecules .
these cells secrete angiogenic factors , such as interleukin-6 ( il-6 ) , vegf , fgf-2 , hepato - cyte growth factor / scatter factor ( hgf / sf ) , transforming growth factor- ( tgf- ) and tumour necrosis factor ( tnf- ) [ 6 , 7 ] . here , we have attempted a fine characterization of the angiogenic response induced by mmec , as compared with monoclonal gammopathy of undetermined significance ec ( mgusec ) in the cam assay .
we investigated the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implantedonthecamsurfacefrom8today12ofincu - bation and we studied the effects of mmec and mgusec on the expression of endogenous levels in the cam of vegf , fgf-2 , angiopoietin-1(ang-1 ) , hypoxia - inducible factor-1 ( hif-1 ) and endostatin by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means 1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator .
gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means 1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
the ability of mmec and mgusec to induce an angiogenic response in vivo was assessed with the cam - gelatin sponge assay .
1 ) . cam implanted with the medium containing fgf-2 or mmec gave significantly higher vessel counts and numerous allantoic vessels converging like spokes toward the sponges were recognizable ( table 1 and fig .
when the sponges were loaded with the medium alone and with mgusec , physiologic angiogenesis was observed in the form of few allantoic vessels partly around and partly converging toward the sponge ( table 1 and fig .
angiogenic response assessed by the chick embryo chorioallantoic membrane ( cam)-gelatin sponge assay p<0.001 versus rpmi-1640 and mgusec angiogenic activity in the chorioallantoic membrane ( cam ) assay . gelatin sponges loaded with fgf-2 ( a ) , mmec ( b ) , rpmi-1640 ( c ) and mgusec ( d ) were implanted on top of the cam on day 8 .
macroscopic view of the cam on day 12 shows numerous allantoic vessels converging like spokes toward the sponge in a and b , while few allantoic vessels are recognizable in c and d. original magnification : a d , x 50 . in order to evaluate the angiogenic effects of mmec and mgusec , mrna expression of pro - angiogenic factors including vegf , fgf2 , ang-1 , hif-1 and mrna expression of anti - angiogenic molecule endostatin
the expression level of each transcript was compared to those detected in control cam . as shown in fig .
2 , treatment of cam with mmec and mgusec did not produce significant difference in the expression of pro - angiogenic factors mrna in comparison with control cam treated with medium alone .
moreover , no statistical difference was found in vegf , fgf-2 , ang-1 , hif-1 and endostatin mrna content between mm and mgus treated cam ( p<0.05 ) .
in contrast , the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam ( mgus od:120.113 and mm od:48.319.6 versus ctrl od:205.84 19.6 ; p<0.05 ) .
expression levels of mrna coding for vegf , fgf-2 , ang-1 , hif1 and endostatin evaluated by semi - quantitative rt - pcr .
transcript levels from the cam assay are referred to ec obtained from 6 mm and 6 mmgus patients and the error band represents the standard deviation of six experiments .
western blot analyses were also performed to study whether the expression of endostatin changes in mmec and mgusec treated cam respect to control . as shown in fig .
3 , according to rt - pcr quantification , endostatin was significantly lower also at the protein level in both mgusec and mmec treated cam respect to control cam ( mgus od : 97.1410 and mm od : 52.313.6 versus ctrl od : 158.715.52 ;
p<0.05 ) . immunoblotting analysis and quantification after western blot analysis of endostatin expression in cam treated with ec obtained from 3 mm and 3 mgus patients .
mm plasma cells secrete vegf , whereas both vegfr-1 and vegfr-2 are markedly elevated in the bone marrow stromal cells , suggesting that a paracrine growth pathway mediated by vegf - activated stromal cells , secretion of il-6 on their part , and subsequent activation of plasma cells may occur . in active mm , we found secretion of the vegf - a isoform by plasma cells , overexpression of vegfr-2 by bone marrow microvessels and isolated ec and of vegfr-1 by the other stromal cells , secretion of vegf - c and vegf - d by stromal cells , and expression of their cognate receptor vegfr-3 by plasma cells , which suggests another paracrine loop of angiogenesis and tumour growth in mm .
parallel studies by kumar et al . found secretion of vegf by several mm cell lines and fresh patients ' bone marrow plasma cells , as well as expression of vegfr-1 and vegfr-2 , which suggests multiple vegf - mediated autocrine pathways of tumour growth and paracrine stimulation of angiogenesis .
when we studied the angiogenic potential of plasma cell suspension obtained from patients with active mm and implanted them onto the cam , we demonstrated a vasoproliferative response , significantly higher than that induced by cell suspension obtained from patients either with non - active mm or mgus .
hgf / sf is yet another angiogenic factor identified in the culture medium of both human mm cell lines and freshly isolated plasma cells .
