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risk benefit analysis with or without concern for legal issues has always been the mainstay of clinical practice .
beneficence , insofar as it is a principle in medicine , has to a greater or lesser degree always been based on this issue . even under the accusation
( and hopefully this argument is resolved and exhausted now ) , whatever decisions doctors used to take was based on what in their opinion was in the patient s best interests and for the patient s good . leaving patients ignorant of their disease and outside the realm of decision - making was considered a therapeutic privilege and done in order to protect the patient from news which he or she did not or should not know in order to keep them from grief which could cause them more harm than good .
the term values is used in order to emphasize that it is not merely a question of relativism or simply because we understand human nature better .
it was indeed the philosophical debate which contributed considerably to increasing the principle ( in the kantian sense ) of respecting autonomy and trumping it over beneficence , until beauchamp and childress1 came around to give us ( or rather , lay down what was in the air ) the four principles .
these evolved into an ethical framework and indeed doctors and health care providers were invoked to use these principles in their decision - making , calling them mid - level principles .
principlism came under attack2 and the defenders went on to invoke methods of using principles , such as specifying and balancing .
specification and balancing were meant to address the particular situation , yet steering away from situational ethics . assessing
there is overlap also with the principle of justice because these decisions invariably also involve the use of resources that can be allocated elsewhere ( without , of course , bowing to utilitarianism , as indeed even a deontologist must consider her duty to society ) . in the meantime
, the field of gerontology and geriatric medicine has taken on new life , and rightly so ; we are not only concerned with the elderly as a specific group , needing special needs with special attention to differences in medical care , just as children need a different approach to adults , but also because our populations , and perhaps cultures , are aging.3 making decisions concerning risk
benefit for the elderly invariably therefore takes on a completely different dimension than for other adults , not only because where the elderly are concerned there is a different physiology and an aging body , but also because we are dealing with perhaps an aging culture , and more persistently so because we have to face the bottom line question : is it worth treating this patient at this stage of life ?
these may be blunt questions , but it is a reality from which discussions of end - of - life decision - making ensue . the issues of cardiopulmonary resuscitation , advance directives , and research into longevity have arisen . because we are discussing an ethical concept , and not merely a clinical algorithm
, what we have at base is our attitude , in philosophical terms , towards our elderly populations .
the first discusses the reality of principles as an ethical framework and whether in essence it is our values towards the above questions which define outcomes .
secondly , it discusses three real - world situations , the third of which discusses how we view elderly in this postmodern society .
decisions are usually based on inherent cultural beliefs from which emanate virtues and general principles .
inherently we are what we are because we have learned basic concepts throughout life which set the scene for our principles . but both the use of and the foundation of these principles are inherently attached to our concept of what is or should be right and wrong . a difference at this level , and you invoke a different reasoning on how the same principles are used to arrive at another different answer .
indeed when we deliberate a moral problem we start with an empathic feeling of what is right and what is wrong .
rational thought about a situation may allow us to change our course of action and perhaps the final decision , but our initial value system remains essentially unchanged .
indeed , it is probable that we have conflict amongst principles because we have different value systems .
gregory tillett perhaps best explains this conflict.4 tillett , in his analyses of resolving conflict distinguishes three concepts , or categories , of differences between ideas , humans , relationships , and between internal ( within oneself ) issues , ie , problems , disputes , and conflicts .
a problem can be resolved by management , ie , by the agreement on how something can or should be done .
we can easily conceive of a difficult clinical situation in which one consultant or family practitioner asks the advice of another , and doctors within a team discuss courses of action between themselves and with patients , especially within a context where there is refusal of a particular kind of treatment .
tillett points out that these problems can indeed become disputes and can generate conflict.4 a dispute occurs when two ( or more ) people ( or groups ) perceive that their interests , needs , or goals are incompatible and they seek to maximize fulfilment of their own interests or needs , or achievement of their own goals ( often at the expense of others ) .
consider , for example , the situation of an elderly woman who is being taken care of at her daughter s home .
this daughter has three brothers , one of whom had distanced himself from his sister due to a dispute with her husband .
the main contention was that this brother convinced the others that their mother should be put in a home because he refused to visit her at their sister s home .
of course , prima facie , the best interests of the patient were for her to remain at home .
however , it transpired that the woman was very depressed because this son , who was evidently her favorite , was not visiting her .
the undue pressure was indeed coercive and the woman conceded to go to the home in order to be able to see all her children , not at all a voluntary choice . in the meantime
after eight months in hospital and no remedy in finding a place at an institution , the decision had to be reversed to take into consideration again her best interests , ie , avoidance of a hospital infection . because the daughter was still willing to take her back , this course of action was taken , and the necessary legal procurements arranged . at each stage
however a value - laden decision was taken , which brings us to the third category , ie , conflict .
conflict , again as defined by tillett , arises when two ( or more ) people ( or groups ) perceive that their values or needs are incompatible whether or not they propose , at present or in the future , to take any action on the basis of those values.4 whilst problems and disputes arise within specific situations , conflicts do not need one .
two parties can be in conflict because of beliefs or because of the values they uphold , and hence the heated debates on such issues as abortion and euthanasia .
but , as in the case described here , we may find that superficially , at least , the people involved had the same values in that they all loved their mother , but when it came to taking a decision which was in the best interest of their mother , it was rather a decision in their own best interests which had become evident , until a situation arose which gave enough strength to override these different values .
the difference in value was between one in which x would rather have his mother in a home and be able to see her ( and perhaps that she would be able to enjoy him ) , and the same person admitting that notwithstanding that his mother would not be able to see him , his sister was in a better position to take care of her and that given the state of existing homes , she may be better off .
the conflict was that between x and the husband of x s sister , and not the situation .
admittedly there can be a hazy line between a dispute and a conflict , as in this case .
one has to delve into the narrative of each participant in order to reveal a conflict and understand that the matter is not simply one of disputes .
in this case , the brothers agreed that the mother be allowed to go to their sister s home on the condition that the sister use her pension only and not the mother s savings in order to take care of her , ie , if she needed a new bed or a commode , they were not willing to contribute to either .
this coercion reveals the underlying conflict between the daughter s husband and the dominant brother , notwithstanding a resolution of the conflict .
it seems that the needs or values of the children trumped the needs of the mother or the value to respect and meet her needs .
certainly this was not a dispute any more but a conflict , which unfortunately the medical practitioner or team may not be able to handle , unless by enforcing legal processes which may not be in the interests of all parties and not so conducive to conflict resolution .
justice is conflict.5 doctors make decisions in the light of beneficence and at the same time doing justice to the person(s ) and the situation at hand . by doing justice , we obviously do not merely mean that the situation is handled well and correctly ( within , that is , the boundaries of standards of care ) , but we seek that justice is done morally .
deciding whose justice and which rationality is certainly not easy in the modern sense , especially in dealing with end - of - life situations , whether to treat or not to treat , whether to leave elderly for acute treatment at their home , or incur a more expensive , perhaps better treatment , but in a hospital setting .
certainly there can be hardly a better exposition of this philosophical reality than that put forward by macintyre.5 whether he does justice to the topic is probable but whether he resolves the dilemma is uncertain .
certainly he speaks extensively of tradition , which is an argument extended through time , in that it can be a medical tradition or a religious tradition or simply a tradition of horse - riding .
solomon6 believes that morality can not be reduced to any strict kantian system of principles .
the very fact that the four principles of health care require further specification and balancing shows that a deeper sense of the real situation is needed , and what this usually boils down to is the narrative of the parties involved who bring forth their values and needs .
solomon does not particularly like macintyre s use of traditions , in the sense that it still leaves open the nostalgia for religion and a sense of community , but goes on to admit that aristotle did invoke a sense of community when speaking about virtues .
neither does he associate virtue with feminist ethics , values not being supposed to be held captive to gender , even though feminist ethics may have a point in contrasting male principles which are hard , oppressive , and impersonal , with the female virtues of warmth and caring .
colloquially , by having principles , we are actually talking about the values a person holds as well .
if one has a so - called value for life and is not in favor of euthanasia , one invokes principles to lead one to this conclusion .
conversely , if one s value system of life includes terminating suffering even by allowing physician - assisted suicide , then one will use the same four principles to reach the opposite goal .
having summarized the philosophical foundations on which risk benefit analysis is based , various relevant clinical situations faced in health care are grouped .
jonsen et al7 classify three forms of disease and goals of medicine that are summarized here with particular reference to care and decisions for the elderly .
these authors say that in the first , the patient suffers from an acute illness that , once diagnosed , can be readily treated and cured . in the second
, the patient experiences a process that causes serious disabilities and which , while some relief can be provided , will progress despite treatment and eventually case death . in the third , the patient suffers a chronic disease that can be effectively treated so as to relieve many of the most debilitating effects . for simplicity they use the acronyms acure ( acute , critical , unexpected , responsive , and easily diagnosed and treated ) , care ( critical , active , recalcitrant , and eventual ) , and cope ( chronic , outpatient , palliative , and efficacious ) . perhaps using simply acute , critical , and chronic would have been quite an accurate substitute , but these acronyms serve the purpose just as well and are applied here .
these authors point out that physicians habitually approach medical problems by attempting to determine the indications for or against medical intervention .
in fact , assessing risk and benefit takes both medical intervention and ethical dilemmas to task , especially when considering elderly patients and when one asks oneself whether one should treat or not in a particular case .
medicine remains a science of uncertainty ; even these acronyms are understood as being probability statements rather than absolute designations .
indeed , uncertainty makes for good decision - making in the face of risk benefit analysis , and not simply one of clinical competence but also one which is value - laden .
these goals apply to acute and relatively straightforward conditions , especially once a diagnosis has been made
. the situations may be critical and unexpected , such as meningitis , reversible and easily treated ( by easy one of course does not mean easy in the strict sense , but that one knows what to do and has an immediate plan of action based on evidence - based medicine and/or standards of care ) .
ethical issues in this situation may range from simply refusal of treatment to families not wishing the elderly patient to know .
recent literature suggests a more culturally sensitive approach in these clinical situations,810 respecting wishes which may be culturally sensitive . in japan for example , it may be considered disrespectful to let an elderly person know of the medical condition , such that the sons and daughters take on the responsibility .
the second category ( care ) describes patients with active , progressive , and deleterious conditions .
one can easily see many elderly people in such situations , although this is by no means restricted to this section of the population . under this category lie most of the ethical dilemmas facing end - of - life decisions , such as withdrawing of life support , decisions not to intubate , decisions not to resuscitate , and irreversible coma / brain death .
so are the mainline ethical discourses of ordinary versus extraordinary treatment , killing versus allowing to die , treating versus tender loving care , etc .
what is in fact ordinary and extraordinary treatment ? standards of care weigh in heavily . even in invoking the principle of
double effect by using high doses of morphine , for example , to treat palliatively , knowing that there is a risk that the patient may die of the dose of morphine and that this was a foreseen but unintended consequence , one must understand the nature of increasing doses of morphine and not be caught in a dispute of hastening death by going about the principle of double effect .
indeed , even though the principle has four basic rules to it , it is heavily burdened on the moral agent to ascertain that it is morally and correctly invoked ( just as it takes a moral agent , for example , not to abuse the abortion act in the uk ) .
there is always a standard of care in increasing dose strengths , although this is a patient - sensitive decision and hence can be externally seen as subjective .
there is no duty to treat when treatment is judged to be useless , nor a duty to treat when treatment is deemed extraordinary .
although today proportionate and disproportionate are terms which may be used instead of ordinary versus extraordinary , the latter are not only still the most commonly used terms , but they are probably the best guideline to make a judgment on risk benefit .
however , one must clearly understand the meaning of extraordinary to make best use of the term , and for this one must look at its original catholic origins .
extraordinary means of preserving life are all medicine , treatments , and operations , which can not be obtained or used without excessive expense , pain or other inconvenience for the patient or for others , or which , if used , would not offer reasonable hope of benefit to the patient.11 a closer look at this definition shows how this covers most , if not all , legal issues that may arise when making moral choices on risk benefit .
when assessing risk , we are indeed facing the particular and singular clinical picture of this patient , including fitness for surgery , side effects and interaction of drugs , wishes of the patient , and physical state of the patient .
the definition clearly does not take into account the state - of - the - art of medicine and technology , and something that is considered extraordinary today may by no means remain so tomorrow .
conversely , something that is quite ordinary treatment in one country , either because of culture or economic status , may indeed be extraordinary in another .
family need not be put to excessive burden , especially if they are required to pay or to go through extraordinary measures to meet the patient s needs . clearly , where insurance and state can not or will not pay , the family can not morally be held responsible to take all measures necessary ( in malta , it has almost become a societal pressure to go to the uk for care when one finds no hope on the island , even if consultants say that no further treatment can be found there ) .
of particular note is that even medicines and excessive expense are mentioned in the definition , along with pain and inconvenience to the patient or family .
hence a treatment which can not be afforded or which will cause prolonged agony need not be given .
these issues are mostly found in outpatient and community settings of family practice , and deal with the chronic and palliative .
although jonsen et al lament that the ethical issues here may be less evident , they in fact may represent the same issues discussed above .
cope is a good acronym because in fact we are working with the patient to
although the same goals of preserving life , preserving function , and reducing pain and suffering are used , they take on a more patient - centered approach and reach compromises with the patient that allow him or her to participate in the treatment . whilst there may be no drama as in life - and - death situations ,
there is a quality - of - life perspective and also end - of - life decision - making to face .
the american college of family physicians have recently paid much attention to helping families cope with end - of - life and also address the cultural aspects of individuals and families.12 this departs considerably from the earlier days of bioethics when autonomy meant revealing everything to the patient and almost burdening the patient with information , giving details in order to allow them to feel they were making a choice , or preferably making the choice themselves .
this renewed cultural sensitivity brings back the onus on the practitioner to be truly patient - centered and to share the burden with the patient who usually indeed seeks the doctors advice .
the bigger burden lies with difficult patients , ie , those who are noncompliant and those who pose problems such as never being fully satisfied . in these situations
, the doctor may be impelled to transfer the care to someone else , because a breakdown of the relationship is perceived to have occurred . yet
patient relationship as well as compromise , and this may in turn translate into breakdown occurring less often .
legal issues may arise with the family , who may not be satisfied with the care imparted to their parents .
however , we need to acknowledge in this regard that the involvement of the family as a community - based approach to treating the elderly is still compatible with the patient - centered approach.13
of course the concept of aging has changed over time . today , with discourse on aging , one may find less difficulty with , eg , an elderly man seeking help for erectile dysfunction .
yet many bioethics committees recommend against women having fertility treatment beyond their menopausal age . with advancing technology , the right to treatments , perhaps with aging populations , will probably give ground to the yearning of the elderly spirit to continue enjoying life beyond the social boundaries thus far accepted .
leon kass , former chairman of the us council on bioethics , found considerable opposition to his general wariness about reproductive technology and efforts to forestall aging.14 therefore , risk benefit analysis can not look at the social concept of aging in a postmodern and perhaps posthuman society .
the concern for us now lies in making the same analysis of legality in risk - benefit not only with regard to medical care but also in terms of research , this being imperative to the advancement of medicine .
we have to decide which research is moral and which not to finance with public funds .
there are strong arguments for the moral imperative to research elderly subjects further , even though research in this realm is both ethically required and ethically suspect.15
do we carry out research in order to increase longevity , to curb the aging process , or even perhaps to stop aging all together ? whereas better cures and medical advances have already brought this out , it was never really a medical imperative .
it was , at most , a welcome , although perhaps foreseen consequence of treatment and care . yet
the line is quickly drawing a distinction between a rise in the elderly population due to better survival and a deliberate search for survivorship and longevity , although such research into the genome and molecular cell biology is rapidly becoming hazy . in discussing aging and the sociology of embodiment , featherstone and hepworth16 give an interesting account worth reflecting upon . at the end of the day
, the body is the bottom line , when we survive to advanced old age.17 elias , in his old age acknowledged , with an air almost of awe , that there is a simple reality of a finite life.18 therefore , the body in decline , for featherstone and hepworth,19 is a central issue for contemporary society to come to terms with .
they assert that this does not mean that historically , when life expectancy was much shorter , people did not strive to prolong their life .
fear of death , intensity of bereavement , and the longing for earthly pleasures are all issues to be faced .
it is precisely in the struggle to reconstruct this cultural inheritance of pessimism that the element of difference between past and present attitudes towards aging through the later period of the life course may be found.19,20 this reconstruction , according to these social authors , lies in moving away from the pessimistic and melancholic processes of aging to one of optimism and a period enriched with distinctive creative possibilities.16 only in this way can the structure of feeling and attitudes towards aging be changed , especially with regard to the aging body .
this social reconstruction ( or better , change in social acceptance ) , will then perhaps illuminate our attitude towards caring for our elderly in a new way and not simply as seeing them one of the vulnerable populations , even though they may be .
we may see new light in research into the elderly and perhaps even in some form of longevity and prevention of body decline , which would bring with it a continuation of activity , be this sexual , educational , or occupational .
we are already seeing countries prolong the retirement age due to improved standards of living and quality of life , and this not merely because elderly populations are on the rise and we need to continue collecting taxes from them . in understanding our ethical and legal attitudes to the risk and benefits of treatment , we must indeed be wary therefore of any desire by groups , such as practitioners of geriatric medicine , to make claims of a specialized form of knowledge and to legitimize the imposition of controls over aging members of the population.21 one should not take this to mean that geriatric medicine should not exist , but to entail a change in concept even of the discipline itself .
, much as family doctors are advocates for seeing the family more consistently in the biopsychosocial dimension , acting as family counselors and advocates for patient rights .
notwithstanding the discipline of geriatrics and the vulnerability experienced by an increased percentage of this sector of the population , we can not continue to see old age as a form of pathology and the body in decline as a medicalized entity , which according to katz , is an accusation often leveled at medicine .
perhaps we need to understand that the stages model of life is a cultural clich , as are expressions like ticking of the clock.22
the implications for the sociology of ageing are clear : it is no longer possible to make adequate generalizations about the aging process that are grounded on biological assumptions about the ages of life . nor is it particularly useful to adopt schemata of the life course based upon loosely conceptualized models of unspecified processes of interaction between the ontologically distinctive entities , body , self and society . as a consequence
contemporary models of ageing into old age must be increasingly post - modern , by which we mean they must anticipate even more advanced forms of biocultural destabilization.16 these authors eloquently argue how our struggle is not really with biologic decline , but with the social construction of aging that has been the center of debate .
hormone replacement therapy , even if it had an element of success , was more about remaining young and active , hidden behind the excuse of better heart and skin outcomes , until the reality of the cancer issue came to light , ie , something which many were very skeptical about from the outset .
the social pressure to take hormone replacement therapy can not be denied , in that many women may have taken it seeking a better skin complexion and less wrinkling , or at least have been encouraged to take the medication for these reasons .
it is interesting that postmodern feminist studies of the interpretation of aging were associated with wisdom . before patriarchal societies ,
the female crone was seen as a naturalistic and matriarchal part of life , with wrinkles being badges of honor.23 history has brought about a separation of the self from the body and society , and the seeking of youth is found at the base of this force . whether this is the result of patriarchal influence which feminism blames is beyond the scope of this paper .
the profound reality is that it is an existential issue and an entity to be reckoned with .
this dualism , ie , the separation of the self from the body , lies in the very nature of the fear of aging in terms of a declining body .
is an expression that perhaps was less heard in primitive society , which adorned their elders with a wisdom and respect much less seen today .
this lies at the heart and concept of the recent change towards end - of - life decision - making .
western culture is learning yet again from populations which lagged behind the materialistic taint and preserve many of their traditional values , and such is the case for eastern countries .
featherstone and hepworth continue to make the argument that whilst accepting the limits of the body and the aging process as having its own rewards , they adopt a negative attitude to technologic advances whereby medical intervention , such as hormone replacement therapy , is a possibility . nowadays , in ethical circles , words like
thus , the main question would be whether hormone replacement therapy is unnatural and a threat to womanhood ?
indeed , there is much sense in not seeing technology as an external factor to human nature , but as a relationship between nature and the human bodily nature ( the creator of the technology itself ) , which is a culturally dynamic process .
we have stopped seeing technology as part of our nature , because it is human nature which creates it , just as a monkey uses a stick , and perhaps moral discourse is more based on a fear of progress rather than a balancing of moral choices .
when seen in this light , we are be forced to accept a change in balancing risk as opposed to benefit . whilst medicine has been attacked for being paternalistic and indeed domineering , in a foucaultian sense
it is the world in general that has strived for technology and improvement of the human condition .
medicine has certainly taken advantage of new technologies for better cure rates , but research is often not carried out by medicine as such , but by corporations who strive to satisfy the thirst of society , and profit of course in the process .
there is a delicate , and sometimes controversial , balance with cultural and religious values .
there is nostalgia , ie , a longing to remain attached to certain roots.24 the reality of the new field of bioethics , as opposed to the centuries old hippocratic tradition , emerged at a time when technology was also blooming .
therefore , we may still be tied to concepts of not treating the elderly because of their age .
the balance between cultural nontelling , and indeed giving a treatment adequate to what the body can withstand ( it would be unwise to opt for surgery , but certainly a long - term course of tamoxifen , even if palliation is not needed ) is not that difficult to conceive .
however , accepting cultural criteria , such as the children of this woman , indeed in their late 60s themselves , of not giving her the bad news , even if she had never explicitly expressed a wish not to know , is the morally ( and perhaps legally ) correct thing to do .
what is probably wrong in our approach to the elderly is to see the changing body as a pathologic process .
even if we can come to accept change in body parts , better appearance , etc , as socially acceptable , there will always be a time of reckoning with death and the human condition .
decline begins very early on in life , perhaps immediately after peak physical growth is reached .
there are certainly problems that affect the elderly more , as there are problems that affect children more .
two years for a 70-year - old is a larger proportion of one s remaining lifespan than the same number of years for a 40-year - old .
certainly balancing risks and benefits may not work out in favor of the elderly person . a new social awareness towards aging helps cultivate an attitude that old age is worthwhile for what it is rather than for what the elderly do or what they have .
whilst looking at the risk and benefit of treatment , we can not ignore the larger risk benefit picture of what research and medicine hold in the future .
advanced research must be respected as part of the human need to ask , invent , and discover .
however , a balance must be struck so as not to uproot people from their cultures because this is what gives people their identities .
simone weil warns against uprooting people , cautioning that uprootedness leads to misery and spiritual lethargy on one hand and to violent efforts to adapt and uproot those not already uprooted . | the ethical dimension of treating the elderly , including risk benefit analysis , focuses mainly on quality of life and end - of - life issues .
these include arguments on advance directives and the concept of extraordinary treatments . this paper looks more closely at the philosophical approach to aging in order to address questions on the direction of research and issues such as longevity and social construction of the aging process .
it is the way society moves to understand the value - laden choices on aging that directs the goals of treatment and research .
whilst these vary culturally , one has to reckon with a postmodern view of aging which may , in turn , reflect on the course of action of future care and research in aging .
the paper canvasses how , in reality , four principles act as guidelines for moral discourse , and discusses how changing values in society decide this course of action . | Introduction
Why principles and why conflict?
Clinical decision-making
Social understanding of aging
Should we strive for technologic advancement of aging?
Treatment and medicalization
Conclusion |
pfeiffer syndrome ( ps ) is a rare autosomal dominant congenital disorder , originally described by pfeiffer in 1964 , and is characterized by an acrocephalic skull , regressed midface , syndactyly of hands and feet , and broad thumbs and big toes , with a wide range of variable severity ( 1 - 3 ) .
ps is known to be caused by mutations in exons iiia or iiic of the fibroblast growth factor receptor ( fgfr ) 2 or fgfr 1 gene ( 4 - 7 ) .
we report with a review of literature the first neonate in korea with ps who had bicoronal craniosynostosis , bilateral syndactyly of the fingers and toes , and broad thumbs and big toes .
a girl was delivered by spontaneous normal vaginal delivery at 38 weeks gestation after an unremarkable pregnancy .
she was 3.5 kg in weight ( 25 - 50 percentile ) , the head circumference was 33 cm ( 25 - 50 percentile ) , and the height at birth was 52 cm ( 25 - 50 percentile ) . her mother is a gravid 4 , para 3 , 40 yr - old korean woman .
the patient is the 2nd among 3 children and is the only affected child . upon birth ,
the infant demonstrated bilaterally fused coronal sutures , mild proptosis , and maxillary hypoplasia ( fig .
the soft tissues of her 2nd , 3rd and 4th fingers were fused bilaterally to the proximal portion of distal phalanx and her bilateral thumbs were broad and deviated radially ( fig .
computed tomogram ( ct ) and magnetic resonance imaging ( mri ) studies of the cranium revealed no other abnormalities except for the presence of craniosynostosis .
three - dimensional reconstruction ct showed 3-dimensional virtual images of the bilaterally fused coronal sutures ( fig .
2a ) but that the sagittal and lambdoid suture were open more than normal to compensate for the small intracranial space , and resulting overall in a brachycephalic head shape ( fig .
plain radiography of her hands and feet showed no bony fusion between the phalanges ( fig .
three - dimensional ct image of choanal air airway showed small and narrow airway of choanal space ( fig .
4 ) . she did not have any visceral abnormality upon ultrasonographic examination of the abdomen and heart .
the patient had a decompressive endoscopic tunnel craniectomy at 4 weeks of age , and is planned for second surgical correction of craniosynostosis , followed by additional correction procedure for the finger and craniofacial anomalies at a later date .
the exact incidence of ps is unknown , but is expected to be 1 in every 100,000 births in the western population , and approximately 60 cases to date have been reported in the past world - wide literature ( 8 , 9 ) .
however , it is rarer in asian population : only several cases were reported in japan ( 10 , 11 ) and no reports as yet in korea .
cohen ( 12 ) in 1993 classified this syndrome into 3 clinical subtypes and suggested that these subtypes might not be classified as separate entities , even though these classifications have important diagnostic and prognostic implications .
type 2 consists of a cloverleaf skull with pfeiffer hands and feet , together with ankylosis of the elbows .
3 ps , ocular proptosis is severe , and the anterior cranial base is markedly short .
early demise is characteristic of both types 2 and 3 , which to date has been reported to occur only as sporadic cases ( 7 , 9 ) .
clinical manifestations of this patient appeared to fit into subtype 1 according to cohen 's clinical classification of ps .
the etiology for this syndrome is autosomal dominant or fresh mutations in type i conditions , and sporadic in types ii and iii .
some cases are linked to mutations at the fgfr 1 gene at chromosome 8p11.22-p12 ( 4 ) .
mutations at the fgfr2 gene , which map at chromosome 10q25-q26 , have also been reported recently ( 9 ) .
these mutations are related to the old age of parents , especially the father , because these mutations are suspected to confer a selective advantage to survival of sperm ( 13 ) .
usually , the parents are not affected and the risk for future children of that couple is minimal .
the offspring of a patient with ps have a 50 - 60% chance of inheriting the syndrome due to the dominant characteristics of this gene .
ps may involve craniosynostosis that is most often of the coronal and lambdoid , and occasionally sagittal sutures . a severe form of craniosynostosis in the ps is kleebattschdel ( cloverleaf anomaly ) .
the abnormal skull shape could result in a small intracranial volume that increases intracranial pressure , which might be commonly presented as headaches or visual changes .
some children with ps may have only one or both mid - face problems and craniosynostosis .
the most common features of the face are regressed mid - face and shallow orbits , which may be present at birth but sometimes become more evident as the childhood progresses .
underdeveloped maxillary bone of the face results in very shallow orbits and exophthalmos or proptosis .
the shallow orbits and proptosis may lead to damage of the cornea from dryness , or exposure keratosis .
maxillary hypoplasia also results in a small larynx and pharynx behind the nose and mouth .
this restricts the passage of air into the trachea and lungs and causes respiratory distress , particularly at night when snoring and snuffling can interrupt sleep .
similarly , the passage of food is restricted , and regurgitation may result in aspiration of food into the lungs .
( 14 ) in 1990 described an infant with ps in whom the trachea showed replacement of the cartilaginous rings by a solid cartilaginous plate extending the full length of the trachea and beyond the carina resulting in tracheal stenosis .
some other features commonly seen in these patients are visual disturbances related to an imbalance of the muscle that move the eyes and hearing loss due to recurrent ear infections .
vallino - napoli ( 15 ) in 1996 reviewed the audiologic and otologic features of 9 patients with ps , ranging in age from 2 to 12 yr .
seven patients had conductive hearing loss and 1 had mixed loss ; none had purely sensorineural loss .
primary ct findings were stenosis and/or atresia of the external auditory canal , hypoplasia of the middle ear cavity , and an enlarged middle ear cavity .
( 3 ) reported upper airway compromise in three of four patients with ps type 2 , and five of seven patients with ps type 3 , and cited this as the cause of death in two cases .
mccarthy et al . ( 16 ) found upper airway anomalies in 5 of 15 patients with ps of unspecified severity , and these were the primary cause of death in two patients .
congenital anomalies of the upper airway are clearly an important cause of morbidity and mortality in ps type 2 and 3 but are rare in ps type 1 ( 14 ) .
the hands and feet in ps are involved to variable degree ( 17 - 19 ) .
. patient may have mild soft tissue webbing of syndactyly between the second , third , and fourth digits of either or both hands and feet .
the digits may be short and misshapen . whilst the fusion of the fingers and toes in apert syndrome always involves fusion of the soft tissues of the second , third and fourth digits ,
this manifestation is similar with ps , but often there is fusion of the bones as well as soft tissues in apert syndrome ( 17 ) .
other anomalies in ps could include a cleft palate , choanal atresia , tracheo- and broncho - malacia , cloverleaf skull , fused vertebrae , arnold - chiari malformation , hydrocephalus , and imperforate anus .
the above case presented by authors is in accordance with type 1 ps because the infant had a craniosynostosis only of bilateral coronal suture and demonstrated broad and radially deviated bilateral thumbs and big toes that are characteristic of ps , even though she also had syndactyly of the bilateral 2nd , 3rd and 4th fingers only with soft tissue webbing .
the diagnosis of the apert syndrome is appropriate rather than ps if a patient has broad thumbs and syndactyly with bony fusion ( 17 ) or syndactyly of the second , third and fourth web spaces ( 19 ) .
however , clinical differential diagnosis has been confusing until now in relation with genetic findings because pfeiffer mutation has been reported to occur in a patient with apert syndrome ( 20 ) , and identical mutations in the fgfr2 gene could cause both ps and crouzon syndrome phenotypes ( 21 ) as illustrated by the demonstration of both phenotypes of apert and of ps in experimental mice ( 22 ) .
some authors have attempted to revise the classification for apert , carpenter , crouzon , jackson - weiss , pfeiffer and saethre - chotzen syndromes on a genetic basis . because of widely intermingling of phenotypic expressions or genetic sharing of the same mutation , as yet there is no definite evidence that phenotypic expressions correlate accurately with genetic mutations ( 10 , 20 - 24 ) .
this is probably because the gene that controls these expressions has not been elucidated to date ( 25 ) .
the prognosis depends on the severity of associated anomalies , mainly the severity of the central nervous system compromise .
patients with types 2 and 3 usually expire early in infant or childhood even though some may survive with aggressive medical and surgical management ( 5 ) .
multiple staged surgeries are the general treatment plan for patients with ps . in the first year of life
it is preferred to release the synostotic sutures of the skull to allow adequate cranial volume and thus allowing for brain growth and expansion . in this patient
, we performed a decompressive endoscopic craniectomy at 4 weeks of age for decompression of the high intracranial pressure due to coronal synostosis and maxillary hypoplasia .
secondary decompressive skull remodeling is planned at 4 - 6 months of age , and additional skull expansion surgery would be repeated as the child grows to about 2 or 3 yr of age .
although significant malformation of the fingers and toes are present , usually these function adequately and do not require the surgical correction .
however , this patient had soft tissue webbing between the fingers and toes , and thus will require surgical dividing procedure after infancy for future active physical therapy .
mid - facial advancement may be performed in order to provide an adequate orbital volume and reduce the exophthalmos , and to correct the dental occlusion to achieve appropriate functional position , but these procedures are not common in patients with ps .
this patient also had severe choanal hypoplasia with respiratory distress from maxillary hypoplasia that will be corrected with mid - facial advancement at 5 - 6 yr of age . | pfeiffer syndrome is as rare as apert syndrome in the western population .
this condition is very rare in the asian population and has not been previously reported in korea .
the authors report with a review of literature the case of a newborn baby with pfeiffer syndrome , manifested by bicoronal craniosynostosis , broad thumbs , and big toes .
the infant also had bilateral syndactyly of the fingers and toes , mild proptosis , choanal hypoplasia and maxillary hypoplasia . | INTRODUCTION
CASE REPORT
DISCUSSION |
expansion of polyglutamine ( polyq ) domains causes inherited neurodegenerative diseases , such as huntingtons , disease , various types of spinocerebellar ataxia , and spinobulbar muscular atrophy ( sbma ) [ 1 , 2 ] .
the formation of intranuclear inclusions of polyq - containing proteins in the brain is one of the pathological features of these diseases .
inhibition of the aggregation of the expanded polyq - containing proteins may therefore provide treatment options for polyq diseases ; however , the relationship between polyq aggregation and cellular toxicity is not well understood .
it was demonstrated in earlier studies in human cells that targeted expression of proteins with an expanded polyq repeat led to nuclear inclusion formation followed by late - onset cell degeneration .
similar observations have been made in drosophila suggesting that the cellular mechanisms underlying human glutamine - repeat diseases are conserved in invertebrates . from a therapeutic view point
, drosophila provides a unique system in which to search for novel genetic factors that suppress cellular toxicity .
previous studies have indicated that qbp1 ( polyq binding peptide 1 ) binds to the expanded polyq stretch and inhibits aggregate formation of the expanded polyq protein in vitro [ 5 , 6 ] .
furthermore , ter 94 , the drosophila homolog of the myeloid leukemia factor 1 , and heat - shock - transcription - factor-1 ( hsf1- ) activating compounds are also reported to have a dominant role in suppressing polyq aggregation [ 79 ] .
alpha - crystallins ( -crystallins ) are major protein components of the vertebrate eye lens .
the -crystallins and especially b - crystallin are also found outside the lens , and show a wide tissue distribution .
the b - crystallin protein is found to be overexpressed in many neurological diseases , and mutations in a- or b - crystallin can cause cataracts and myopathy .
the b - crystallin protein is a member of the small heat shock protein family , and it has a chaperone - like function [ 1215 ] .
therefore , b - crystallin can bind to the unfolded proteins and inhibit nonspecific aggregation .
consistent with this it has been shown to prevent the fibrillar aggregation of proteins implicated in human diseases and limit the onset of misfolding diseases [ 1618 ] .
furthermore , accumulation of b - crystallin is an important mechanism for the prevention of cellular damage associated with heat shock and oxidative stress conditions in the trabecular meshwork [ 19 , 20 ] .
-synuclein is a presynaptic protein whose major role in neurodegeneration was recognized in parkinson disease where a mutation in the -synuclein gene was associated with autosomal dominant inheritance of the disease .
it is reported that b - crystallin interacts with -synuclein to protect against amyloid aggregation and limit the onset of misfolding diseases in vitro . in the present study
, we investigate the ability of the molecular chaperone , b - crystallin , to inhibit the aggregation of protein induced by ectopic expression of polyq and -synuclein in fly model .
our results indicate that b - crystallin suppresses the neurodegenerative effects induced by overexpression of polyq as well as -synuclein .
the transgenic fly line carrying gmr - gal4 on x chromosome was described earlier as was the transgenic fly line carrying gmr - q92 [ 4 , 7 ] .
the uas - lacz flies used in this study were obtained from bloomington drosophila stock center . wild - type flies : w ; uas- syn ; + , was obtained from .
, the full coding sequence of human b - crystallin cdna ( accession number s45630 ) corresponding to amino acids ( aa ) 1 to aa175 was amplified by pcr with primers carrying bglii site and xhoi site ( ab - crystallin - bglii : 5-gca gat ctg aca tcg cca tcc acc acc , ab - crystallin - xhoi : 5-cgc tcg agc tat ttc ttg ggg gct gc ) . the pcr products were digested with bglii and xhoi and inserted into the plasmid puast - ha ( lablife ) .
the p element - mediated germ line transformation with puas-b - crystallin - ha was carried out as describe earlier , and f1 transformants were selected on the basis of white - eye color rescue . the established transgenic fly lines and their chromosome linkages are listed in table 1
. proteins were extracted from eye imaginal discs of third - instar larvae , applied to a sds - polyacrylamide gel containing 12% acrylamide and transferred to polyvinylidene difluoride membranes ( bio - rad ) in transfer buffer ( 50 mm borate - naoh , ph 9.0 , and 20% ethanol ) by using iblot system ( invitrogen ) .
the blotted membranes were incubated with monoclonal antibodies to ha ( ha124 , invitrogen ) at 1 : 400 dilution or anti-tubulin igg ( sigma ) at 1 : 2.000 for 16 h at 4c .
the bound antibodies were detected with peroxidase - conjugated goat anti - mouse igg and the ecl system ( ge healthcare ) according to the manufacturer 's recommendations .
the images were analyzed with a lumivision prohsii image analyzer ( aisin seiki , aichi , japan ) .
adult flies were anesthetized , mounted on stages , and observed under a ve-7800 scanning electron microscope ( keyence inc . ) in the high vacuum mode . in every experiment ,
the eye phenotype of at least five adult flies of each line was simultaneously examined by scanning electron microscopy , and these experiments were repeated 3 times . in the experiments , no significant variation in eye phenotype among the five individuals was observed .
third instar larvae were dissected in pbs , and tissues were fixed in 3% paraformaldehyde / pbs for 30 min at 25c . after washing with pbs/0.3% tritonx-100 ( pbs - t ) , the samples were blocked with pbs - t containing 10% normal goat serum for 20 min at 25c and incubated with an anti - ha ( invitrogen ) , anti - flag monoclonal antibody ( sigma ) , or anti--galactosidase monoclonal antibody ( developmental studies hybridoma bank , dshb ) at a 1 : 200 dilution at 4c for 16 h. after extensive washing with pbs - t , the tissues were incubated with an anti - mouse igg conjugated with alexa 594 or alexa 488 ( molecular probes ) at a 1 : 400 dilution each at 37c for 1 h. after extensive washing with pbs - t and pbs , samples were mounted in fluor guard antifade reagent ( bio - rad ) for microscopic observation under a fluorescence bx-40 microscope ( olympus , tokyo , japan ) equipped with a cooled ccd camera ( orca - er ; hamamatsu photonics k. k. , shizuoka , japan ) .
we first established lines of transgenic flies carrying the uas - human-b - crystallin . in order to confirm that human-b - crystallin is functional in drosophila
, we used the gmr - gal4 driver strain to provide ectopic expression of b - crystallin - ha from the morphogenetic furrow to the posterior end of the drosophila eye imaginal disc . by inspection with the scanning electron microscope ,
the compound eyes of adult flies carrying the gmr - gal4 alone showed normal eye morphology ( figures 1(a ) and 1(b ) ) , while those of flies carrying both the gmr - gal4 and the uas-b - crystallin exhibited a rough - eye phenotype ( figures 1(d ) and 1(e ) ) . to further confirm the expression of b - crystallin carrying an ha tag in its c - terminal end in eye imaginal discs , we carried out immunostaining of eye imaginal discs from third instar larvae using anti - ha antibody . only gmr - gal4 ; uas-b - crystallin flies showed ectopic b - crystallin - ha signals from the morphogenetic furrow to the posterior end of eye discs ( figure 1(f ) ) .
in contrast , the gmr - gal4 line that was used as the negative control showed no signal ( figure 1(c ) ) .
the expression of b - crystallin - ha was further confirmed by the western immunoblot analysis of eye imaginal disc extracts with anti - ha antibody , detecting a single b - crystallin - ha band at around 20 kda ( figure 1(g ) ) . to examine the effect of b - crystallin protein on polyq - induced neurodegeneration in drosophila , we crossed gmr - q92 fly lines expressing a q92 peptide in the eye discs under the direct control of the gmr promoter [ 5 , 7 ] , and gmr - gal4 > uas-b - crystallin - ha ( uas-b - ha ) flies in which b - crystallin - ha expression is driven by gmr - gal4 .
expression of the flag - tagged 92-glutamine peptides induced compound eye degeneration such as rough eye and loss of red eye pigment ( loss - of - pigment phenotype ) as described ( figures 2(a ) and 2(d ) ) [ 5 , 7 ] and the eye phenotype , especially the loss - of - pigment phenotype , was effectively rescued by coexpression of b - crystallin - ha ( figures 2(c ) and 2(f ) ) .
in contrast , expression of the control lacz protein did not affect the polyq - induced phenotype ( figures 2(b ) and 2(e ) ) . in order to investigate the ability of b - crystallin to inhibit polyq aggregation in vivo , we examined polyq aggregate formation in the eye imaginal discs of third instar larvae in the gmr - q92 fly lines with and without expression the b - crystallin - ha .
polyq forms inclusion bodies in the region posterior to the morphogenetic furrow in eye discs of the gmr - q92 flies and many of these aggregate formations could be detected by immunostaining with anti - flag antibody ( figures 3(a1 ) and 3(c1 ) ) .
expression of b - crystallin - ha completely suppressed formation of the polyq aggregates in the line exhibiting a mild eye phenotype ( gmr - q92 ) ( figure 3(b1 ) ) and dramatically suppressed it in the line exhibiting a severe eye phenotype ( 2xgmr - q92 ) ( figure 3(d1 ) ) , while expression of control lacz protein exerted no suppressing effect ( figures 3(a1 ) and 3(c1 ) ) .
these results suggest correlation between formation of the polyq aggregates and neurodegeneration . a previous in vitro study indicated that b - crystallin was very effective in suppressing fibrillization of both -synuclein wild - type and a30p -synuclein mutant .
therefore , in this study , to evaluate ability of b - crystallin to suppress the rough - eye phenotype induced by ectopic expression of -synuclein , we performed coexpression of b - crystallin and -synuclein in fly eyes and compared the results with those of control flies coexpressing lacz and -synuclein .
as noted previously , overexpression of wild - type -synuclein induced rough eye phenotype ( figures 4(g ) and 4(h ) ) .
the rough eye phenotype was significantly suppressed by coexpressing b - crystallin ( figures 4(a ) and 4(b ) ) , while no effect on the rough eye was observed by coexpressing control lacz ( figures 4(c ) and 4(d ) ) .
these results indicate that b - crystallin protein effectively suppresses the events induced by overexpression of -synuclein . in this study
, we investigated a role of b - crystallin on polyq aggregation in a drosophila model to gain insight into pathways which could be useful in the treatment of neurodegenerative diseases .
we used immunohistochemical methods to show that the gmr - gal4/uas-b - crystallin flies expressed an abundant b - crystallin protein in the region from the morphogenetic furrow to the posterior end of eye discs .