still other factors may be responsible for bone marrow angiogenesis in mm : the plasma cell secretion products , such as tgf- , il-1 , il-6 and il-8 ; granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) and tnf- , secreted by bone marrow microenvironment cells recruited and activated by plasma cells .
since human mm cell lines produce ang-1 that up - regulates the tie-2 cognate receptor in bone marrow ec , involvement of the ang-1/tie-2 loop in mm neovascularization has been demonstrated .
conversely , mmec secrete angiogenic cytokines , such as fgf-2 , vegf and ang-1 as well as matrix metalloproteinase-2 and -9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
other studies , however , showed that the expression levels of vegf , fgf-2 , and their receptors were similar among plasma cells from mgus and mm , suggesting that increasing angiogenesis from mgus to mm is , at least in part , explained by increased tumour burden rather than increased expression of vegf / vegfr-2 by plasma cells .
another possibility is that the inhibition of angio - genesis in mgus is lost with progression , hence that the switch from mgus to mm may involve a loss of an anti - angiogenic activity .
our data are in accord with this statement , because the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam .
the phenotypic switch to angiogenesis in tumour growth involves more than simple up - regulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators , and our study suggests that angiogenic switch in mm , investigated by using an in vivo experimental model such as the chick cam , may involve loss of an endoge - nous angiogenesis inhibitor , namely endostatin . | abstractwe have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells ( mmec ) by using the chick embryo chorioallantoic membrane ( cam ) assay and by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
results showed that in the cam assay mmec induced an angiogenic response comparable to that of a well - known angiogenic cytokine , namely fibroblast growth factor-2 ( fgf-2 ) , while rt - pcr demonstrated that the expression of endostatin mrna detected in mm treated cam was significantly lower respect to control cam .
these data suggest that angiogenic switch in mm may involve loss of an endogenous angiogenesis inhibitor , such as endostatin . | [
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six cases were reported from 2007 to 2012 to the oral and maxillofacial unit of annaswamy mudaliar general hospital , bangalore , out of which four patients were males with the lesion occurring between the second decade of life and sixth decade of life [ figures 1a3c ] . in all cases ,
the mandible was involved with the greatest size of tumor seen being 11 cm 9.6 cm [ figure 1a ] .
all cases exhibited enlargement of cortical plates with perforation , pain and tenderness with regional lymph node enlargement , with three cases showing fixed submandibular groups of lymph nodes ( level ib ) and in one case parasthesia of inferior alveolar nerve .
five cases showed unilocular radiolucency and one case showed multilocular radiolucency with erosion of bone , including inferior border of mandible in one case [ figure 1c ] .
maximum follow - up was for 39 months with one patient being lost to the follow - up [ table 1 ] .
all patients had a chest x - ray and an abdomen ultrasound done to avoid missing distant metastasis as we were aware of the potential of this lesion to metastasize .
incisional biopsy was done for diagnosis for all cases and was diagnosed as features suggestive of ameloblastoma with areas showing dysplastic changes in the epithelium suggestive of a neoplastic change .
( a ) patient exhibiting large extra oral tumor with draining sinus , ( b ) intra oral view of tumor : ( c ) orthopantomogram showing bicortical involvement of mandible
in one patient , wide excision was done as the lesion was small and the patient being young did not wish aggressive measures that may have had postsurgical cosmetic deformity [ figure 2a c ] .
he chose the wait and watch policy and there is no evidence of disease after 32 months .
( a ) preoperative view showing tumor involving left mandible and sulcus , ( b ) preoperative orthopantomogram , ( c ) intra oral view of tumor being excised two patients underwent hemimandibulectomy and neck dissection because of increased involvement of mandible with palpable level ib nodes where fine - needle aspiration cytology showed positive for carcinoma , as also one more patient who had underwent a wide excision with hemimandibulectomy and neck dissection due to skin involvement and palpable fixed nodes at ib level [ figure 1a c ] .
one patient had a segmental resection of the mandibleone patient had a hemimandibulectomy [ figure 3a c]all the resected specimens were sent for histological examination .
one patient had a segmental resection of the mandible one patient had a hemimandibulectomy [ figure 3a c ] all the resected specimens were sent for histological examination .