these results indicate that the human b - crystallin protein is functional in flies .
previous studies showed that b - crystallin can act as a molecular chaperone to prevent fibril formation and aggregation of -synuclein , 2-microglobulin , and -casein in vitro [ 10 , 12 , 1517 , 2729 ] .
moreover , chaperone activity sites of b - crystallin were identified in the sequences from residues aa73-aa85 and aa101-aa110 [ 3032 ] , suggesting that expression of full length or a part of chaperone sequences of this protein is capable of suppressing polyglutamine - induced protein aggregation . in the present study
, we found that the full - length b - crystallin could inhibit aggregation of the 92-polyq peptide in vivo .
the results support the hypothesis that interactive domains in b - crystallin play a role in prevention of protein aggregation .
in contrast , the recent study shows that b - crystallin does not inhibit or modify the fibrillar aggregation of spacq52 in vitro . because glutamine is a hydrophilic amino acid , the surface hydrophobicity of polyq
furthermore , the polyq region is thought to be in a condensed disordered state in the native conditions .
therefore , the physiochemical properties of the polyq tract may lead it to interact with chaperonin differently in vitro compared with in vivo .
the effectiveness of suppression of polyq disorders may also depend on the other factors , such as the temperature , concentrations of chemical compounds , or the inhibitor proteins [ 33 , 35 , 36 ] .
here , our data indicate that inhibition of polyq - aggregation by b - crystallin in flies carrying one copy of the q92 gene is complete , but flies carrying two copies of the q92 gene show only partial suppression , suggesting the importance of optimization to determine the effective level of inhibitors for therapies for polyq diseases .
more interestingly , it is reported that b - crystallin is a potent in vitro inhibitor of the fibrillization of -synuclein , a protein involved in neurodegenerative diseases , both wild - type and the two mutant forms ( a30p and a53 t ) [ 15 , 16 ] .
the data we present here suggests that this is achieved by the interaction of b - crystallin and -synuclein inhibiting further aggregation .
the present study has demonstrated an essential role of b - crystallin in suppressing the neurodegeneration induced by polyq expansion and ectopic expression of -synuclein in vivo and suggest a possible target for the development of therapeutics for neurodegenerative diseases .
moreover , the assay system to suppress neurodegeneration using transgenic flies described in the present study should be a powerful tool to evaluate the in vivo effect of peptides carrying potential chaperone activities , and the b - crystallin - overexpressing flies established in this study might provide an invaluable standard for the evaluation . | disorganisation and aggregation of proteins containing expanded polyglutamine ( polyq ) repeats , or ectopic expression of -synuclein , underlie neurodegenerative diseases including alzheimer 's , parkinson , huntington , creutzfeldt diseases .
small heat - shock proteins , such as b - crystallin , act as chaperones to prevent protein aggregation and play a key role in the prevention of such protein disorganisation diseases . in this study , we have explored the potential for chaperone activity of b - crystallin to suppress the formation of protein aggregates .
we tested the ability of b - crystallin to suppress the aggregation of a polyq protein and -synuclein in drosophila .
we found that b - crystallin suppresses both the compound eye degeneration induced by polyq and the -synuclein - induced rough eye phenotype .
furthermore , by using histochemical staining we have determined that b - crystallin inhibits the aggregation of polyq in vivo .
these data provide a clue for the development of therapeutics for neurodegenerative diseases . | 1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusion |
it is important to understand the mechanism of the oxidatively generated damage to guanines in dna .
the reason is that the damage causes mutations and cancer , and it is an obstacle to use dna as a molecular device.(1 ) in the case of the damage due to a one - electron oxidant , the reactions occur as follows .
it then migrates to a nearby guanine base , because a guanine base has the lowest ionization potential ( ip ) among the dna bases .
the trapped hole escapes to other guanine bases or triggers the chemical reactions which cause the damage to the guanine .
it was found that the longer guanine run in a duplex dna is damaged more than the shorter ones .
it was also reported that the attachment of a phenyl group to a guanine suppresses the damage not only at the phenylated guanine but also at nearby guanines in the duplex dna.(18 ) to understand the mechanism underlying these experimental results , we need to know how the hole transfer reactions occur in dna .
one of the important parameters determining the charge transfer rate is the free energy difference between the reactant and the product .
thus , in the present work , we theoretically analyzed the ip of duplex dna corresponding to the free energy of the transferring hole on the duplex dna . since the above experiments were carried out in the presence of a solvent ( water ) , we took into account the solvent ; this turned out to be crucial to understanding the above experimental results .
one of our goals is to resolve the discrepancy between the theoretical results and the experimental one;(21 ) these results are about the free energy dependence of the transferring hole on the length of the guanine runs .
the ips ( corresponding to the free energy of the hole ) obtained by the ab initio hartreefock ( hf ) molecular orbital ( mo ) calculations are smaller for the longer guanine run .
these calculations were performed for the dna oligomers without a backbone in the gas phase .
this result of the ip is in qualitative agreement with the experimental results as follows .
if the free energy of the hole on the longer guanine run is smaller , then the probability of the hole to be there is larger and thus the longer one is damaged more .
however , the calculated ip difference between g and gg ( ggg ) of 0.47 ( 0.68 ) ev is too large compared to the corresponding free energy difference of 0.052 ( 0.077 ) ev obtained from the time - resolved data for dna oligomers in water.(21 ) that is , if the previously calculated value was close to the real value , then the hole transfer from the longer guanine run to the shorter one hardly occurred and thus the distribution of the damage in the dna would become considerably different from the real one . here
, we neglect the entropy difference in the free energy difference between the reactant and the product of the hole transfer reaction by assuming that the potential functions of the reactant and the product are harmonic around the equilibrium structures and their second derivatives are similar .
another question to be answered in the present paper is about the effect of the chemical modifications of dna oligomers on the hole transfer reactions . when a neutral functional group is attached to a guanine in a dna oligomer in water ,
this removal was found to reduce the free energy of the hole on the guanine.(22 ) this might sound paradoxical .
that is , since a polar solvent usually reduces the free energies of ions , the removal of nearby solvent molecules might be expected to increase the free energy of the hole
it will turn out that this mechanism also explains the discrepancy mentioned in the previous paragraph .
the free energy reduction of the hole mentioned above was confirmed by the ab initio and the semiempirical hf calculations for various neutral functional groups , i.e. , the phenyl , the benzyl , and the tert - butyl groups with the two structures of the typical b - dna structures.(22 ) this free energy reduction was observed even when the phenyl group attached was replaced by an artificial h2 cluster mimicking the solvent - accessible surface of the phenyl group .
thus , it is considered that this free energy reduction is due to the removal of the solvent molecules.(22 ) this result agrees with the experimental one(18 ) that the damage to guanines near the phenylated guanine is suppressed as follows .
the phenyl group attachment to a guanine reduces the free energy of the hole on it , and thus , the hole is trapped there ; therefore , the guanines near the phenylated guanine are less damaged .
we briefly review the relevant works on the electrostatic interaction and the solvent effects on dna , since they turned out to play crucial roles in the present cases .
the electrostatic potential in dna has been studied for many years in order to understand its reaction with other molecules .
it was pointed out that the effect of the electrostatic interaction on the ip of a guanine doublet is important.(25 ) as for the solvent effects , the hydration effect on the ip of the small fragments of dna , namely , the watsoncrick ( wc ) base pairs , nucleotides , and a phosphorylated dinucleotide , was investigated .
recently , the solvent effects on the much - larger - sized dna were investigated .
these studies showed the importance of the solvent effects on dna . in the present work
, we calculated and analyzed the ip of the duplex dna by taking into account the solvent , where this ip corresponds to the free energy of the hole on the duplex dna .
on the basis of the same mechamism , we answer the above two questions as follows . since the electrostatic interaction between the hole and a nucleotide pair in the duplex dna is attractive , the ip of the longer guanine run is smaller than that of the shorter one .
however , this attractive interaction is reduced in water , which results in the small difference of the ip between the longer guanine run and the shorter one in water .
this mechanism explains why the theoretical results without taking into account the solvent were too large compared to the experimental one.(21 ) the effect of the chemical modification is understood as follows .
a neutral functional group attached to a guanine in a duplex dna in water expels the nearby water molecules which reduce the electrostatic interaction .
thus , the attractive electrostatic interaction stabilizing the hole on the guanine increases and the hole is stabilized .
this is why the free energy of the hole decreases when nearby water molecules are removed .
we calculated the ip of the duplex dna oligomers in the gas phase and in water as follows .
we used the two structures of the typical b - dna structures , namely , the 55th(37 ) and the 4th(38 ) fiber models , to ensure that the result is independent of the structure deviation within the b - dna structure .
their global structures are similar ; the helical twists are 36 for both of the two models and the rises are 3.39 and 3.38 for the 55th and 4th fiber models , respectively .
these model structures are determined by x - ray diffraction , and thus , the coordinates of the h atoms are missing . therefore , we added these coordinates and optimized them using the electronic structure calculations described in the next paragraph .
we calculated these optimized coordinates for dna oligomers , which are in the neutral state in vacuum and in water , and in the ionic state in water .
we calculated the electronic structures using the austin model 1 ( am1 ) hamiltonian(39 ) implemented in the mopac2002 v1.0 program.(40 ) ( we did not use the linear scaling calculation program mozyme . ) we included the solvent as the dielectric continuum , namely , the conductor - like screening model ( cosmo ) with the dielectric constant of water , 78.4 , and the vdw parameters optimized for the density functional theory.(42 ) these optimized parameters are expected to produce better results than the default ones implemented in the software;(43 ) this expectation was confirmed in the case of the ip of the nucleosides.(22 ) we calculated the ip as the difference between the total energies after and before one electron is removed . for these calculations
there was no spin contamination in the case of an even number of electrons . in the case of an odd number of electrons ,
the spin contamination was less than about 3% and thus the error in the energy due to the spin contamination was calculated to be less than a few tenths of an ev . as for the ip in water , we calculated it for dna oligomers in the neutral state and the ionic state ; in the latter state , the phosphate backbones are completely ionized .
the actual ip in water is considered to be between these ips , since the ionic ( neutral ) state corresponds to the case when the counterions are far away from ( very close to ) the dna oligomers.(22 ) as for the validity of the method , we calculated the relative ips of the nucleosides in water ( the relative ops ) , and compared them to the experimental result.(44 ) the root - mean - square ( rms ) deviation between the computational and the experimental result(44 ) was 0.14 v ( table 1 ) , which is similar to the experimental error.(44 ) in addition , the method described here was successfully used to calculate the ips and the electron affinities of nucleic acid bases in the gas phase , where the rms deviations between the computational results and the experimental ones are 0.12 and 0.20 ev , respectively .
the ips calculated by the method include the reorganization energies due to the relaxation of the solvent and h atoms of the solute but not that due to the relaxation of the heavy atoms of the solute .
our conclusions are not affected by this approximation , as will be discussed in section .
the solvation energy for the solvent change acetonitrile / h2o is calculated to be 0.02 ev using the born equation,(44 ) and thus , the correction to the relative op for the solvent change is negligible . in order to interpret the ips obtained by the electronic structure calculations
, we used the net charges and dipoles of atoms and the electrostatic interaction calculated from them .
the charge distribution of the hole is defined as the difference in the charge distributions between after and before the removal of an electron .
we calculated the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 1a ) ; we found that the ip in the gas phase significantly decreases with the increasing number of base pairs , n , whereas the ip in water ( or op ) only slightly does ( figure 1b ) .
the decrease in the ip in the gas phase from n = 1 to 3 is more than about 1 ev , but that in water is less than 0.2 ev .
the latter result is consistent with the experimental result for the dna oligomers in water.(21 ) the difference between the ip decreases in the gas phase and in water is much larger than the rms deviation ( 0.14 v ) between the computational and the experimental results obtained in section .
the values of the ips in the gas phase rapidly decrease for n 3 and converge for n 5 or 6 .
about 90% of the charges of the hole is in a certain guanine and 5% is in the sugar covalently bonded to this guanine both in the gas phase and in water .
we confirmed the above results using the two structures of the typical b - dna structures .
note that the actual ip in water is between the solid and dashed lines , as described in section .
( a ) the structure of the duplex dna d[5-(g)6 - 3]d[3-(c)6 - 5 ] .
( b ) the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs for the 55th fiber model structure and the 4th fiber model structure , as indicated .
solid line with closed circles : the dna oligomer in the neutral state in water . dashed line with closed diamonds : the one in the ionic state in water .
dotdashed line with closed squares : the one without a backbone in the gas phase .
we calculated the ip of the dna oligomers , d(5-aagaa-3)d(3-ttctt-5 ) and d(5-aggga-3)d(3-tccct-5 ) , where the lengths of the guanine runs are different but the total number of base pairs is fixed ; we again found that the ip difference between the two sequences in water , 0.11 ( 0.10 ) ev , is smaller than that in the gas phase , 0.21 ( 0.15 ) ev , for the 55th fiber model ( the 4th fiber model ) .
as shown in figure 2 , the ips calculated above ( the closed symbols ) are close to the electrostatic energy of the hole both in the gas phase ( the open triangles ) and in water ( the open circles ) .
it is also shown that this energy in the gas phase decreases more strongly when we replace the charge distribution of the dna oligomer in the gas phase by that in the neutral state in water ( open inverted triangles ) .
we define the electrostatic energy of the hole as the electrostatic interaction between the hole and all atoms except for the atoms of the guanine where the hole is localized plus a constant ; this constant is such that the value of this electrostatic energy coincides with the ip in the gas phase for n = 1 . in the case of the dna oligomer in water
, we added the electrostatic interaction between the hole and the water ; we obtained this interaction as the dielectric energy,(49 ) which is considered to be the free energy but not the energy .
we neglected the charge distribution of the hole outside the guanine where the hole is localized .
we used the charge distribution after the removal of an electron as the charges which interact with the hole .
we confirmed the above results using the two structures of the typical b - dna structures .
comparison of the electrostatic energy of the hole ( defined in the text ) with the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs for the 55th fiber model structure and the 4th fiber model structure , as indicated .
the symbols and lines are the same as those in figure 1b except that the open symbols denote the electrostatic energy of the hole .
open inverted triangles : the dna oligomer in the gas phase but with the charge distribution of the dna oligomer in the neutral state in water . to understand the electrostatic interactions in the dna oligomers ,
we calculated the charge distributions of the dna oligomer d[5-(g)53]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water ( figure 3 ) ; we found that the bases and sugars have negative partial charges and the phosphates have positive partial charges both in the gas phase and in water ( figure 4 ) .
( the phosphate backbones in the neutral state are not ionized . ) about 90% of the charges of the hole is in a certain guanine both in the gas phase and in water , as described earlier .
we observed the same features in other sequences of the dna oligomers ( not shown ) .
charge distributions in the dna oligomer d[5-(g)5 - 3]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water , as indicated .
g , c , s , and p denote guanine , cytosine , sugar , and a part of the phosphate , respectively .
simplified charge distributions in the dna oligomer d[5-(g)5 - 3]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water , as indicated .
same as in figure 3 except that the net charges of the fragments are shown .
the fragment from which the largest fraction of an electron is removed is shown by the bold fonts and bold lines .
the net charge of the fragment after removing an electron is indicated by an arrow .
the fragments of sugars and bases are divided at the n9c1 bond for guanines and the n1c1 bond for cytosines .
the fragments of the phosphates and sugars are divided at the po5 and po3 bonds .
we calculated the ip of the dna oligomers with other sequences , where a guanine is embedded in the center of the contiguous adenines or thymines , and obtained the same result with that of d[5-(g)n-3]d[3-(c)n-5 ] ( figure 5 ) .
that is , the ip significantly decreases with the increasing n in the gas phase , whereas it does only slightly in water .
the values of the ip decreases are slightly smaller than the corresponding values of the contiguous guanines in figure 1b , both in the gas phase and in water .
ip of the dna oligomers as a function of the number n of base pairs , where a guanine is embedded in the center of the contiguous adenines or thymines .
( a ) n = 1 , 2 , 3 , 4 , 5 , and 6 are for d(5-g-3)d(3-c-5 ) , d(5-ag-3)d(3-tc-5 ) , d(5-aga-3)d(3-tct-5 ) , d(5-aaga-3)d(3-ttct-5 ) , d(5-aagaa-3)d(3-ttctt-5 ) , and d(5-aaagaa-3)d(3-tttctt-5 ) , respectively .
( b ) same as in figure 5a except that a and t are exchanged in the sequences of the dna oligomers . to understand the dependence of the obtained ips on n
, we calculated the ip of the system made of two gc base pairs ( the inset in figure 6 ) as a function of the rise r ; we found that the ip of this system ( the solid line in figure 6 ) is close to the ip of the single base pair plus the electrostatic interaction between the hole and the base pair where the hole does not reside ( the dashed line in the same figure ) .
the values of the ip rapidly increase for r 5 and converge for r 10 .
we used the method described in section except that we removed the sugarphosphate backbones ( except the c1 atom ) from the structure of the dna oligomer d(5-gg-3)d(3-cc-5 ) , capped the dangling bonds with the h atoms , and optimized the coordinates of h atoms for single base pairs .
that is , the structure of the system in the cationic ( neutral ) state is made of the optimized structure of the gc base pair of the 5-side in the cationic ( neutral ) state and that of the gc base pair of the 3-side in the neutral state .
we calculated the electrostatic interaction between the hole and the base pair where the hole does not reside ; this interaction is the electrostatic interaction between one base pair in the cationic state and the other in the neutral state , minus that between the two base pairs both in the neutral state .
more than 97% of the charges of the hole is in the guanine of the 5-side in the calculated electronic structures of the system .
ip of the two gc base pairs as a function of the rise r. the inset shows the structure of the two gc base pairs .
solid line : ip of the system made of the two gc base pairs . dashed line :
ip of the single gc base pair plus the electrostatic interaction between the hole and the base pair where the hole does not reside ( defined in the text ) .
to understand the reduction in the ip decrease with the increasing n due to the solvent , we developed simple models of dna oligomers ( figure 7a ) ; we showed that these models reproduce the ips of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 7b ) . in this figure ,
the open symbols are for the simple models and the others are the same as those in figure 1b .
a duplex dna oligomer is modeled as a box with the simplified charge distribution of a duplex dna oligomer in the neutral state ( the hole is localized on a certain guanine ) and the water is replaced by a conductor ( figure 7a ) .
the reason is that the dielectric constant of water is large ( 78.4 ) and the solvation energies of the dielectric continuum scale as ( 1)/( + x ) with the dielectric constant , where 0 x 2.(50 ) ( a ) charge distributions of the simple models for dna pentamers with and without backbones , as indicated .
one electron is removed from the filled circle marked by the outer circle , when the dna is oxidized .
the size of the box in the x- and y - directions is 18 .
charge distributions for dna oligomers with different numbers of base pairs are defined in the same way as for the pentamers .
( b ) comparison of the ips of the simple models with those obtained by the mo calculations in section for the 55th fiber model structure ( figure 1b ) .
the symbols and lines are the same as those in figure 1b except that the open symbols are for the simple models of the dna oligomers in the gas phase , those in water in the neutral state , and those without a backbone in the gas phase , as indicated
. to discuss the obtained ips , we calculated the vertical ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 8) , and obtained a similar result with that of the ip ( figure 1b ) .
that is , the vertical ip more significantly decreases with the increasing n in vacuum than in water .
since , by definition , the vertical ip does not include the reorganization energy , we obtained the vertical ip as the energy level of the highest occupied molecular orbital ( homo ) with the opposite sign using the restricted hf ( rhf ) method with koopmans theorem .
the vertical ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs .
we calculated the ip of the modified nucleotide - containing dna oligomer , d[5-t1a2g3g4t5a6 - 3]d[3-a12t11c10c9a8t7 - 5 ] , where a phenyl group is attached to g3 ( figure 9a ) ; we found that the attachment of a phenyl group reduces the ip in water but not in the gas phase ( figure 9b ) .
for the phenylated dna oligomer in water , the hole is always localized on the phenylated guanine g3 .
we used the dna sequence of the six base pairs around the phenylated guanine in the experiment.(18 ) we used the method described in section except that we optimized the coordinates of the phenyl group atoms by the electronic structure calculations .
as for a single base , the op of n - phenyldeoxyguanosine g relative to that of deoxyguanosine was 0.03 v in the experiment,(18 ) and the calculated one is 0.09 v , where the geometries are fully optimized . in the experiment,(18 )
the solvent was dimethylformamide ( dmf ) ; the solvation energy for the solvent change dmf / h2o is calculated to be 0.02 ev using the born equation and thus the correction to the relative op for the solvent change is negligible .
( a ) structure of the modified nucleotide - containing dna oligomer , d[5-t1a2g3g4t5a6 - 3]d[3-a12t11c10c9a8t7 - 5 ] , where a phenyl group is attached to g3 .
the dna sequence is the sequence of the six base pairs around the phenylated guanine in the experiment.(18 ) ( b ) the effect of the phenyl group attachment on the ip of the dna oligomer in the gas phase , in the neutral state in water , and in the ionic state in water , for the 55th fiber model structure and the 4th fiber model structure , as indicated .
the ips before and after a phenyl group is attached are indicated by g and g , respectively .
figure 1b shows that the large decrease ( in the order of 1 ev ) of the ip in the gas phase with the increasing dna length is reduced to about a tenth of an ev in water .
this reduction in the ip decrease is due to the solvent , since the computational error is less than about a tenth of an ev ( section ) .
we can now understand why the calculated ip difference between g and gg ( ggg ) ( in the order of 1 ev ) is much larger than the experimental one ( in the order of 0.1 ev).(21 ) this is because the solvent is not included in the calculations . in the present work , we used the two structures of the typical b - dna structures , i.e. , the 55th(37 ) and the 4th(38 ) fiber models .
these models do not include the sequence dependence of the dna structure ; namely , the structure parameters of a base are independent of nearby bases . since figure 1b shows that the general features of the ips are similar for both structures , we consider that the effect of the structure deviation within the b - dna structure on the ip is minor and
so is that of the sequence dependence of the dna structure . from figure 2 , it is clear that the decrease of the electrostatic energy of the hole causes the ip decrease with the increasing number of base pairs , n. since the electrostatic energy of the hole decreases with the increasing n in figure 2 , the electrostatic interaction between the hole and a nucleotide pair is attractive in a duplex dna ; this nucleotide pair includes the two phosphates bonded to it .
we now understand the obtained dependence of the ips on n as follows . because of the attractive interaction between the hole and a nucleotide pair , the ip in the gas phase decreases with the increasing n. this attractive interaction is reduced in water , and so is the ip decrease with the increasing n. when we replace the charge distribution of the dna oligomer in the gas phase by that in the neutral state in water , the electrostatic energy of the hole in the gas phase decreases more strongly ( figure 2 ) .
the reason is that the polarization of the dna oligomer is induced in water ( figure 4 ) . from the simplified charge distribution of the dna oligomer in figure 4
, we understand why the electrostatic interaction between the hole and a nucleotide pair is attractive in the duplex dna ; this nucleotide pair includes the two phosphates bonded to it .
the reason is that the hole is on a guanine and thus it is closer to nucleobases and sugars , which have negative partial charges , compared to phosphates , which have positive partial charges .
we consider that the simplified charge distribution can be used to understand the above reason because the hole is delocalized over a guanine base and the rough electrostatic potential is smoothed .
the ip in the gas phase decreases more strongly than that without a backbone ( figure 1b ) , because the positive partial charges are separated more from the hole for the dna oligomer with backbones compared to that without . in water ,
the ips of the dna oligomers in the ionic states are smaller than those in the neutral states ( figure 1b ) .
this is because the pn s , which have positive partial charges in the neutral state of dna oligomers , become almost neutral in the ionic state of the dna oligomers and the repulsive interaction between pn s and the hole is reduced in the ionic state .
the charge distribution of the dna oligomer in figure 4 is in accord with the result that the electrostatic potential around the guanine is lower in the interior than at the end of the sequence.(51 ) figure 5 shows that the similar dependence of the ip is observed also for the different sequences .
we consider that the reason is the same as that for the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] , since the charge distribution has the same features , as mentioned above .
figure 6 shows that the attractive electrostatic interaction between the hole and a base pair causes the ip dependence of the rise r ; this supports that this attractive interaction causes the dependence of the ip on n in figure 1b .
the ip in figure 6 rapidly increases for r 5 and converges for r 10 . from this result
, we can understand that the ips in the gas phase in figure 1b rapidly decrease for n 3 and converge for n 5 or 6 by considering that the hole is usually in the middle of the dna oligomer and the rise in the duplex dna is 3.4 .
the simple models of dna oligomers ( figure 7a ) illustrate that water molecules around the duplex dna ( but not between the base pairs ) reduce the electrostatic interaction in the duplex dna ( figure 7b ) .
as shown in figure 8 , the vertical ip more significantly decreases with the increasing n in vacuum than in water , similarly to the ip in figure 1 .
as for the dielectric shielding , when we use koopmans theorem , the hole is not shielded by the solvent , but other charges are shielded , and thus , the attractive electrostatic interaction between them is reduced in water .
thus , this reduction in the attractive electrostatic interaction leads to the result that the vertical ip in water is larger than that in the gas phase .
because of the reorganization energy , the ips in water are more than a few ev smaller than the corresponding vertical ips .
the result of the ips include the electronic energy reduction due to the charge redistribution in dna after an electron is removed .
however , that of the vertical ip does not . this is because we used koopmans theorem to calculate the vertical ip .
we also observed a slightly smaller decrease in the vertical ip in the gas phase with the increasing n and the slightly larger one in water compared to the corresponding ips .
the reorganization energy due to the relaxation of the dna atoms and the electronic energy reduction mentioned above increase with the increasing n and these are included in the ips but not in the vertical ips .
thus , the ip in the gas phase decreases slightly more than the corresponding vertical ip . as for dna in water ,
when n is increased , a part of the dna replaces a part of the solvent , where the dielectric constant of the former is smaller than that of the latter , and thus , the total reorganization energy decreases .
therefore , the ip in water decreases less than the corresponding vertical ip . since we did not include the reorganization energy due to the relaxation of the heavy atoms of dna , the above decrease in the reorganization energy with the increasing n was overestimated .
thus , the decrease in the exact ip with the increasing n should be between those of the ip and the vertical ip obtained in the present work . as shown in figure 9 , when the neutral functional group is attached to the guanine in the duplex dna in water , the free energy of the hole on the guanine is reduced ( but not in the gas phase ) .
this result is counterintuitive , since the attached group expels the nearby water molecules , which are usually expected to stabilize ions .
thus , when nearby water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases .
as for a single base , guanosine , the calculated op of n - phenyldeoxyguanosine g relative to that of deoxyguanosine is small and positive , i.e. , 0.09 v , which is close to the experimental value of 0.03 ev.(18 ) this means that the free energy of the hole ( or op ) is not reduced by attaching the phenyl group .
the reason is that there is no other bases and sugars which could attractively interact with the hole in the case of a single base .
this result of the free energy reduction agrees with the previous one.(22 ) the difference between the former and the latter is that the former includes the reorganization energy due to the relaxation of the solvent but the latter does not .
the stabilization of the guanine radical cation by the attached phenyl group similarly affects its chemical reactions , namely , the hydration and deprotonation processes,(52 ) as follows .
this stabilization makes the possibility that the hole injected is on the phenylated guanine greater and that for guanines nearby less .
thus , the quantum yields of both of the chemical reactions of the nearby guanines decrease .
in addition to that , this stabilization reduces the free energy of the initial state for both of the chemical reactions of the phenylated guanine radical cation .
this result agrees with the experimental result that the damage to the guanines near the phenylated guanine is suppressed.(18 ) here , we have to be careful about the chemical yield data in the experiment(18 ) as follows . in this experiment ,
thus , the product of the deprotonation was detected , but the products of the hydration processes , namely , 8-oxo-7,8-dihydroguanine ( 8-oxogua ) and 2,6-diamino-4-hydroxy-5-formanido phyrimidine ( fapygua ) were not .
the ratio of these two kinds of damage is sequence dependent,(53 ) and 8-oxogua , which is the main product of the hydration process , is highly susceptible to secondary one - electron oxidation reactions .
however , if the probability of the damage of one kind is larger ( or smaller ) at a site , then so is that for the other kind,(53 ) and this feature is not changed by the secondary one - electron oxidation reactions .
thus , if the probability of the damage of one kind is smaller at a site , then so is the other .
the miscellaneous points about the stabilization of the guanine radical cation by the attached phenyl group are as follows .
according to the above mechanism , the ip of the modified nucleotide - containing dna oligomer in the neutral state in water is supposed to be between the ip of the dna not modified in the neutral state in water and that in the gas phase . however , it is not , as shown in figure 9 .
one of the reasons is that when the nearby water molecules are removed , the ip of the modified nucleotide - containing dna oligomer in the neutral state in water approaches not simply the ip of the dna in the gas phase but that when the dna is polarized by water . in water ,
the ip of the modified nucleotide - containing dna oligomer in the ionic state is smaller than that in the neutral state .
the reason is the same as that for the dna oligomers which are not modified .
there are the computational results obtained by neglecting the solvent , where the ip dependences on the dna sequence are smaller than those obtained by the ab initio hf mo calculations . for example , the calculated ip differences between the dna oligomers , 5-aga-3 and 5-ggg-3 , are 0.10.2 ev , which are closer to the corresponding experimental result , 0.077 ev.(21 ) however , in the case of the different sequences , 5-tgt-3 and 5-ggg-3 , the calculated ip differences are 0.30.5 ev , which are larger than the value(58 ) ( less than 0.1 ev ) calculated from the experimental results . in these computations , the semiempirical nddo - g method(56 ) and the density functional theory(57 ) were used . in both of them
, the backbones of the dna oligomers were neglected ; this makes the ip dependence on the dna sequence smaller ( figure 1b ) . in the latter,(57 )
the charge distribution of the hole was calculated from the homo obtained by diagonalizing the reduced hamiltonian with the elements i|h|j , where h is the kohnsham ( ks ) hamiltonian , i and j are homos of individual nucleobases , and the total system is made of the stacked three base pairs without a backbone .
however , the vertical ionization potential was obtained not as the energy of the homo of the total system but as the diagonal element of the reduced hamiltonian at the middle guanine site in the base pairs . the reason was not described . about 90% of the charges of the hole is always in a certain guanine in our calculations .
the inclusion of the neglected reorganization energy due to the relaxation of the heavy atoms of dna will further localize the hole and not change the situation . the damage observed in the experiments is rather localized on a certain guanine in the g run .
this fact also supports that the hole is localized . by neglecting the reorganization energy and the solvent effects ,
more than 95% of the charges of the hole is in a certain guanine for the stacked guanine bases and 70% is in a certain guanine for the stacked gc base pairs.(19 ) more than 80% is in a middle guanine for the stacked three ( n = 3 ) gc base pairs with backbones , but it is delocalized over two guanines for n = 4.(51 ) the reason for the difference in the extent of the localization between these results and ours is mainly that the reorganization energy was neglected in these calculations .
the calculated ip of the stacked ga is smaller than that of g in the gas phase , and the difference between them is reduced in water.(27 ) this result is similar to that in figure 1 and is understood in the same way .
the calculated vertical ips of the dna tetramers with 65 water molecules are larger than those in the gas phase by a few ev,(34 ) where the b - dna structure model is different from the ones used here .
this result is similar to that in figure 8 and is understood in the same way .
thus , it supports that the effect of the structure deviation within the b - dna structure on the ip is minor and so is that of the sequence dependence of the dna structure .
it was discussed that the discrepancy between the theories and the experiment on the ip of the guanine run is reduced by taking into account the solvent effects;(29 ) in this work , the previously calculated ips(19 ) were used as the values for the hole fully delocalized over the guanine run and this delocalization was assumed to be the origin of the ip dependence on n in vacuum . however ,
more than 95% of the hole is localized on a certain guanine in the results(19 ) used there , and the origin of the ip dependence on n is not the delocalization of the hole , as described above .
if the origin of the ip dependence on n was the delocalization of the hole , the average of the homo and homo-1 of the guanine doublet was close to the homo of the single guanine .
we examined the ionization potential ( ip ) corresponding to the free energy of a hole on a duplex dna using the am1 method(39 ) with cosmo .
the electrostatic interaction between the hole and a nucleotide pair in the duplex dna is attractive . due to this attractive interaction ,
the ip in the gas phase significantly decreases with the increasing number of base pairs of the dna oligomer . on the other hand , this attractive electrostatic interaction is reduced in water .
the guanine runs , this is the reason why this ip dependence calculated by neglecting the solvent(19 ) was much larger than that obtained from the time - resolved data.(21 ) including the solvent makes this ip dependence consistent with the experimental result . as for the effect of the chemical modifications of dna ,
when a neutral functional group is attached to a guanine in a dna oligomer in water , nearby water molecules are removed ; this removal was found to reduce the free energy of the hole on the guanine.(22 ) one might naively have expected the opposite case , since a polar solvent usually stabilizes ions .
when some water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases , and thus , the hole is stabilized . in order to design the hole energetics by a chemical modification of dna , | we examined the ionization potential ( ip ) corresponding to the free energy of a hole on duplex dna by semiempirical molecular orbital theory with a continuum solvent model .
as for the contiguous guanines ( a guanine run ) , we found that the ip in the gas phase significantly decreases with the increasing number of nucleotide pairs of the guanine run , whereas the ip in water ( op , oxidation potential ) only slightly does .
the latter result is consistent with the experimental result for dna oligomers in water .
this decrease in the ip is mainly due to the attractive electrostatic interaction between the hole and a nucleotide pair in the duplex dna .
this interaction is reduced in water , which results in the small decrease in the ip in water .
this mechanism explains the discrepancy between the experimental result and the previous computational results obtained by neglecting the solvent . as for the chemically modified guanine , the previous work showed that the removal of some solvent ( water ) molecules due to the attachment of a neutral functional group to a guanine in a duplex dna stabilizes the hole on the guanine .
one might naively have expected the opposite case , since a polar solvent usually stabilizes ions .
this mechanism also explains this unexpected stabilization of a hole as follows .
when some water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases , and thus , the hole is stabilized . in order to design the hole energetics by a chemical modification of dna
, this mechanism has to be taken into account and can be used . | Introduction
Methods
Results
Discussion
Conclusions |
stabilization exercise is an effective intervention method for relieving the pain and
dysfunction associated with low back pain and for decreasing its recurrence .
stabilization
exercise programs typically include the motor control training of abdominal muscle . among
the abdominal muscles ,
the transversus abdominis ( tra ) is of particular interest to physical
therapists as a spinal stabilizer because of its anatomical characteristics1 .
this is because the upper fibers of tra
provide stability to the thorax , the middle fibers increase the tension of the thoracolumbar
fascia ( tlf ) for controlling the spine , and the lower fibers provide compression for
decreasing the laxity of the sacroiliac joint and supporting the internal organs of the
abdomen2 .
independent contraction of tra is achievable through the abdominal drawing - in maneuver
( adim)3 .
the adim re - educates the
functions of this muscle and is therefore effective at relieving lumbopelvic pain and
dysfunction4 .
for this reason , the adim
is frequently used as a basic element in stabilization exercise programs . however , learning
and teaching an accurate adim can be time consuming and difficult5 .
for this reason , feedback tools such as the pressure
biofeedback unit ( pbu ) , electromyography ( emg ) , and real - time ultrasound imaging ( rusi ) are
often used .
surface emg is a non - invasive method , but it is limited in its ability to detect fine
activities of the deeply located tra
. fine - wire emg can be used to observe these fine
activities , but this is an invasive method and can cause pain and inflammation . on the other
hand , rusi is a non - invasive method that enables observation of the fine activities , but its
high cost is prohibitive .
in contrast , a pbu is a non - invasive method that is more
economical than either of these other feedback tools , and it can be easily used anywhere
since it is portable .
pbus are used for clinical evaluation of the abdominal and cervical muscles , but it can
also provide feedback to subjects who are receiving motor control training6 . in the case of adim ,
the prone and supine
positions can be employed when using a pbu . in the prone position
, the pbu is placed between
the navel and the anterior superior iliac supine and air is infused into the bulb to create
a pressure of 70 mmhg .
a decrease of 4 mmhg in pressure in performance of the adim is
believed to indicate a successful result of the exercise7 , whereas a 410 mmhg pressure decrease indicates independent
contraction of the tra8 .
obese patients ,
patients with respiratory diseases , and pregnant women must avoid prone positions and
perform adim in the supine or other positions8 .
in the supine position , the pbu is placed below the lumbar lordosis and air is infused into
the bulb to create a pressure of 40 mmhg . however , unlike the prone position , no validated
pressure variation values have been published for the performance of adim in the supine
position .
rather , slight pressure increase or maintaining the pressure of 40 mmhg has been
recommended for the performance of adim8 .
this lack of definitive information has led to the use of diverse levels of pressure when
using a pbu , depending on the researchers intentions9,10,11,12 .
for this reason , the aim
of the present study was to determine the appropriate pressure variation for performing a
successful adim , by measuring thickness variations in the abdominal muscles and their
contraction ratios in response to pressure variations of a pbu during performance of adim in
the supine position .
the subjects of the present study were 14 healthy males and 9 healthy females who were
given an explanation of the purpose and method of the study , and who voluntarily agreed to
participate in the study .
subjects who had experienced low back pain within the last six
months , who had pain while performing adim , who showed deformation such as scoliosis , who
had received a surgical intervention , who had neurological disease , or who had previous
experience of adim training using a pbu were excluded .
three participants ( 2 males , 1
female ) could not perform adim at any of the four pressure variations and were excluded from
the analysis of the present study .
therefore , the final study subjects were 20 persons ( 12
males , 8 females ) and their mean age , height , weight and bmi were 23.603.72 years ,
169.158.49 cm , 60.3011.85 kg , and 20.912.80 , respectively . a pbu ( chattanooga group inc .
hixson , tn37343 , usa ) was placed between the lumbar lordosis
and the ground , with subjects in the supine position with knees bent at 90 before
performing adim .
the bulb was then inflated to 40 mmhg of pressure and maintained while the
subject performed the adim .
the subject used an adim method in which the pelvic floor
muscles were also contracted in order to increase the contraction of the tra , as described
by critchley13 .
all of the subjects were
taught to pull the lower abdomen in slowly , without moving the spine , ribs , or pelvis , while
simultaneously contracting the pelvic floor muscles .
the subjects performed adim in the
supine position targeting randomized pressure increase of 0 , 2 , 4 , or 6 mmhg from the
initially maintained 40 mmhg .
variations in tra and the thicknesses of the internal oblique
abdominal muscle ( io ) and the external oblique abdominal muscle ( eo ) were measured using
ultrasonography . to verify the accuracy of the pbu , a weight of 4.54 kg was placed on each
pbu and the pbus were observed for 24 hours14 .
the units that showed decreases of 0.5 mmhg or less were used in
the study . a sonoace
the effects of breathing were
controlled by collecting all data at the end - point of expiration15 using a 7.5 mhz linear transducer .
abdominal muscle
thicknesses were measured by placing the transducer transversely on the middle abdominal
region between the border of the 11th costal cartilage and the iliac crest16 .
the abdominal muscle thicknesses were
first measured at rest , and were subsequently measured every time the pressure changed ,
while the subjects were performing adim .
muscle contraction ratios were calculated using
equations presented in previous studies16,17,18 . tra contraction ratio = tra thickness in
contraction / tra thickness at rest eo contraction ratio = eo
thickness in contraction / eo thickness at rest io + eo
contraction ratio = io + eo thickness in contraction / io + eo thickness at
rest tra preferential activation ratio = ( tra in
contraction / tra + io + eo in contraction ) ( tra at rest / tra + io + oe at
rest ) differences in abdominal muscle thickness variations in relation to pressure variations and
the contraction ratios of individual muscles were compared using one - way anova .
statistical processing was conducted using spss 12.0 for windows , and the
significance level , , was chosen as 0.05 .
unit : mm ; * significant difference ( p<0.05 ) ; values with different
superscripts within the same columns are significantly different at p<0.05 .
values : meanstandard deviation ; cr : contraction ratio ; pcr : preferential contraction
ratio ; values with different superscripts within the same columns are
significantly different at p<0.05 .
the effects of pressure variations on abdominal muscle thicknesses at rest and during adim
are shown in table 1 .
significant differences
were found in muscle thickness between the resting and adim conditions for the io and the
eo . thickness variations in the io showed significant differences between pressure increases
of 0 and 6 mmhg , and between 2 and 6 mmhg .
thickness variations in the eo also showed
significant differences between pressure increases of 0 and 6 mmhg , and between 2 and 6
mmhg .
variations in abdominal muscle contraction ratios in relation to pressure variations showed
significant differences among the io+eo contraction ratios , the eo contraction ratios , and
the tra selective contraction ratios ( table 2 ) .
the io+eo contraction ratios showed significant differences between 0 and 4 mmhg , 0 and 6
mmhg , and 2 and 6 mmhg .
the tra selective contraction ratios showed significant differences between 0
and 6 mmhg , and 2 and 6 mmhg .
the reliability within the measurers of the measurements of the thicknesses of each muscle
was tested using icc ( 3,1 ) .
the reliability values for tra , io , and eo were 0.96 , 0.96 , and
0.97 , respectively .
the present study was conducted to determine the appropriate pressure variation for
performing successful adim in the supine position , using a pbu .
increases in pressure of 02
mmhg resulted in significant decreases in the thicknesses of both the io and the eo compared
to the changes seen following increases of 6 mmhg . in this study
, 02 mmhg increases showed
respective decreases of 1.131.54 mm , and 0.510.66 mm in io and eo thicknesses compared to
6 mmhg . additionally , although the 4 mmhg increase was resulted in io and eo thicknesses
that were not significantly different from the 02 mmhg increase , the 02 mmhg increase
showed respective decreases of 0.540.95 mm , and 0.250.40 mm in io and eo thicknesses from
their values at 4 mmhg increase .
increases in pressure of 02 mmhg may be more effective for
decreasing the thicknesses of both io and the eo compared to other pressure variations .
increases of 46 mmhg resulted in increased io and eo thicknesses similar to those reported
in a previous study19 in which no pbu was
used , and this means that adim was not performed successfully . in the present study , the tra contraction ratio showed no significant differences in
relation to pressure variations , but the tra preferential activation ratios between 02 mmhg
and 6 mmhg pressure increases showed significant differences .
this is because there was a
significant difference between the io+eo contraction ratio and the eo contraction ratio .
successful performance of adim is determined by the selective activity of tra rather than
the more superficially located abdominal muscles , such as the rectus abdominis ( ra ) , io , and
eo20 .
in particular , unlike the case
for ra , activity can not be easily suppressed in eo while adim is being performed because of
the anatomical characteristics of eo21 .
in this study ,
02 mmhg increases resulted in significant decreases in the eo contraction
ratio compared to the 6 mmhg increase .
the variation in eo contraction ratio resulting from
02 mmhg increases was similar to results reported in previous studies17 , 18 in which adim
was performed in the supine position using a feedback tool .
however , the 02 mmhg increase
resulted in an eo thickness that was 0.350.50 mm lower than that reported in a previous
study19 , in which adim was performed
using the same method as in the present study , but without a pbu .
pressure increases of 02 mmhg also resulted in significant decreases in the io+eo
contraction ratio compared to the 6 mmhg pressure increase and a significant increase in the
tra preferential activation ratio . in the present study , while variations in tra thickness
and the response of the tra contraction ratio to pressure variations did not show any
significant differences between the resting state and adim , the variations in the tra
preferential activation ratio showed significant differences between pressure increases of
02 mmhg and 6 mmhg . given these results , pressure variations appear to regulate the
thicknesses and contraction ratios of superficial muscles , such as io and eo , rather than
those of tra , causing an indirect increase in the preferential activation ratio of tra . for 02 mmhg increases ,
the tra preferential activation ratio was higher than previously
reported in studies using a feedback tool17 , 18 and those shown by other pressure
variations in the present study .
therefore , 02 mmhg increases are appropriate pressures for
successful performance of adim . among the subjects of the present study
, 3 participants
could not perform the exercise following a 6 mmhg increase and they were excluded from the
final tests . among the final subjects , only 5 participants ( 3 males , 2 females ) succeeded in
performing the exercise after an 8 mmhg increase .
the other subjects had difficulty in
performing adim after an 8 mmhg increase , since the movements of the pelvis and other joints
were affected .
in addition , almost all of the participants in the present study used many
other movement strategies to perform adim when pressure was increased by up to 10 mmhg from
40 mmhg10,11,12 .
therefore , for
successful adim , we recommend performing the exercise at a slightly increased pressure of
about 02 mmhg .
since different movement strategies can be adopted at the same pbu pressure , the
quantification of tra contraction using pbus has limitations11 . to solve this problem ,
all of the subjects in the present study
performed the exercise in the same order and used the same method of contraction .
however ,
since abnormal movement strategies were visually observed , we consider the lack of kinematic
information for objective control of movement strategies is a limitation of the present
study .
we hope that future studies will account for these limitations and that diverse study
tools will be applied to the investigation of abdominal muscle contraction ratios in
relation to pressure variations for adim performance in the supine position . | [ purpose ] the aim of this study was to determine the appropriate pressure variation for
performing a successful abdominal drawing - in maneuver ( adim ) . the abdominal muscle
thickness variations and contraction ratios were examined in relation to pressure
variations using a pressure biofeedback unit ( pbu ) during an adim in the supine position .