( a ) preoperative view of tumor involving left mandible , ( b ) intra oral view of tumor , ( c ) orthopantomogram showing involvement of left mandible
the largest diameter of the tumors was 10 cm 9 cm [ figure 1a ] showing increased vascularity with numerous cystic spaces and areas of necrosis .
typical features found were multiple pieces of tissue with predominant tumor islands within connective tissue seen under scanner . under higher magnification tumor islands appearing epithelial odontogenic in origin showed infiltrative pattern with each island showing peripheral columnar cells with central stellate reticulum exhibiting feature of ameloblastoma , but with atypical features such as bizarre mitosis , altered nuclear chromatin ratio , hyperchromatisim , and mild pleomorphism .
individual cell keratinization with keratin pearl formation with stroma showed predominant inflammatory component and endothelial lined blood vessels [ figure 4 ] .
immunohistochemical findings showed that ameloblastic carcinomas reacted strongly with antibodies directed against cytokeratin chm and aei and ae3 .
histopathological picture showing bizarre mitosis , altered nuclear chromatin ratio , hyperchromatisim , mild pleomorphism , and central stellate reticulum confirming ameloblastic carcinoma no evidence of disease or metastasis was seen postsurgery in any of the patients .
it is interesting to note that up to date of completion of this study 71 cases of ameloblastoma had reported to our department between february 2007 and may 2012 . in this series ,
ameloblastic carcinoma is a lesion with histologic behavior that dictates a more aggressive surgical approach than that of a simple ameloblastoma .
mean age for occurrence is 30.5 years , male : female ratio is 1.5:1 , and 80% are located in the mandible posterior region .
involvement of the maxilla is less frequent than that of the mandible . the clinical symptoms of ameloblastic carcinoma are more aggressive than conventional ameloblastoma , with swelling , pain , rapid growth , trismus , dysphonia , expansion of the jaws , and frequently perforation of the cortex . these were mirrored in our series also .
radiographic appearance of ameloblastic carcinoma is consistent with that of ameloblastoma except for occasional presence of some focal radiopacities , reflecting dystrophic calcification .
histologically , ameloblastic carcinoma does not show uniform proliferation , and has pleomorphic and hyperchromatic cells arranged in the form of sheets and chords with reversed polarity .
an additional consideration in the differential diagnosis is the squamous cell carcinoma arising in the lining of an odontogenic cyst .
histologically , this lesion tends to more closely resemble oral squamous cell carcinoma ameloblastic carcinoma .
the squamous odontogenic tumor may also be mistaken for ameloblastic carcinoma , being composed of islands of squamous epithelium that lack stellate reticulum like zones , peripheral palisading , microcystic changes and dystrophic calcifications .
however , the epithelium of the squamous odontogenic tumor lacks any cytologic evidence of malignant disease .
thus , the term ameloblastic carcinoma can be applied to our series , where all showed evidence of malignant disease including cytologic atypia and mitoses with indisputable features of classic ameloblastoma .
whether ameloblastoma may transform biologically and histologically from a classic ameloblastoma to a malignant lesion is controversial .
many authors have shown that metastasizing ameloblastomas are histologically indistinguishable from classic ameloblastomas and others have identified malignant features in the tumor , usually after repeated surgical excisions .
the high rate of recurrence maybe due to the mode of growth and surgical mismanagement rather that any inherent malignant properties and metastases are exceedingly rare .
wide local excision is the treatment of choice as most investigators have recommended , which we had followed in all our cases .
regional lymph node dissection should be performed selectively as we did in three of our cases where in the regional lymph node was palpable .
the efficacy of adjuvant radiation or chemotherapy as a postsurgical treatment is not clear and seems to have a limited value .
lanham treated a patient with doxorubicin , cisplatin , cyclophosphamide , dacarbazine , and 5 fu ; but , the tumor failed to respond .
yoon et al . have reported distant metastasis as early as 4 months and as late as 47 months after surgery .
none of our patients showed recurrence , but one patient was lost to follow - up .
none of our patients were sent for radiotherapy or chemotherapy . in our case series ,
least follow - up until date is 22 months and maximum follow - up being 39 months .
this small series of cases illustrates the malignant portion in the spectrum of ameloblastomas and it is possible that ameloblastoma may show a wide variety of histologic and biologic behavior ranging from benignity to frank malignancy .
cases of ameloblastoma should be studied carefully , correlating their histologic pattern with biologic behavior to detect changes in histology that may predict aggressive behavior . when a case is diagnosed as ameloblastic carcinoma , assessment of nodal metastasis and evidence of distant metastasis
treatment of ameloblastic carcinoma is wide surgical resection and if evidence of nodal metastasis is present , neck dissection should also be considered . | ameloblastic carcinoma is a rare odontogenic tumor exhibiting not only features of ameloblastoma , but also features of carcinoma .
clinical dissemination of this lesion is more aggressive and rapid than that of ameloblastoma and it can metastasize to the lung or regional lymph node .
histologically , there are features of both ameloblastoma and carcinoma .
< 50 cases have been reported until 2011 .
we report a series of six cases with our treatment modalities . | [
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This repository contains model predictions generated by AutoTrain for the following task and dataset:
- Task: Summarization
- Model: google/bigbird-pegasus-large-arxiv
- Dataset: scientific_papers
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Contributions
Thanks to @Blaise_g for evaluating this model.
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