[ methods ]
the pbu was placed identically between the lumbar lordosis of 20 healthy
subjects ( 12 males and 8 females ) and the pressure of the pbu was maintained at 40 mmhg .
then , while the subjects performed adim at four random pressure variations ( 0 , 2 , 4 , or 6
mmhg ) , the thicknesses of the transversus abdominis ( tra ) , the internal oblique abdominal
muscle ( io ) , and the external oblique abdominal muscle ( eo ) were measured using
ultrasonography .
[ results ] pressure increases of 02 mmhg resulted in significant
decreases in io and eo thicknesses compared to pressure increases of 6 mmhg .
increases of
02 mmhg also resulted in significant decreases in io+eo and eo contraction ratios
compared to pressure increases of 6 mmhg , while the preferential activation ratio of the
tra was significantly increased .
[ conclusion ] compared to the other pressure increases , an
increase of 02 mmhg effectively regulated the thicknesses and contraction ratios of
superficial muscles such as io and eo , rather than the thickness and contraction ratio of
the tra , showing high and indirect preferential activation ratios for tra .
therefore , for
successful adim , rather than using large pbu pressure increases , exercises that promote
slight increases of around 02 mmhg from a baseline of 40 mmhg are desirable . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
polychlorinated biphenyls
( pcbs ) are a class of industrial chemicals
and unintentional byproducts of industrial processes banned under
the stockholm convention on persistent organic pollutants .
pcbs remain
an environmental and human health concern because of their ongoing ,
inadvertent production , their environmental persistence , and the presence
of pcbs in the environment , diet , and in human serum and tissues .
nineteen pcb congeners and various hydroxylated ( and other ) metabolites
with three or four ortho chlorine substituents and an unsymmetrical
substitution pattern in both phenyl rings are chiral .
these pcb derivatives exist as nonsuperimposable rotational
isomers , called atropisomers , which are mirror images of each other .
chiral pcb congeners , in particular congeners with a 2,3,6 substitution
pattern in one phenyl ring , have been linked to neurodevelopmental
toxicity in humans and laboratory animals and shown to cause effects
on neurotransmitter functions in the central nervous system and alter
cellular processes related to calcium signaling .
chiral pcbs are present in commercial pcb mixtures as racemates
( i.e. , a 1:1 mixture of atropisomers ) and , due to pcbs chemical and
thermal stability , are released into the environment as racemates .
studies of the atropisomeric enrichment of chiral pcbs reveal near
racemic signatures in diet , house dust , and air , but highly variable
atropisomeric enrichment in wildlife , especially at higher tropic
levels , and humans ( reviewed in ref ( 5 ) ) .
because physical and chemical transport ( e.g. ,
passive diffusion ) and transformation processes ( e.g. , photodegradation )
are not atropselective , the variable atropisomeric enrichment of pcbs
in environmental samples is due to atropselective biotransformation
and/or biological transport processes .
pcbs can undergo atropselective
bacterial biodegradation and are atropselectively
metabolized in plant and animal models
( reviewed in ref ( 5 ) ) .
laboratory studies have shown that cytochrome p450 enzymes , such
as different cyp2b isoforms , can atropselectively
metabolize pcbs to oh - pcbs and , thus , contribute to nonracemic signatures
of pcbs at higher trophic levels .
oh - pcbs are found in many
species , including humans , and represent an environmental
and human health concern . in mammals ,
oh - pcbs can adversely affect
neurodevelopment by altering processes related to calcium signaling or thyroid function .
activity
relationship studies show that the oh - pcb metabolites of chiral pcbs
are ryanodine receptor- ( ryr-)active and display different modes of
action depending on the position of the hydroxyls group on the biphenyl
moiety in vitro .
oh - pcb
profiles are highly species dependent and can display
interindividual variability .
these differences
in oh - pcb profiles are due to differences in the isoform composition ,
expression , and activities of pcb and oh - pcb metabolizing enzymes
( e.g. , p450 enzymes , sulfotransferases , glucuronosyl transferases ,
and others ) .
furthermore , differences in the composition and oh - pcb
binding affinity of various transport proteins ( e.g. , transthyretin )
may contribute to species and interindividual differences in oh - pcb
profiles in vivo . since pcbs are atropselectively
metabolized by p450 enzymes to oh - pcbs , it is likely that , similar
to the parent pcbs , p450 enzyme - mediated
metabolism contributes to differences in the atropisomeric enrichment
of chiral oh - pcbs in wildlife and humans .
consistent with this hypothesis ,
we have recently reported differences in the atropselective formation
of oh - pcbs in rats and mice in in vitro metabolism
studies ; however , systematic laboratory
and environmental studies of the atropselective formation of oh - pcbs
by p450 enzymes from different species have not been reported to date .
the present study investigates the atropselective formation of
oh - pcb from 2,2,3,3,6,6-hexachlorobiphenyl
( pcb 136 ) by liver microsomes from humans and several other mammalian
species . pooled liver microsomes from nave animals were
used to assess representative oh - pcb profiles and chiral signatures
in the species investigated .
pcb 136 was selected for this study as
a prototypical chiral pcb congener of environmental relevance .
untreated beagle dog ( female ) , cynomolgus
monkey ( male ) , new zealand rabbit ( male ) , golden syrian hamster ( male ) ,
hartley albino guinea pig ( male ) , and human liver microsomes pooled
from 50 donors with mixed age , sex , and race ( hlms ) were purchased
from xenotech ( lenexa , ks , usa ) .
microsomes obtained from female instead
of male beagle dogs were used because of the higher enzymatic activity
of the respective microsomal preparations .
mouse liver microsomes
were prepared by pooling livers from saline and corn oil treated male
c57bi/6 mice and characterized as described previously .
the microsomal cytochrome p450 content is described
in the supporting information ( tables s1
and s2 ) .
dimethyl sulfoxide ( dmso ) , sodium phosphate
dibasic ( na2hpo4 ) , sodium phosphate monobasic
( nah2po4 ) , magnesium chloride ( mgcl2 ) , tetrabutylammonium sulfite , sodium sulfite , and pesticide grade
solvents were obtained from fisher scientific ( pittsburgh , pa , usa ) .
nicotinamide adenine dinucleotide phosphate reduced ( nadph ) was purchased
from sigma - aldrich co. ( st . louis , mo , usa ) .
racemic pcb 136 was synthesized
by the ullmann coupling of 2,3,6-trichloro-1-iodobenzene , and the atropisomers of pcb 136 were separated
using two serially connected nucleodex -pm columns ( macherey - nagel ,
dren , germany ) .
the enantiomeric fractions ( ef = area(+)-pcb 136 /(area(+)-pcb 136 + area()-pcb 136 ) ) of ( )-pcb
136 and ( + ) -pcb 136 were 0.01 and 1.00 , respectively .
2,2,3,3,6,6-hexachlorobiphenyl-4-ol
( 4136 ) , 2,2,3,3,6,6-hexachlorobiphenyl-5-ol
( 5136 ) , 4,5-dimethoxy-2,2,3,3,6,6-hexachlorobiphenyl ,
and 2,2,3,4,6,6-hexachloro-3-methoxybiphenyl
were prepared as described elsewhere .
recovery standards ( 2,3,4,4,5,6-hexachlorobiphenyl , pcb
166 ; 2,3,4,5,6-pentachlorobiphenyl , pcb 117 ; 2,3,3,4,5,5-hexachlorobiphenyl-4-ol ,
4-159 ) and the internal standard ( 2,2,3,4,4,5,6,6-octachlorobiphenyl ,
pcb 204 ) were purchased from accustandard ( new haven , ct ) .
incubation conditions were initially
optimized for microsomal protein content and napdh concentration using
human and dog liver microsomes as described previously .
subsequently , time - course experiments were performed
in triplicate with the optimized experimental condition .
briefly ,
an incubation mixture ( 12 ml ) consisting of phosphate buffer ( 0.1
m , ph 7.4 ) , nadph ( 0.5 mm in hlms or 1.5 mm in all animal microsomes ) ,
magnesium chloride ( 3 mm ) , and hepatic microsomal protein ( 1.0 mg / ml
for all animal microsomes or 0.5 mg / ml for human microsomes ) was preincubated
for 5 min at 37 1 c in a shaking water bath
. pcb 136
in dmso ( 0.5% ) was added with a final concentration of 50 m .
these incubation conditions , including the high pcb 136 concentrations ,
were selected to ensure the formation of sufficient oh - pcb quantities
for atropselective analyses .
experiments with hlms used ( )- ,
( )- , or ( + ) -pcb 136 .
an aliquot ( 2 ml ) of the
incubation mixture was removed after 5 , 10 , 15 , 20 , 25 , and 30 min .
a total of 2 ml of ice cold sodium hydroxide ( 0.5 m ) was added to
each aliquot to stop the reaction . for the 0 min time point ,
a separate
sample ( 1990 l ) was preincubated for 5 min as described above ,
followed by sequential addition of the sodium hydroxide ( 2 ml ) and
pcb 136 solution ( 10 l ) .
control incubations without microsomes
or nadph or containing heat - inactivated microsomes were performed
in parallel .
extraction of pcb 136 and its hydroxylated metabolites
was performed
using a published method . in short , surrogate
standards
( 500 ng of pcb 117 in animal microsomes or pcb 166 in human
microsomes ; 274 ng of 4-159 ) were added to each sample , followed
by hydrochloric acid ( 6 m , 1 ml ) and 2-propanol ( 3 ml ) .
the samples
were extracted with hexane - mtbe ( 1:1 v / v , 5 ml ) and hexane ( 3 ml ) .
the combined organic extracts were washed with an aqueous kcl solution
( 1% , 3 ml ) .
after removal of the organic phase , the kcl phase was
re - extracted with hexane ( 3 ml ) , and the combined extracts were reduced
under a gentle stream of nitrogen to 1 ml .
the hydroxylated
metabolites were derivatized with diazomethane and subjected to a
sulfur cleanup as described previously .
pcb 204 ( 200 ng ) was added as an internal standard prior to analysis .
levels of oh - pcb
136 metabolites were determined using an agilent 6890n gas chromatograph
with a ni-ecd detector and a db1-ms capillary
column ( 60 m 0.25 mm i d 0.25 m film thickness ;
agilent , santa clara , ca , usa ) .
relative rates
of oh - pcb formation were determined in the linear range of metabolite
formation ( i.e. , 5 min ) by adjusting the amount of oh - pcb by the total
p450 content .
the limits of detection
and background pcbs levels are listed in table
s3 . to further verify the formation of specific metabolites ,
samples from 30 min incubations
were analyzed on an agilent 7890a
gas chromatograph with a 5975 c mass selective detector in both total
and selective ion monitoring modes with an hp-5 ms column ( 30 m
0.32 mm i.d .
atropselective analysis of the derivatized
hydroxylated pcb 136
atropisomers was performed using an agilent 7890a gas chromatograph
with a ni ecd detector .
the atropisomers of 5136
and 4136 were separated on chirasil - dex ( cd column , 25 m
0.25 mm i d 0.25 m film thickness ; varian , palo alto ,
ca , us ) and cyclosil - b columns ( cb column , 30 m 0.25 mm i d
0.25 m film thickness ; agilent , santa clara , ca , us ) ,
respectively , following a published method .
enantiomeric fractions ( efs ) were determined as ef = a2/(a1 + a2 ) , where a1 and a2 are the peak areas of the first and second eluting atropisomers ,
respectively .
the species dependent formation
of metabolites was studied using one way anova and proc in the statistical
analysis package sas ( version 9.3 , sas institute , cary , nc , usa ) .
metabolites formation and ef values were compared by tukey s
studentized range ( hsd ) test .
a paired t test was
used to compare the ef values of 5136 and 4136 to
racemic standards and the formation rates of 4136 and 5136
between each species .
a p value < 0.05 was used
to indicate a significant difference between species .
racemic pcb 136 or its atropisomers
were incubated with hlms to investigate if potentially neurotoxic
oh - pcbs are formed atropselectively in humans .
only a small percentage
( < 1% ) of the total pcb 136 was converted to oh - pcbs under the incubation
conditions ( table s4 ) . 4136 and
5136 were the major metabolites for racemic pcb 136 and pure
pcb 136 atropisomers , with
more 4136 being formed ( 5136/4136
ratio = 0.39 after 5 min , table s5 ) .
4,5136
and the 1,2-shift product of pcb 136 ( 3150 ) were minor metabolites .
one unknown metabolite peak ( m / z = 420.0 ) was observed at a later retention time , indicating the
formation of a second dihydroxylated metabolite ( figure s1 ) .
the amounts of 3150 , 4136 , 5136 ,
and 4,5136 increased with time in all hlm incubations and
depended on the pcb atropisomer composition ( figure 1 ) . for all metabolites , the rate of formation followed the
order ( + ) -pcb 136 > racemic pcb 136 > ( )-pcb 136 ( figure 1 and table s4 ) .
the time - dependent
formation of ( a ) 3150 , ( b ) 5136 ,
( c ) 4136 , ( d ) 4 , 5136 , and ( e ) oh-136 followed
the rank order ( + ) -pcb 136 > ( )-pcb 136 > ( )-pcb
136
in incubations with human liver microsomes .
( f ) the relative rates
of oh - pcb formation showed a significantly slower metabolite formation
in the incubations with ( )-pcb 136 compared to incubations
with ( + ) -pcb 136 and ( )-pcb 136 .
different letters indicate
statistically significant differences in the oh - pcb formation rates
( p < 0.05 ) as determined by a tukey student range
test using sas . * p = 0.05 for comparison of the 4,5136
formation rates between incubations with ( + ) - and ( )-pcb 136 .
racemic pcb
136 was incubated with liver microsomes obtained from different species
( i.e. , male monkey , guinea pig , mouse , hamster , and rabbit ; female
dog ) to explore differences in typical metabolite profiles and chiral
signatures between humans and toxicologically relevant mammalian species .
only a small percentage of pcb 136 ( < 3% ) was converted to oh - pcbs
( table s4 ) .
similar to experiments with
hlms , 4136 was the major metabolite formed in incubations
with microsomes obtained from male monkeys and rabbits ( figure 2 ) , with 5136/4136 ratios of 0.17
and 0.64 at 5 min , respectively ( table s5 ) .
in contrast , 5136 was the major metabolite in incubations
using microsomes from dogs , guinea pigs , mice , and hamsters .
after
a 5 min incubation time , the 5136/4136 ratios were
14 , 10 , 2.2 , and 6.9 for incubations with dog , guinea pig , mouse ,
and hamster microsomes , respectively ( table s5 ) .
4,5136 was a minor metabolite observed in the incubations
with microsomes prepared from dogs , guinea pigs , mice , hamsters , and
rabbits .
the formation of 3150 , but not 4,5136 , was
observed in microsomes obtained from monkeys .
the formation of these
oh - pcb metabolites was confirmed by gc - ms ( figure
s1 ) .
time- and species - dependent formation of oh - pcbs in incubations
of pcb 136 with liver microsomes from ( a ) humans ( pooled ) , ( b ) dog ,
( c ) monkey , ( d ) guinea pig , ( e ) mouse , ( f ) hamster , and ( g ) rabbit .
4136 was the major metabolite in incubations using human ,
monkey and rabbit liver microsomes .
5136 was the major metabolite
in experiments with dog , guinea pig , mouse , and hamster liver microsomes .
the 1,2-shift metabolite , 3150 , was only observed in incubations
using human and monkey liver microsomes .
the relative rates
of formation of 5136 and 4136 were determined for
the 5 min incubation time by expressing oh - pcb levels per nanomole
of total p450 content .
this adjustment accounts for the differences
in total p450 content between different microsomal preparation and
allows a comparison across species ( figure 3 ) .
the rates of formation of 5136 followed the order rat
( estimated based on published data , see ref ( 30 ) ) > dog guinea
pig hamster > human > monkey mouse
rabbit .
however , the rate of 5136 formation by hlms was only significantly
different compared to incubations using microsomes obtained from dogs
and guinea pigs .
the rate of 5136 formation by hlms was similar
compared to the rate observed in incubations with microsomes from
hamster , monkey , mouse , and rabbit .
the rate of 4136 formation
by hlms was significantly faster compared to experiments using microsomes
from other species and followed the order human > monkey > rabbit
rat > dog guinea pig mouse
hamster
( figure 3 ) .
the formation rate of 5136
was significantly different from that of 4136 in all species
after paired t test within species .
comparison of the formation
rates of ( a ) 5136 and ( b ) 4136
in incubations with liver microsomes obtained from different species .
different letters indicate statistically significant
differences in the oh - pcb formation rates ( p <
0.05 ) as determined by a tukey student range test using sas .
the formation
rate of 4136 was significantly different from that of 5136
within each species ( p < 0.05 ; paired t test ) .
the atropisomeric
enrichment of oh - pcb 136 metabolites was determined in microsomal
incubations with racemic pcb 136 using atropselective gas chromatography .
the objective was to determine if oh - pcbs are formed atropselectively
in incubations with hlm and how this enrichment differs compared to
toxicologically relevant species .
the second eluting atropisomers
of 5136 ( e2 - 5136 ) , which is formed from
( + ) -pcb 136 , was enriched in incubations using human , dog , monkey ,
guinea pig , and rabbit microsomes ( figure 4a ) .
the atropselective formation of the 5136 resulted in
near constant ef values with time ( data not shown ) .
therefore , ef
values at 5 min were statistically analyzed and presented in figure 4c .
the atropisomeric enrichment of 5136 and 4136
formed
from liver microsomes is species - dependent .
( a ) representive chromatograms
showing an enrichment of the second eluting 5136 atropisomer
in incubations with human ( pooled ) , dog , monkey , guinea pig , and rabbit
liver microsomes and an enrichment of the first eluting 5136
atropisomer in experiments with mouse and hamster liver microsomes .
( b ) representive chromatograms showing an enrichment of the second
eluting 4136 atropisomer in incubations with human ( pooled ) ,
dog , monkey , guinea pig , hasmster , and rabbit liver microsomes and
an enrichment of the first eluting 4136 atropisomer in experiments
with mouse liver microsomes .
different letters indicate statistically significant
differences in the ef values ( p < 0.05 ) as determined
by a tukey student range test using sas . * ef values significantly
different from control ( p < 0.05 , paired t test ) .
ef values of 4136 in incubations
with dog microsomes showed a trend to significance from control ( p = 0.054 ) .
the extent of the atropisomeric enrichment of 5136
in microsomal
incubations followed the order dog guinea pig > monkey
human rabbit ( figure 4c ) .
interestingly ,
the first eluting atropisomer of 5136 ( e1 - 5136 ) ,
the 5136 metabolite formed from ( -)-pcb 136 , displayed atropisomeric
enrichment in experiments with mouse and , to a lesser extent , hamster
microsomes .
the ef values of 5136 were significantly different
from the racemic standard , with the exception of incubations using
hamster microsomes .
similar to 5136 , the second eluting atropisomers
of 4136 ( e2 - 4136 ) , a metabolite formed
from ( + ) -pcb 136 , was enriched in incubations using human , dog , monkey ,
guinea pig , hamster , and rabbit microsomes ( figure 4b ) .
the extent of the atropisomeric enrichment of 4136
formed in microsomal incubations followed the order human
monkey > guinea pig hamster rabbit > dog .
in contrast ,
the first eluting atropisomer of 4136 ( e1 - 4136 ) ,
which is formed from ( )-pcb 136 , was enriched in experiments
with mouse microsomes .
the ef values of 4136 were significantly
different from racemic standards , with incubation using dog microsomes
displaying only a trend of e2 - 4136 enrichment ( p = 0.054 ; figure 4d ) .
the present study uses hepatic microsomes to gain insights into
typical oh - pcb 136 metabolite profiles and chiral signatures formed
by p450 enzymes in different mammalian species , including humans .
4136 and 5136 were the two major monohydroxylated
pcb 136 metabolites formed atropselectively by hlms , which is consistent
with an earlier study by schnellmann and co - workers .
in addition , a few other mono- and dihydroxylated pcb metabolites
were observed as minor metabolites .
this includes 3150 , a
1,2-shift product of pcb 136 formed via an arene oxide intermediate .
the formation of such a 1,2-shift metabolite by hlms has not been
reported previously .
the 5136/4136 ratios in our study
ranged from 0.4 to 0.8:1 ( for incubation times from 5 to 30 min ) .
on the basis of our re - evaluation of the published mass spectra ,
this difference in the metabolite profile is
most likely due to differences in the p450 enzyme composition of the
respective hlms .
the relative rate of formation of all oh - pcbs was
different for incubations using ( + ) -pcb 136 , ( )-pcb 136 , and
racemic pcb 136 , with ( + ) -pcb 136 being more rapidly oxidized compared
to ( )-pcb 136 .
these atropisomer - specific differences in the
oh - pcb formation rates explain the atropisomeric enrichment of pcbs
observed in in vitro studies and are consistent with a role of p450 enzymes in their atropisomeric
enrichment observed human samples .
analogous to hlms , 5136 , 4136 , and 4,5136
were formed by most animal microsomal preparations studied .
there
is considerable evidence that cyp2b enzymes are involved in the oxidation
of pcb 136 and structurally related pcb congeners in the meta position .
studies with recombinant enzymes demonstrate that rat cyp2b1 and dog cyp2b11 selectively oxidize pcb 136 to 5136 .
cyp2b1 also
metabolizes 4-oh - pcbs and 5-oh - pcbs to the corresponding 4,5-dihydroxylated
metabolites , such as 4,5136 .
warner and
co - workers demonstrated that pcb 136 is oxidized by human cyp2b6 to
a single , unidentified oh - pcb .
this oh - pcb
metabolite is most likely 5136 because cyp2b6 oxidizes other
pcbs in the meta position .
the p450 isoforms responsible for the formation of 4136
remain elusive , as cyp3a enzymes are probably not involved in its
formation in rats or mice .
we also observed no change in 4136 levels in liver microsomes
after induction of cyp1a enzymes in rats pretreated with pcb 126 ,
which suggests that 4136 is not formed by cyp1a enzymes ( wu
and lehmler , unpublished data ) .
while essentially the same metabolites
were formed by liver microsomes
from different species , the ratios , relative formation rates , and
chiral signatures of the oh - pcbs differed considerably depending on
the species .
experiments with hlms displayed the fastest formation
rate for 4136 and one of the lowest formation rates for 5136
of all microsomal preparations investigated .
it is important
to emphasize that our result represents an average oh - pcb profile
formed by a pool of liver microsomes from 50 individual donors ; however ,
there can be considerable interindividual variability in humans due
to genetic polymorphisms , diseases , and exposure to other xenobiotics .
for example , a recent pcb 146 metabolism experiment with hlms from
individual donors revealed considerable interindividual metabolism
of pcb in humans associated with cyp2b6 activity .
4136 was also the major metabolite in incubations
using
microsomes from monkeys and rabbits .
in contrast , 5136 was
the major metabolite in experiments with microsomes from dogs , guinea
pigs , mice , hamsters , and , as reported previously , rats .
the faster formation of 5136 in rat
compared to dog microsomes in the current study is consistent with
the differences in the oxidation of pcb 136 reported by waller et
al . for recombinant rat cyp2b1 and dog cyp2b11 . 5136 and 4136 are also the major pcb 136
metabolites formed in rats after intraperitoneal administration of
pcb 136 , with a rank order of 5136
> 4136 .
these species differences in the oh - pcb ratios
and
formation rates are not surprising because of the considerable interspecies
differences in the constitutive expression and catalytic activity
of p450 enzymes .
it is important to emphasize
that our results represent oh - pcb ratios and formation rates obtained
with pooled microsomal preparations from nave animals .
as with humans
, genetic and environmental factors can result in interindividual
variability in the oh - pcb profiles and formation rates in these species .
however , these differences are likely small in toxicologically relevant
animal models because the animals under investigation are inbred and
maintained under rigorously controlled environmental and dietary conditions .
more significant interindividual variability in the p450 enzyme - mediated
oxidation of chiral pcbs is likely to occur in wildlife .
the
present study also revealed considerable differences in the
atropisomeric enrichment of 5136 and 4136 formed by
microsomal preparations obtained from different species .
interestingly ,
the e2 - 5136 and e2 - 4136 were
enriched in incubations with liver microsomes from most species , albeit
to a different extent .
microsomes from mice were a notable exception ,
because e1 - 5136 and e1 - 4136
were enriched .
the same direction of the atropisomeric enrichment
of 5136 and 4136 has been observed in mouse tissue
slices .
it is currently unclear to which
extent our in vitro metabolism studies predict chiral
oh - pcb signatures in vivo .
in particular , subsequent
metabolism ( e.g. , sulfation and glucuronidation ) and transport of
oh - pcbs may modulate the atropisomeric enrichment of oh - pcbs in vivo ; however , the atropselectivity of these biological
processes has not been investigated to date . both e1 - 5136 and e1 - 4136
consequently ,
most mammalian species , including humans , metabolize and eliminate
the ( )-pcb 136 atropisomer less rapidly than the ( + ) -pcb 136
atropisomer .
in contrast , mice metabolize ( )-pcb 136 more
rapidly , at least compared to the other mammalian species investigated
in our study .
consistent with this interpretation , ( + ) -pcb 136 undergoes
atropisomeric enrichment in mice , whereas
only a slight enrichment of ( )-pcb 136 is observed in rats .
this observation is important because ( )-pcb
136 , but not ( + ) -pcb 136 , is a potent sensitizer of ryrs and alters
neuronal connectivity via a ryr - dependent mechanism .
it is therefore possible that differences in the metabolism of pcb
136 atropisomers across species , sex , or individuals play a role in
the developmental neurotoxicity of pcb 136 and structurally related
congeners .
the toxicological relevance of the atropisomeric
enrichment of
oh - pcb metabolites of pcb 136 and other chiral pcbs is currently unknown
and warrants further investigation .
like the parent pcbs , pure oh - pcb atropisomers may display atropselectivity toward cellular
targets and , thus , cause atropselective toxicity in wildlife and humans .
developmental neurotoxicity is a particular concern in humans because
oh - pcbs cross the placenta and accumulate
in fetal target tissues .
oh - pcbs have
several modes of action and , for example , disrupt cellular calcium
homeostasis by mechanisms involving ryrs or cause thyroid dysfunction .
oh - pcb 136 metabolites
and structurally related , chiral oh - pcbs have not been detected in
humans , partly because suitable analytical standards are not readily
available ; however , their parent compounds can be present at high
levels in indoor air , including in school buildings
in the united states .
it is therefore
likely that potentially neurotoxic , chiral oh - pcbs are present in
humans , especially in school children and other susceptible human
populations .
similar to the parent pcbs , our observation
that the atropisomeric enrichment of oh - pcbs is highly species - dependent
will be useful for source apportionment studies of oh - pcb .
oh - pcbs
have not only been detected in laboratory animals and humans , but also in species at different
tropic levels , such as fish , sea birds , marine mammals , and plants .
although studies with liver microsomes demonstrate that p450 enzymes
are involved in the formation of oh - pcbs in many species , several studies demonstrate the presence of oh - pcbs in abiotic samples .
oh - pcbs are also formed by the reaction of oh
radicals with pcbs and have been detected in surface water and precipitation . as with pcbs , chiral oh - pcb formed by abiotic
processes
will be racemic , whereas oh - pcbs in biological samples will
be nonracemic due to atropselective biological transport and biotransformation
processes .
consequently , chiral signatures can be used to distinguish
abiotic from biotic oh - pcb sources .
furthermore , species - dependent
differences on chiral signatures may be useful to study how oh - pcbs
move through aquatic and terrestrial food webs .
similarly , chiral
signatures are a powerful tool to study the movement of chiral pcbs
through aquatic and terrestrial food webs . | chiral polychlorinated biphenyls
( pcbs ) display variable atropisomeric
enrichment in wildlife and animal models , especially at higher trophic
levels .
these differences in pcbs chiral signatures are , at
least in part , due to species - dependent oxidation of pcbs to hydroxylated
pcb metabolites ( oh - pcbs ) . here
, we investigate the hypothesis that
the cytochrome p450 ( p450 ) enzyme - mediated oxidation of chiral pcbs
results in species - dependent differences in the chiral signatures
of oh - pcbs ( i.e. , the direction and extent of oh - pcbs atropisomeric
enrichment ) . to investigate this hypothesis , we incubated pcb 136 ,
a representative chiral pcb , with pooled human liver microsomes ( hlms )
or liver microsomes from male guinea pig , hamster , monkey , mouse ,
and rabbit or female dog and determined average profiles and chiral
signatures of the oh - pcbs . 2,2,3,3,6,6-hexachlorobiphenyl-4-ol
( 4136 ) was the major metabolite in incubations with hlms and
monkey and rabbit microsomes
. 2,2,3,3,6,6-hexachlorobiphenyl-5-ol
( 5136 ) was the major metabolite formed by microsomes from
all other species .
both 4136 and 5136 were formed
atropselectively in all microsomal incubations ; however , the direction
and extent of the atropisomeric enrichment of both oh - pcb metabolites
showed considerable differences across microsomal preparations obtained
from different species .
these differences in oh - pcbs atropisomeric
enrichment may not only be toxicologically relevant but may also be
useful to study sources and transport of oh - pcbs in the environment . | Introduction
Experimental
Section
Results
Discussion |
in children , the main causes of hypoparathyroidism include digeorge syndrome ( 22q11.2 deletion syndrome ) , parathyroid agenesis , mutations of the calcium - sensing receptor ( casr ) or parathyroid hormone ( pth ) genes , and pseudohypoparathyroidism .
the disease is characterized by low parathyroid hormone ( pth ) levels , hypocalcaemia , and hyperphosphatemia , and can be revealed by tetany , irritability , and seizure .
patients with autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ( apeced ) also suffer from hypoparathyroidism along with chronic mucocutaneous candidiasis and addison 's disease ( adrenocortical insufficiency ) , although the presence of two of this classic triad is diagnostic 1 .
a spectrum of other autoimmune endocrine and ectodermal disorders such as type 1 diabetes mellitus , thyroid autoimmunity , hypogonadism , pernicious anemia , keratitis , and vitiligo may also manifest 1 .
the disease is caused by loss - of - function mutations in the autoimmune regulator ( aire ) gene , which prevent the aire transcription factor from acting to ensure negative selection of autoreactive t cells in the thymus 2 .
due to the failure of immunological tolerance , affected organs are infiltrated by autoreactive t lymphocytes , and antibodies against tissue - specific proteins such as nacht leucine - rich - repeat protein 5 ( nalp5 ) , calcium - sensing receptor ( casr ) , and tyrosine hydroxylase can be present in patients ' sera 3,4 .
the first disease components , most usually chronic mucocutaneous candidiasis and hypoparathyroidism , normally emerge in childhood , but the heterogeneity of apeced in terms of its initial clinical presentation can make it challenging to identify and , typically , the condition is diagnosed after the first decade of life 1,5 .
more recently , the presence of antibodies against interferon ( ifn)- and ifn-2 has been shown to aid diagnosis due to their sensitivity and relative specificity for apeced 6,7 .
anti - interleukin ( il)-22 , anti - il-17a , and anti - il-17f antibodies are also common 8 .
in addition , mutational analysis of aire enables more than 95% of cases to be identified 2 . here , we report on an unusual clinical presentation of apeced in a young girl who had an early - onset of hypoparathyroidism and chronic keratitis , and who had experienced previously a mild and transient mucocutaneous candidiasis .
a 3-year - old girl was admitted to our pediatric emergency unit after having repeated carpopedal spasms , dyskinesia , and disequilibrium .
the girl 's medical history showed that she had presented with her first episode of spasm with fever some months before experiencing a mild and transient mucocutaneous candidiasis at 1 year of age , and needed an ophthalmological examination at 2 years because of photophobia and lacrimation at which time she was diagnosed with bilateral keratoconjunctivitis of unknown etiology .
she was born from healthy consanguineous parents without any neonatal problems , and had one healthy sibling .
the patient 's physical examination was unremarkable , but ocular assessment revealed chronic keratitis with progressive corneal scars ( fig.1 ) .
the patient 's spasms were indicative of hypocalcaemia and , indeed , biochemical analyses revealed a severely low serum calcium level of 1.28 mmol / l ( normal range , 2.22.7 mmol / l ) .
this was coupled with a high serum phosphate level of 2.56 mmol / l ( normal range , 1.31.85 mmol / l ) indicating profound hyperphosphatemia .
these evaluations , together with a low blood pth concentration of <3 pg / ml ( normal range , 1080 pg / ml ) and a decreased urine calcium : creatinine ratio of 0.06 mmol / l ( normal range , 0.20.7 mmol / l ) , suggested that the patient had hypoparathyroidism .
the serum level of 25-hydroxyvitamin d at 24 nmol / l ( normal range , 80250
creatinine at 34 mol / l ( normal range , 2337 mol / l ) and magnesium at 0.83 mmol / l ( normal range , 0.751.0
serum levels of thyroid - stimulating hormone ( tsh ) at 2.2 miu / l ( normal range , 0.43.1 miu / l ) , and free t4 ( the active form of thyroxine ) at 18.6 pmol / l ( normal range , 11.120.6
the absence of dysmorphy , cardiac malformation , and immunodeficiency ruled out the diagnosis of digeorge syndrome as a cause of the patient 's hypoparathyroidism .
lack of mutations of the casr gene also precluded this as the basis for the patient 's severe hypocalcaemia .
ultrasonography examination also showed normal parathyroid glands . despite the absence of the classic disease triad and lack of autoimmunity in her parents or sibling , we considered apeced as the possible diagnosis based on the association of hypoparathyroidism with a minor apeced component chronic keratitis , and a previous mild and transient mucocutaneous candidiasis . after obtaining written consent ,
blood samples were collected from the child and her parents for genetic analysis of the aire gene using peripheral blood mononuclear cells ( pbmcs ) as a source of dna .
subsequently , the diagnosis was confirmed by an aire gene frameshift mutation c.931delt ( p.c311fsx376 ) affecting codon 311 of exon 8 .
this mutation leads to the production of a downstream stop codon at codon 376 and thus , a shortened and a nonfunctional aire protein lacking both plant homeodomain ( phd ) 1 and 2 zinc fingers ( fig.2 ) .
schematic representation of the aire protein . shown are selected mutations , including the c311fsx376 ( c.931delt ) variant ; the caspase recruitment domain ( card ) ; the sp100 , aire1 , nucp41/p75 , deaf1 domain ( sand ) ; two plant homeodomain ( phd ) zinc finger motifs ; and the proline - rich region ( prr ) .
amino acid residues of the aire protein are numbered from the amino ( n ) terminus to the carboxy ( c ) terminus .
credence was added to the diagnosis by the detection of antibodies against ifn-2a ( antibody index , 5.2 ; normal range , 0.621.72 ) , ifn- ( antibody index , 2.79 ; normal range , 0.971.08 ) , interleukin ( il)-22 ( antibody index , 44.2 ; normal range , 0.871.39 ) , il-17f ( antibody index , 16.2 ; normal range , 0.861.19 ) .
however , the patient was sero - negative for anti - il-17a ( antibody index , 0.97 ; normal range , 0.781.61 ) , anti - ifn- ( antibody index , 0.89 ; normal range , 0.851.20 ) , and anti - ifn-1 antibodies .
further serological investigations indicated the patient was positive for antibodies against the casr ( antibody index , 33.2 ; normal range , 0.971.22 ) , but in casr functional assays , these did not activate the receptor .
anti - nalp5 antibodies were not detected ( antibody index , 1.12 ; normal range , 0.791.19 ) .
the patient had no humoral autoimmune response against the adrenal glands : anti-21-hydroxylase antibodies that are markers for the prevalent apeced component addison 's disease were absent .
similarly , the patient was sero - negative for the major markers of type 1 diabetes mellitus , namely anti - islet cell , anti - glutamic acid decarboxylase ( gad65 ) , and anti - tyrosine phosphatase - like protein ia2 antibodies .
thyroid autoimmunity was not apparent as indicated by normal function tests and an absence of anti - thyroid peroxidase and anti - thyroglobulin antibodies .
antibodies directed against actin , mitochondria , liver kidney microsomes , the nucleus , and dsdna were not present .
treatment of hypoparathyroidism consisted of combining calcium ( 0.5 g three times daily ) , 1,25-dihydroxyvitamin d3 ( 0.5 g once daily ) associated with 25-hydroxyvitamin d3 ( 100,000 iu every 3 months ) , and phosphate binders sevelamer hydrochloride , in order to alleviate spasms and return serum calcium and phosphate levels to 2.20 mmol / l and 1.66 mmol / l , respectively .
however , the ocular lesions progressed and led to the loss of visual acuity and irreversible corneal scarring in spite of a topical treatment with steroids .
at the time of diagnosis , the 3-year - old patient did not have the classic apeced disease triad of chronic mucocutaneous candidiasis , hypoparathyroidism , and adrenocortical insufficiency , and familial evidence of the disorder was lacking .
in addition , aside from hypoparathyroidism and chronic keratitis , none of the many other apeced - associated diseases were evident either clinically or immunologically . the diagnosis was therefore confirmed by detecting anti - ifn- and anti - ifn-2 antibodies , and sequencing of the aire gene for potential mutations . as part of the syndrome ,
chronic mucocutaneous candidiasis is apparent in 70% of cases by age 10 rising to 97% by age 30 and is the earliest and most common sign of apeced 1 .
prior to the diagnosis , our patient had suffered from only a single episode of mild and transient mucocutaneous candidiasis . the patient presented with hypoparathyroidism , the most frequent endocrine feature of apeced with a prevalence of 66% by age 10 and 85% by age 30 1 . like other endocrinopathies in apeced ,
sporadic case reports of idiopathic hypoparathyroidism have documented lymphocytic and plasma cell infiltration of the parathyroid 9 , as well as complement - mediated damage of the glands 10 . however , our patient had a normal parathyroid examination negating immunologically damaged glands as a cause of her hypoparathyroidism .
anti - casr antibodies which can have an etiological role in hypoparathyroidism by activating the casr and thus inhibiting pth secretion , have been identified in a small number of cases of isolated hypoparathyroidism and apeced 1113 . in our patient , although a humoral autoimmune response against the casr was detected , the antibodies did not appear to stimulate the receptor .
conceivably , anti - casr - activating antibodies may have been at too low level to be demonstrated with our assay .
interestingly , the patient was negative for anti - nalp5 antibodies which are reported markers for hypoparathyroidism in apeced 4
addison 's disease appears most commonly following mucocutaneous candidiasis and hypoparathyroidism , and by age 10 in 40% of patients and by age 30 in 78% of patients 1 .
there was no clinical or immunological evidence of adrenocortical insufficiency in the current case , but diagnosis of adrenal failure is crucial to avoid the risk of a fatal adrenal crisis .
the potential development of addison 's disease will therefore be monitored as a diagnosis of apeced , meaning it is highly likely that this will occur later in the patient 's lifetime given her young age .
the only nonendocrine disease identified in the patient at diagnosis was keratitis , the onset of which was rapid , probably before hypoparathyroidism .
keratitis is the first manifestation in only 314% of apeced cases , but is usually an early component and affects 2550% of patients 1 .
symptoms include photophobia , blepharospasm , conjunctival redness , and decreased visual acuity , all of which were noted in our patient 14 .
although the pathophysiology of keratopathy is not fully understood , immune cells seem to play an essential role : in aire - deficient mice , corneal and conjunctival epitheliums , stroma and meibomian glands are infiltrated by cd4 + and cd8 + t cells 15 .
meibomian gland destruction and the loss of goblet cells result in the loss of tear film and may trigger inflammation 16 , although the subsequent progression of epithelial edema and superficial opacity can be reversed under topical steroid therapy .
recurrences are frequent and lead to irreversible scarring ( nodule and pannus formation ) , deep vascularisation , and blindness 14,16 .
crucial to the diagnosis of apeced in this unusual case was the detection of typical anti - ifn- and anti - ifn-2 antibodies 6,7 .
the th17 cell - associated cytokines , il-17a , il-17f , and il-22 , play a key role in protecting against candida infection 17 .
high levels of antibodies to the il-17 family of cytokines are typically produced by patients with apeced and are suggested to predispose to candidiasis by virtue of their neutralizing activities 18 . indeed , apeced patients with no or mild candidiasis have low or undetectable levels of antibodies against il-17a , il-17f , and il-22 18,19 .
compatible with this theory , our patient was sero - negative for anti - il-17a antibodies .
however , she did have high levels of anti - il-22 and anti - il-17f antibodies , an observation analogous to a report of five apeced patients in which a lack of candida infection correlated only with an absence of antibodies against il-17a 19 .
the final confirmation of apeced came with the identification of homozygous aire gene mutation c.del931t , a rare variant with only two previously documented cases 2,20 .
the first was described in a french patient analyzed as part of a series of 112 apeced patients from various ethnic backgrounds 2 .
although the exact clinical details of the patient were not given , the individual had at least two of the classic apeced triad of hypoparathyroidism , chronic mucocutaneous candidiasis , and adrenocortical insufficiency .
a second case with this aire gene mutation was reported in a young male sicilian 20 .
onset was at 6 years of age and , similar to our patient , he experienced candidiasis and hypoparathyroidism .
in contrast , he suffered also from malabsorption , alopecia , enamel hypoplasia , and nephrocalcinosis , but not from keratitis .
overall , the c.del931t mutation does not appear to correlate with a specific clinical apeced phenotype .
early diagnosis of apeced , a potentially life - threatening condition , is crucial . however , because of the clinical heterogeneity of the disorder , there is often a delay between the first symptoms and identification of the disease with a mean gap of 10.2 years 5 . as seen from this study , even in the absence of the main triad of disorders of chronic mucocutaneous candidiasis , hypoparathyroidism , and adrenocortical insufficiency , apeced
should be considered in young patients who have hypoparathyroidism and chronic keratitis , and a past medical history showing a mild and transient candida infection .
an initial diagnosis can be confirmed quickly by serological testing for anti - ifn- and anti - ifn-2 antibodies , and sequencing of the aire gene where available .
long - term follow - up will be mandatory to screen for additional organ - specific autoimmunity which could develop over the following years and decades . | key clinical messageearly diagnosis of potentially life - threatening autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ( apeced ) is crucial , but is often delayed due to the clinical heterogeneity of the disorder . even in the absence of the classic disease triad of chronic mucocutaneous candidiasis , hypoparathyroidism , and adrenocortical insufficiency , a diagnosis of apeced should be considered in children who have hypoparathyroidism and chronic keratitis , with a past medical history showing a mild and transient candida infection . | Introduction
Case History and Examination
Laboratory Evaluations
Differential Diagnosis and Investigations
Treatment and Follow-up
Discussion
Conflict of Interest |
at low levels of central hypovolemia elicited by mild lbnp in humans , msna increases in the absence of alterations in heart rate ( hr ) or blood pressure ( sundlof and wallin , 1978a ; victor and leimbach , 1987 ) .
this initial increase in msna occurs in a similar fashion whether measured from the radial nerve in the arm or the peroneal nerve in the leg , and is therefore a reflection of generalized sympathetic activation to muscle vascular beds ( rea and wallin , 1989 ) .
importantly , the response in msna to relatively low levels of lbnp ( i.e. , 10 mmhg ) is identical to the msna response to blood loss of 450 ml , demonstrating the effectiveness of lbnp as an experimental model of hemorrhage ( figure 1 ; rea et al . , 1991 ) .
comparison of relationships between central venous pressure ( cvp ) and muscle sympathetic nerve activity ( msna ) during 10 mmhg lbnp and 450 ml hemorrhage in nine human subjects .
circles and lines represent mean se values . * p < 0.05 compared with baseline . from cooke et al .
because arterial pressure was unchanged , the increase in msna elicited by low levels of lbnp or hemorrhage was originally ascribed to deactivation of cardiopulmonary baroreceptors alone , without contribution from arterial baroreceptors ( rea et al . , 1991 ) .
in fact , so many investigators have used low levels of lbnp ( 20 mmhg ) to selectively unload cardiopulmonary baroreceptors that this has recently been referred to as dogma
subsequent work , however , has conclusively demonstrated that deactivation of arterial baroreceptors also contributes to sympathoexcitation induced by non - hypotensive hypovolemia .
first , non - hypotensive hypovolemia reduces the diameter of both the ascending thoracic aorta ( taylor et al . , 1995 ) and the carotid artery ( lacolley et al . , 1992 ) , sites of the stretch - sensitive aortic and carotid arterial baroreceptors .
( 2001 ) demonstrated that , during lbnp of only 5 mmhg , msna increased while parasympathetic modulation of hr ( assessed via power spectral analysis ) decreased , which is a manifestation of arterial baroreceptor unloading .
third , msna increases during mild hypovolemia in both intact control subjects and in cardiac transplant patients , indicating a greater contribution to sympathoexcitation of sinoaortic baroreflexes than ventricular receptors ( jacobsen et al . , 1993 ) .
2009 ) recently observed that transient reductions in stroke volume and blood pressure [ both systolic ( sap ) and diastolic ( dap ) pressures ] occur at the onset of even mild levels of lbnp .
taken together , it has become clear that the sympathoexcitation produced by even low levels of central hypovolemia is a result of reflex - mediated deactivation of both cardiopulmonary and arterial baroreceptors .
additionally , recent evidence suggests that reflexes from muscle afferents stimulated during venous distension may also directly evoke increases in msna ( cui et al . , 2009 , 2011a ) .
while venous distension should not occur during hemorrhage , the possibility that reflexes arising from distension of leg veins contribute to the sympathoexcitation observed during lbnp can not be discounted .
once initiated , msna has been proposed to linearly increase during progressive central hypovolemia , although much of the data supporting this suggestion until recently has been accrued using lower levels of lbnp ( 30 mmhg ; convertino and cooke , 2002 ; cooke et al . , 2004 ) .
this is because of the inherent difficulty in maintaining nerve recordings during higher levels of negative pressure , as the microelectrode is often displaced during the lbnp procedure . to mitigate this issue , khan et al .
( 2002 ) applied negative pressure to only one leg while performing microneurography in the other . using this protocol
, these investigators demonstrated that progressive reductions in lbnp ( up to 50 mmhg ) produce concomitant graded increases in msna , but they did not measure the degree of central hypovolemia induced by lbnp in order to quantify the relationship between volume loss and sympathetic activation ( khan et al . , 2002 ) . over the past 6 years
, we have created a large database of human experiments ( > 200 subjects ) in which progressive central hypovolemia has been used to produce hemodynamic decompensation ( i.e. , presyncope ) . in each subject
, chamber decompression was applied for 5 min at 15 , 30 , 45 , and 60 mmhg , and then additional increments of 10 mmhg were used until the onset of decompensation . in the course of this work , we have obtained microneurography recordings in a subset of subjects in which stroke volume , a measure of central hypovolemia , has also been assessed .
we were able to maintain msna recordings throughout lbnp to either the point of presyncope or to a high ( 80 mmhg ) lbnp level in 20 subjects .
this unique data set has provided the opportunity to develop new understandings of the msna response to intense levels of central hypovolemia .
for example , figure 2 shows msna recordings from a representative subject exposed to progressively higher levels of lbnp .
average values across subjects demonstrate that increases in msna burst frequency are inversely related to stroke volume levels during central hypovolemia in humans ( figure 3 ) , thereby confirming the linearity of the relationship between central hypovolemia and the induced msna response .
this same relationship between central hypovolemia and direct measurement of renal sympathetic nerve activity ( rsna ) is also observed in anesthetized sheep during graded hemorrhage ( batchinsky et al . , 2007b ) .
it should be noted that progressive increases in sympathetic activation during lbnp are evident whether msna is expressed as frequency ( bursts / min ) , incidence ( bursts/100 heartbeats ) , or total activity ( which takes into account burst amplitude ; cooke et al .
muscle sympathetic nerve activity ( msna ) in a representative subject during progressive lower body negative pressure . from cooke et al .
relationship between stroke volume ( sv ) and muscle sympathetic nerve activity ( msna ) during lbnp in 20 human subjects .
in addition to increases in absolute msna , progressive central hypovolemia also changes the pattern of bursting activity . even at rest
, sympathetic nerve bursting is characterized by a rhythmic pattern that exists in the low frequency ( lf ) range ( 0.040.15 hz ) ; this oscillatory pattern in sympathetic nerve activity contributes to the 10-s arterial pressure mayer waves ( preiss and polosa , 1974 ) . while it is beyond the scope of this review ,
there is a continued controversy regarding whether the oscillatory pattern of sympathetic nerve activity and mayer waves is produced via a central oscillator mechanism or through the action of the baroreflex ( for a review of this issue , see julien , 2006 ) . in animal models of hemorrhage ,
the amplitude of lf oscillations in blood pressure and sympathetic nerve activity increase ( guyton and harris , 1951 ; malpas et al . , 1998 ) . in humans ,
the amplitude of lf oscillations in msna ( msnalf ) increase concomitantly with burst frequency and total activity during stresses that reduce venous return such as head - up tilt ( cooke et al .
, 1999 ; furlan et al . , 2000 ; kamiya et al . , 2005 ) and lbnp ( ryan et al . , 2011 ) .
in addition to increases in the frequency and amplitude of sympathetic nerve firing , it is therefore possible that alterations in the oscillatory patterns of that firing may contribute to the ability of an individual to adequately compensate for progressive central hypovolemia ( see below ) .
sympathetic nerves normally fire in response to reductions of arterial pressure ( particularly , dap ) and are silenced with the consequent sap upstroke , resulting in almost constant latencies from preceding r - waves ( sundlof and wallin , 1978b ; fagius and wallin , 1980 ) . at high levels of lbnp , however , an interesting phenomenon occurs in some subjects .
figure 4 depicts msna from a subject at baseline , during 60 and 90 mmhg of lbnp ( cooke et al . , 2009 ) .
at 60 mmhg , coupling of bursts occurred followed by fusing of multiple bursts with greater central hypovolemia ( i.e. , at 90 mmhg ) . in our study , burst fusion occurred in 10 of the 17 subjects available for study at the time of publication ( cooke et al . , 2009 ) .
burst fusion did not appear to be artifactual , although we can not completely discount the possibility that the 0.1-s time constant used to integrate msna may have contributed to their appearance ( cooke et al . , 2009 ) .
similar alterations in the typical pulse synchronous burst pattern of msna had previously been noted in subjects susceptible to fainting during head - up tilting ; during the presyncopal ( i.e. , decompensation ) phase , burst reflex latencies were shortened and burst durations were lengthened , effects which were reversed on return to the fully conscious state ( iwase et al .
our data extend this observation to the earlier compensatory phase of central hypovolemia , when blood pressure is still well - maintained .
importantly , fusion of rsna has also been observed in anesthetized sheep made hypotensive by severe hemorrhage ( batchinsky et al . , 2007b ) .
we have suggested that this phenomenon may represent sympathetic baroreflex deafferentation ( cooke et al . , 2009 ) , as the fused bursts observed during intense lbnp are similar in both their pulse asynchrony and their structure to those observed after bilateral blocks of glossopharyngeal and vagus nerves ( fagius et al . , 1985 ) .
it is also possible that loss of pulse - synchrony and continuous burst firing may occur in some individuals as a strategy to continue to maintain blood pressure during severe reductions in venous return . in support of this concept ,
( 2011 ) have recently shown that extreme baroreceptor unloading requiring high sympathetic outflow ( induced by 80 mmhg lbnp ) recruits a subpopulation of large postganglionic axons in some but not all individuals .
coupling ( 60 mmhg ) and then fusing ( 90 mmhg ) of muscle sympathetic nerve activity ( msna ) in one subject during lower body negative pressure . from cooke et al .
( 1944 ) observed that the onset of fainting produced by experimental hemorrhage in man was preceded by a profound vasodilation in the forearm and a decrease in systemic vascular resistance , suggesting inhibition of the compensatory sympathetic activation that had occurred before this point .
as it became possible to directly measure sympathetic nerve activity in humans , a variety of case reports and experimental studies appeared showing an inhibition of sympathetic activity associated with the development of presyncope in both healthy humans ( burke et al . , 1977 ; sanders and ferguson , 1989 ; scherrer et al .
iwase et al . , 2000 ; cooke and convertino , 2002 ) and in patient populations ( wallin and sundlof , 1982 ; yatomi et al . , 1989 ; converse et al . , 1992 ; jardine et al . , 1996 , 1998 , 2002 ; morillo et al . , 1997 ) .
on the basis of these data , it was concluded that hemodynamic decompensation ( i.e. , presyncope ) was associated with and possibly caused by sympathetic neural withdrawal ( convertino and cooke , 2002 ; cooke et al . , 2004 ) .
recently , however , we have observed that sympathetic withdrawal does not occur in all subjects at the point of hemodynamic decompensation ( figure 5 ) ; in fact , 41% of our subjects did not demonstrate any diminution of msna despite the onset of hypotension and presyncopal symptoms ( cooke et al . , 2009 ) .
2010 ) showed that increases in msna induced by orthostatic stress were preserved through the point of syncope in 90% of patients previously diagnosed with vagovagal syncope .
it is therefore apparent that sympathetic withdrawal is not an absolute requirement for the onset of hemodynamic decompensation in both healthy humans and in patients with a history of syncope . indeed ,
careful perusal of some of the literature frequently cited to support the proposition that sympathetic withdrawal precipitates cardiovascular collapse reveals that blood pressure begins to decrease while msna remains elevated during the compensatory phase of hypovolemia ( wallin and sundlof , 1982 ; sanders and ferguson , 1989 ; converse et al . , 1992 ; jardine et al .
, 1996 , 1998 ; mosqueda - garcia et al . , 1997 ; iwase et al . , 2000 ) , an observation confirmed more recently ( kamiya et al . ,
, these data refute the concept that cessation of sympathetic nerve activity precedes and causes hypotension , the subsequent reduction in cerebral perfusion pressure , and syncope in all subjects .
arterial pressure ( ap ) and muscle sympathetic nerve activity ( msna ) for three representative subjects 2 min before the onset of presyncope . the lowest arterial pressure ( bp ) recorded for each subject
a185 , msna decreased in the last 2 min of lbnp ( 120 to 60 s , 82 bursts / min ; 60 s to presyncope , 64 bursts / min ) , but was still maintained at high levels relative to the pre - lbnp control ( 36 bursts / min ) .
subject a075 displayed burst fusion , elevated msna over control ( 25 bursts / min ) , and no withdrawal of msna at presyncope ( 120 to 60 s , 53 bursts / min ; 60 s to presyncope , 54 bursts / min ) .
subject a199 ( a low tolerant subject ) did not display large increases in msna from the pre - lbnp control value ( 17 bursts / min ) and also did not demonstrate msna withdrawal at presyncope ( 120 to 60 s , 22 bursts / min ; 60 s to presyncope , 21 bursts / min ) . from cooke et al .
another possible mechanism that may contribute to the onset of cardiovascular collapse is resetting of baroreflexes , resulting in a loss of synchrony between arterial blood pressure and compensatory responses such as sympathetic activation . during
mild to moderate central hypovolemia , the operating point of baroreflex - mediated control of sympathetic nerve activity is shifted upward without a change in gain , such that sympathetic nerve activity is increased at any given dap ( ichinose et al .
additionally , there is a tighter coupling between oscillations in arterial blood pressure and msna , as quantitated using cross - spectral analysis by an increase in the coherence function between these variables ( furlan et al . , 2000 ;
this increase in coherence reflects greater baroreflex modulation of sympathetic activity compared with the baseline state and both the increase in coherence and operating point may be beneficial in mounting an appropriate compensatory response to hypovolemia .
evidence exists , however , to suggest that this tight coupling of blood pressure to sympathetic nerve activity may be lost at the point of hemodynamic decompensation . based on their observation of sympathoinhibition ( as measured by plasma catecholamines ) despite hypotension , jacobs et al .
( 1995 ) first suggested that the vasodepressor response at presyncope was due to a sudden central resetting of baroreflexes .
( 2006 ) using data from subjects who either exhibited presyncope or did not during central hypovolemia induced by lbnp .
approximately 12 min prior to the onset of hemodynamic decompensation , the gain of the baroreflex function relating dap to msna was substantially reduced in those subjects exhibiting presyncope but was unchanged in non - presyncopal subjects ( ichinose et al . , 2006 ) .
likewise , we have also demonstrated a loss of linearity between the change in dap and the change in msna just before the onset of presyncope , suggesting disruption in the normal baroreflex - mediated coordination between arterial pressure and sympathetic activation ( cooke et al . , 2009 ; convertino et al . ,
taken together , these results suggest that impairment of arterial baroreflex control over sympathetic vasomotor activity may contribute to the onset of hemodynamic decompensation .
such a sudden attenuation of baroreflex function before hemodynamic decompensation has also been noted for cardiovagal reflexes ( ogoh et al .
as mentioned above , there is also evidence to suggest that the pattern of msna firing may be as important in determining the ability to withstand central hypovolemia as the absolute level of msna . in an elegant study ,
( 2005 ) determined the temporal occurrence of events immediately preceding presyncope . during central hypovolemia induced by head - up tilt
, both the absolute levels of msna ( expressed as burst frequency and total activity ) and the amplitude of lf oscillations in msna ( msnalf ) increased markedly ; increases in msnalf were reflected in increases in lf oscillations of mean arterial pressure ( maplf ) . as subjects moved toward presyncope , msnalf and maplf decreased and
were associated with a decrease in map , despite the maintenance of msna burst frequency and total activity at their elevated level .
msna , msnalf , and maplf were maintained and did not decrease in those subjects who did not exhibit hypotension and presyncope ( kamiya et al . ,
we have observed a similar phenomenon in a subject whose tolerance to central hypovolemia was improved through the use of inspiratory resistance breathing ( figure 6 ) . in this experiment , subjects were exposed to lbnp to the point of presyncope in separate experiments performed at least 2 weeks apart . in one experiment , subjects breathed through a device that did not provide resistance to inspiration ( sham ) , while in the other experiment , subjects breathed through a device that provided resistance to inspiration ( active ) . because resistance breathing improved lbnp tolerance , data were analyzed at the end of lbnp during the sham experiment and at this same absolute time point during the active experiment ( i.e. , well before the onset of presyncope during inspiratory resistance breathing ) . during breathing with the sham device , msna increased from 12 bursts / min at baseline to 44 bursts / min at the end of lbnp , while msna increased from 10 to 54 bursts / min at this same absolute time point ( i.e. , prior to presyncope ) during resistance breathing .
importantly , msnalf increased from 1.15 to 22.4 au at presyncope under the sham resistance breathing condition ; with resistance breathing , msnalf increased from 2.23 to 40.1 au at this same absolute time point .
moreover , the coherence between dap and msna , a measure of the strength of the relationship between these two variables , was increased from 0.80 in the sham condition to 0.94 with resistance breathing at this same time point .
thus , in this one subject , it is apparent that inspiratory resistance breathing increased the amplitude of msna oscillations and coherence between msna and arterial blood pressure , and that this alteration in oscillatory pattern was associated with improved tolerance to central hypovolemia .
these data are consistent with the concept that the maintenance of an increase in lf oscillations of both msna and map may be associated with the defense of blood pressure during severe central hypovolemia .
blood pressure ( bp ) , muscle sympathetic nerve activity ( msna ) and end - tidal co2 ( et co2 ) tracings for a single subject under conditions of breathing with a sham ( top ) or active ( bottom ) device that provided resistance to inspiration .
thus , there are several possible mechanisms involving alterations in activation of sympathetic nerve activity to explain hemodynamic decompensation during severe hypovolemia . since sympathetic withdrawal occurs in some individuals before presyncope but not in others , it is no longer thought to be a prerequisite for the ensuing hypotension ( cooke et al . , 2009 ) .
it is also possible that there is a central resetting of arterial baroreflex function that alters sympathetic outflow ( ichinose et al . , 2006 ) ; this scenario may be especially prominent in those subjects in whom sympathetic inhibition occurs despite progressive hypotension . finally , there is evidence to suggest that an increase in lf oscillations in both msna and map may be protective during central hypovolemia and that loss of these oscillations might precipitate hypotension ( kamiya et al . , 2005 ) .
it is important to note that these mechanisms are not mutually exclusive and the contributions of each to the process have yet to be fully revealed .
furthermore , it is also possible and even probable that different mechanisms may predominate in different individuals . because of the scope of this review , we have chosen to focus on those mechanisms preceding presyncope that involve loss of compensatory alterations in msna , but it is likely that loss of other compensatory responses may also contribute to the inability to maintain blood pressure during severe hypovolemia .
1997 ) is that marked peripheral vasodilation is a major contributor to the fall in arterial pressure preceding vasovagal syncope . in this regard ,
it is of interest that , during prolonged lbnp at a low level ( 15 mmhg ) , vasodilation of the forearm musculature was observed despite the continued presence of a sustained compensatory increase in msna , suggesting that sympathetic escape occurs ( joyner et al . ,
implicit in the preceding discussion is the notion that there are individual differences in the ability of healthy humans to tolerate central hypovolemia before reaching the point of cardiovascular collapse .
indeed , it has been known for many years that there is a great deal of variability in the ability of patients ( davis , 1949 ) and animals ( chien , 1967 ; kim and shoemaker , 1970 ) to survive traumatic hemorrhage ; we have recently learned that there is a genetic basis underlying this variability ( klemcke et al . , 2008 , 2011 ) .
likewise , individual differences in tolerance to central hypovolemia induced by lbnp have also been described ( sather et al . , 1986 ) and attributed to differences in the release of vasoactive hormones ( convertino and sather , 2000b ; greenleaf et al . , 2000 ) , compensatory tachycardia and vasoconstriction ( convertino and sather , 2000a , b;greenleaf et al . , 2000 ) , cardiac baroreflex gain ( convertino and sather , 2000a ; convertino et al .
, in press ) , baroreflex gain of sympathetic nerve activation ( wijeysundera et al . , 2001 ) , and central blood volume and cerebral blood velocity ( levine et al . , 1994 ) . in this regard
, we have recently shown that subjects demonstrating high tolerance ( ht ) to lbnp have a greater ability to increase msna than subjects with low tolerance ( lt ; convertino et al .
, in press ) . additionally , the ability to sustain the compensatory mechanisms described above involving both the baroreflex modulation of sympathetic activation and the oscillatory component of that activation may also act to determine tolerance to central hypovolemia .
msna baroreflex in subjects who became presyncopal ( i.e. , those demonstrating lt ) during central hypovolemia but did not observe this resetting in those who did not ( ht ) .
( 2005 ) observed a loss of msnalf and maplf power in lt subjects before presyncope elicited by head - up tilt , but no such loss in ht subjects .
; convertino and sather , 2000a , b ) , we recently confirmed and expanded on these results using our large database of subjects in which lbnp was applied to the point of presyncope in all individuals .
we classified subjects as ht if they completed at least the 60-mmhg level of lbnp , and lt if they did not complete this level ( rickards et al . , 2011 ) . as in the previous study using head - up tilt ( kamiya et al . , 2005 ) ,
maplf increased in both lt and ht groups during early stages of lbnp , but maplf decreased to baseline levels in the lt group at 60 mmhg while it continued to increase in the ht group ( rickards et al . , 2011 ) .
a similar relationship between the ability to increase orthostatic tolerance and the ability to increase blood pressure oscillations has previously been shown ( gulli et al . , 2001 ) . in our study ,
measurement of middle cerebral artery velocity ( mcav ) by transcranial doppler revealed a similar oscillatory pattern to that observed with map , indicating that the continued increase in amplitude of lf arterial pressure oscillations observed in ht subjects was transferred to the cerebral vasculature ( rickards et al . , 2011 ) .
although we did not report msna in our paper , figure 7 shows these responses during lbnp in a ht and a lt subject . in the ht
subject , msnalf increased from baseline ( 4.4 au ) to a maximum of 85.8 au just prior to presyncope .
in contrast , msnalf did not increase in the lt subject at all ( 2.4 to 2.3 au ) .
thus , we propose that the ability to increase lf oscillations in msna , map , and mcav is an inherent characteristic associated with improved tolerance to central hypovolemia and therefore protects against the onset of hemodynamic decompensation during severe hypovolemia .
representative tracings from a high tolerant and low tolerant subject at baseline ( bl ) , sub - maximal lbnp ( i.e. , the lbnp level before the level at which presyncope was reached ; sm ) and immediately before presyncope ( indicated by arrow ; ps ) .
because the autonomic nervous system serves to maintain cardiovascular homeostasis under a variety of physiological and pathological stresses , a non - invasive surrogate for sympathetic and/or vagal activation has long been sought for diagnostic use ( goldstein et al . , 2011 ) .
measures of hr variability ( hrv ) have received a great deal of attention , as some of the time and frequency domain measures have been associated with autonomic function .
specifically , power spectral analysis of intervals between r - waves ( rri ) of the ecg yields lf ( 0.040.15 hz ) and hf ( 0.150.4 hz ) powers ; lf power was originally thought to contain components of both cardiac sympathetic and vagal ( parasympathetic ) function , while hf power predominantly reflected parasympathetic function ( akselrod et al . , 1981 ) .
many investigators have also suggested that the ratio of lf / hf represents sympathovagal balance ( malliani et al . , 1991 ; goldstein et al . , 2011 ) and that these or other hrv metrics might be useful in disclosing dysautonomia in such clinical conditions as myocardial infarction , cardiac arrhythmias , diabetes , and renal failure ( acharya et al . , 2006 ;
montano et al . , 2009 ) . indeed , a pubmed search on the term heart rate variability yields more than 14,300 references at the time of this writing ( february 2012 ) , yet we are unaware of any pathophysiological condition in which hrv is currently used as a standard of care for diagnosis and/or treatment .
the use of hrv is appealing because calculation requires only non - invasive collection of a standard ecg .
because increases in sympathetic activation occur in a linear fashion with decreases in stroke volume ( figure 3 ) , we proposed that hrv metrics might be useful non - invasive surrogates of msna for assessing the degree and progression of hemorrhage in trauma victims ( cooke and convertino , 2005 ) . in 2008
, we demonstrated using our lbnp model of simulated hemorrhage that , on average , rrihf power and some time domain metrics associated with parasympathetic function are inversely related to direct measurement of msna .
rrilf power , on the other hand , was not associated with msna ( cooke et al . , 2008 ) ; it has been made clear more recently that rrilf is not a measure of cardiac sympathetic activity ( billman , 2011 ; goldstein et al . , 2011 ) .
from these data , we concluded that hrv might be of clinical utility in assessing autonomic function during hemorrhage , although we and others noted that correlations between msna and hrv in individual subjects were not as strong as those derived from group means ( floras et al .
application of power spectral analysis to ecg recordings collected from actual trauma patients during air transport to the hospital further suggested that the use of these metrics might be appropriate for clinical assessment of the severity of hemorrhage , as group means of some hrv metrics differed between patients who lived and those who died up to 24 h later ( cooke et al .
subsequent studies extended these observations to the use of non - linear hrv metrics to predict mortality ( batchinsky et al .
, 2007a ) and to discriminate between trauma patients who required a life - saving intervention and those who did not ( cancio et al . ,
importantly , all of the conclusions delineated above were developed based on standard analyses of group mean data .
as our understanding evolved , however , we began to take the next step to assess whether hrv metrics could be useful in determining the physiological status of individuals rather than a group as a whole , a requirement that must be met for successful application of any metric for diagnosing individual patients .
in doing so , we became aware of a number of challenges with all of the hrv metrics that we examined ( time domain , frequency domain , and non - linear metrics ) .
first , there are issues of large inter - individual variability and poor reproducibility within subjects during the same recording session ( rickards et al . , 2010a ) as well as across days ( tan et al . , 2009 )
second , accurate determination of hrv requires long ( in some cases , up to 800 beats ) segments of ecg recordings that do not contain electromagnetic noise or ectopic beats , but these occur more frequently in trauma patients than in healthy individuals ( sethuraman et al .
third , increases in hr will always be associated with decreases in hrv , purely as a mathematical function of the curvilinear nature of the relationship between hr and rri ( sacha and pluta , 2008 ) .
decreases in hrv are therefore not specific to central hypovolemia but may be elicited by any physiological stressor that induces tachycardia such as physical movement ( rickards et al . , 2008 ) , pain , or anxiety , which are common in conscious trauma patients .
fourth , hrv metrics are not able to discriminate between ht and lt subjects early in the progression of hypovolemia ; an effective triage tool should be able to alert medical personnel to those patients that will progress to hemodynamic decompensation more quickly ( hinojosa - laborde et al . , 2011 ) . finally ,
although group means of hrv metrics are highly correlated with decreases in stroke volume during central hypovolemia , analysis of the individual trajectories for these metrics demonstrated poor and inconsistent correlations with stroke volume at the individual subject level , even under controlled laboratory conditions ( ryan et al .
armed with this new understanding , re - analysis of ecg data collected from trauma patients in the prehospital phase demonstrated that , while means of some of these hrv metrics differed between groups that received a life - saving intervention and those that did not , there was such high overlap of individual patient hrv values between groups that it would have been impossible to accurately classify patients on the basis of hrv alone ( figure 8 ; rickards et al . , 2010b ) .
, 2007a ; cancio et al . , 2008 ) in which differences existed between groups even in standard vital signs , only those trauma patients with normal vital signs were chosen for analysis in order to ascertain whether the use of hrv metrics provided added discriminatory value over standard vital signs .
based on these data , hrv metrics on their own fail to be useful for assessing the severity of hemorrhaging trauma patients in the prehospital and emergency department phases of diagnosis and treatment .
individual values of four representative heart rate variability metrics for life - saving intervention ( lsi ; n = 32 ) and no - lsi ( n = 127 ) patient groups ( each point represents a single patient ) . for each hrv metric
, group means statistically differ between lsi and no - lsi groups ( p 0.03 ) .
the percentage of individual lsi patient values that fall within the range of the no - lsi group are presented in each plot .
r intervals root mean squared sd ( a time domain metric ) ; rrihf , r
r intervals high frequency ( a frequency domain metric ) ; sampen , sample entropy ( a non - linear metric ) ; dfa - l , detrended fluctuations analysis long range correlations ( a non - linear metric ) . from rickards
the amplitude of the lf oscillations in sap has also been suggested as a potential non - invasive surrogate for sympathetic vasomotor tone and nerve activation ( pagani et al . , 1986 ) .
linear relationships between saplf and direct measurement of msna were later described in human subjects during sympathetic activation induced by sodium nitroprusside infusion ( pagani et al . , 1997 ) and head - up tilt ( furlan et al . , 2000 ) . on the basis of such observations , saplf has been used as a non - invasive metric for the determination of dysautonomia in hypertension ( lucini et al . , 2002 ) and type 1 diabetes ( lucini et al . , 2009
however , this viewpoint has been the subject of continued controversy ( parati et al . , 2006 ; taylor and studinger , 2006 ) .
data that argue against the use of saplf as a non - invasive metric of msna include the inability of saplf to adequately correlate with levels of msna in humans at rest ( taylor et al . , 1998 ) or during various physiological or pathophysiological conditions ( radaelli et al . , 1999 ; cui et al .
we have recently investigated this question using our database of msna recordings obtained during central hypovolemia ( ryan et al . , 2011 ) .
when group mean data were considered , there was a strong direct relationship ( r = 0.98 ) between the increase in msna and saplf , but it was curvilinear rather than linear as had been previously described ( ryan et al . , 2011 ) .
when we took the next step to determine correlations between saplf and msna within individual subjects , however , we found poor correlations in a significant portion of our subjects ( range of r : 0.090.96 ; ryan et al . , 2011 ) .
because a strong relationship between these variables is not universally applicable to all healthy human subjects , saplf should not be used as a non - invasive surrogate of msna .
therefore , no reliable and accurate metric has been identified that is based on non - invasive recordings and that may be reliably used in the place of direct recordings of msna to assess levels of sympathetic activation during physiological stressors or disease states .
hemorrhage continues to be a major cause of morbidity and mortality in both military and civilian settings , and research efforts continue to provide better means of both diagnosing the severity of blood loss and guiding resuscitation efforts .
while important work continues in animal models , human experimental models such as lbnp have provided valuable insight as to the mechanisms of compensatory physiological responses to central hypovolemia without the confounding factors of anesthesia , species differences , and tissue injury . during even mild lbnp , both arterial and cardiopulmonary baroreceptors
are unloaded , resulting in activation of the sympathetic nervous system to increase hr and vasomotor tone .
importantly , we now know that sympathetic activation entails not only an increase in sympathetic nerve firing but also an alteration in the pattern of firing , such that the amplitude of lf oscillations in msna and , consequently , blood pressure is also increased ; both of these responses seem to be protective in that they are associated with improved tolerance to central hypovolemia . at some point , which occurs at different levels of central hypovolemia for individual subjects , this compensation fails and hypotension ensues .
although it was once thought that sympathetic withdrawal always precipitated hemodynamic decompensation and hypotension , it is now clear that a diminution of absolute levels of msna firing is not required in all individuals .
instead , it is possible that loss of the compensatory increase in msnalf may also be involved , particularly in those subjects demonstrating ht to central hypovolemia .
additionally , there may be an acute resetting of the baroreflex at the level of the cns such that coherence between arterial blood pressure and sympathetic nerve activity is lost .
determination of the mechanisms underlying the development of presyncope in both healthy human subjects and patients with diseases characterized by episodes of fainting continues to be an ongoing area of research .
the search for a non - invasive surrogate of msna for both research and clinical purposes continues .
certainly , an easily obtainable non - invasive metric of sympathetic activation could be of great importance for assessment of the severity of hemorrhage .
however , metrics based on determination of variability in both rris and arterial blood pressure do not fulfill the necessary criteria to perform effectively in this role . before implementation of any such metric
, it is essential that investigators determine whether the metric will be able to reliably track msna within individual subjects rather than simply rely on analyses based on group mean data .
importantly , gender differences in the msna response to central hypovolemia must also be taken into account ( fu et al . , 2005 ; carter et al . ,
, it will also be necessary for laboratory determination of the efficacy of the metric in the face of blood loss combined with other physiological stressors which can impact sympathetic activation , such as heat stress ( cui et al .
, 2004a , 2011b ) , dehydration ( kimmerly and shoemaker , 2002 , 2003 ; fu et al . ,
2005 ) , mental or emotional stress ( carter et al . , 2008 ) , and ingestion of alcohol ( carter et al . , 2011 ) , nicotine , or other drugs .
all of these stressors are commonly observed in conjunction with traumatic hemorrhage in civilian and/or military settings .
because of the complexity of this problem , it is possible that clinical assessment of the severity of hemorrhage during the acute phase may be better realized using artificial intelligence technologies that reflect the integration of the sympathetic nervous response to hemorrhage with changes in circulatory pressure and volume ( convertino et al . , 2011 ) .
the opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the department of the army or the department of defense .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | hemorrhage remains a major cause of mortality following traumatic injury in both military and civilian settings .
lower body negative pressure ( lbnp ) has been used as an experimental model to study the compensatory phase of hemorrhage in conscious humans , as it elicits central hypovolemia like that induced by hemorrhage .
one physiological compensatory mechanism that changes during the course of central hypovolemia induced by both lbnp and hemorrhage is a baroreflex - mediated increase in muscle sympathetic nerve activity ( msna ) , as assessed with microneurography .
the purpose of this review is to describe recent results obtained using microneurography in our laboratory as well as those of others that have revealed new insights into mechanisms underlying compensatory increases in msna during progressive reductions in central blood volume and how msna is altered at the point of hemodynamic decompensation .
we will also review recent work that has compared direct msna recordings with non - invasive surrogates of msna to determine the appropriateness of using such surrogates in assessing the clinical status of hemorrhaging patients . | MSNA during the Compensatory Phase of Central Hypovolemia
MSNA at Hemodynamic Decompensation
Non-Invasive Surrogate for MSNA for Assessing the Severity of Hemorrhage?
Conclusion
Disclaimer
Conflict of Interest Statement |
lesions in the thalamus and those extending into the midbrain can cause various movement disorders such as dystonia , asterixis , and ballism - chorea .
convulsive movements have been reported in bilateral paramedian thalamic and midbrain infarction [ 2 , 3 , 4 , 5 , 6 , 7 ] .
we here report on the case of a patient with clonic movements of both arms that were observed at the onset of infarction and offer a review of the literature .
a 71-year - old man , with a ten - year history of chronic hemodialysis due to diabetic nephropathy , was brought to the emergency room .
spontaneous symmetrical clonic movements of both arms lasting for 10 to 20 s were observed at 2-to 3-min intervals .
the results of laboratory tests on the following were normal : blood cell count , electrolyte level , glucose level , arterial blood gas , and cerebrospinal fluid .
brain magnetic resonance imaging ( mri ) showed acute bilateral paramedian thalamic and midbrain infarction ( fig .
we report on the case of a patient with clonic movements of both arms at the onset of bilateral paramedian thalamic and midbrain infarction . in the literature
[ 2 , 3 , 4 , 5 , 6 , 7 ] , convulsive movements were observed mostly at the onset of infarction .
clonic movements appear frequently in the limbs , particularly in both arms ( table 1 ) .
lesions in the thalamus and those extending into the midbrain can cause various movement disorders such as dystonia , asterixis , and ballism - chorea . the thalamus relays inputs from peripheral sensory pathways , the basal ganglia , and the cerebellum to the cerebral cortex . lesions in specific thalamic nuclei and dysfunction of the networks may contribute to these abnormal movements .
however , dystonia , asterixis , and ballism - chorea are unlikely because of the clonic nature of the movements in our patient .
although the cerebral cortex is considered important for the development of seizures in general , previous experimental studies have suggested that electrical or chemical stimulation of the brainstem tegmentum , particularly the mesencephalic reticular formation ( rf ) , induces convulsive seizures [ 9 , 10 , 11 , 12 ] .
autopsy and mri show the involvement of the midbrain tegmentum in patients with convulsive movements [ 2 , 3 , 4 , 5 , 6 , 7 ] .
however , to the best of our knowledge , such movements have never been observed in bilateral paramedian thalamic infarction without midbrain lesions .
these findings suggest that the mesencephalic rf has a crucial role in the generation of convulsive seizures in bilateral paramedian thalamic and midbrain infarction .
convulsive seizures induced by electrical stimulation of the mesencephalic rf are not eliminated by precollicular transection [ 10 , 11 ] , but pontine tegmentum lesions attenuate the convulsions of brainstem origin .
these findings suggest that the generation of the motor component in convulsive seizures is induced by the activation of the descending rf .
furthermore , seizures can take a variety of motor patterns depending on the intensity of stimulation .
a high intensity of stimulation causes tonic movements , whereas a low intensity of stimulation causes clonic movements of the forelimbs , and the subsequent increases in stimulation intensity produce a spread of clonic movements down to include the hind limbs .
it is hypothesized that the rostrocaudal spread observed in convulsions reflects the rostrocaudal spread of discharge down the polysynaptic descending pathways within the intermediate zone of the spinal gray matter .
this can explain why clonic movements were sometimes observed only in both arms , but have never been observed in the legs without the involvement of the arms in bilateral paramedian thalamic and midbrain infarction
. in summary , convulsive movements , particulary clonic movements of both arms , can develop in bilateral paramedian thalamic and midbrain infarction as an initial symptom .
physicians should recognize this condition , because not only seizure control but also early management of ischemic stroke is required . | although some previous reports have described convulsive movements in bilateral paramedian thalamic and midbrain infarction , little is known about their nature . a 71-year - old man presented with impaired consciousness and clonic movements of both arms .
each series of movements lasted 10 to 20 s and occurred at 2-to 3-min intervals , which disappeared after intravenous administration of diazepam and phenytoin .
magnetic resonance imaging showed acute bilateral paramedian thalamic and midbrain infarction . a review of the literature revealed that convulsive movements were observed mostly at the onset of infarction .
clonic movements appeared frequently in the limbs , particularly in both arms .
clinical observations and results of animal experiments suggest that these seizures might originate from the mesencephalic reticular formation .
physicians should recognize this condition , because not only seizure control but also early management of ischemic stroke is required . | Introduction
Case Report
Discussion |
subclinical hypothyroidism ( sch ) defined as normal free thyroxine ( t4 ) and elevated thyroid stimulating hormone ( tsh ) is primarily a biochemical diagnosis with or without clinical symptoms .
sch is associated with several long term effects including dyslipidemia , hypertension , subfertility and may be an independent risk factor for cardiovascular morbidity . circulating tsh shows a normal circadian rhythm with a peak between 11 pm-5 am and a nadir between 5 pm-8 pm .
secretory pulses occur every 2 - 3 hours and are interspersed with periods of tonic non - pulsatile tsh secretion . although the tsh secretion is pulsatile , the low amplitude of the pulses and the long half - life of tsh result in only modest circulatory variations .
it is generally observed that tsh in early morning fasting states were higher than tsh levels measured later in the same day . in routine clinical practice not much importance is being given to the timing of the sample or the fasting / non - fasting status of the patient .
however , an entity like sch which heavily relies on tsh values may be under or overdiagnosed based on a single value .
further , in the recent past , narrower and stricter cut - offs for tsh has been advocated for defining euthyroidism in special situations like pregnancy . hence uniformity in testing under standard conditions is necessary . with this background
, we proposed this study to evaluate whether tsh measured in fasting state or postprandially would make a difference .
the study was conducted in the government stanley medical college hospital , chennai , tamilnadu where the thyroid functions are usually done in fasting state only .
fifty - seven adult ambulatory patients were selected from our laboratory database and were divided into group a ( normal freet4 and tsh ) , group b ( sch with increased tsh and normal free t4 ) and group c ( overt hypothyroidism with low free t4 and high tsh ) . the lab reference ranges ( given below ) were used to define low and high values of freet4 and tsh .
the study was approved by the institutional review board , government stanley medical college , chennai and informed consent was obtained prior to phlebotomy from the patients .
phlebotomy was performed after an 8 - 12 hour overnight fast between 7:30 - 8:30 am for free t4 and tsh measurements and the patients returned 2 hours after breakfast for their samples to be rechecked between 10:30 - 11:00 am on the same day .
samples were analyzed by the electrochemiluminesence immunoassay intended for use on eleccsys and cobas immunoassay analyzers .
machine was calibrated and the serum was collected and processed according to manufacturer 's instructions .
the methodology had an analytical sensitivity of 0.005 iu / ml and a functional sensitivity of 0.014 iu / ml ( coeffecient of variation 1.4% )
. suggested normal values for tsh were 0.27 - 4.2 iu / ml and these values correspond to the 2.5 and 97.5% of results obtained from a total of 516 healthy test subjects examined .
suggested normal values for free t4 was 0.80 - 1.8 ng / ml and the values correspond to the 2.5 and 97.5% of results from a total of 801 healthy test subjects studied .
differences in free t4 and tsh levels between fasting and non - fasting state were analyzed by paired student - t test .
differences in free t4 and tsh levels between fasting and non - fasting state were analyzed by paired student - t test .
tsh values were lowered after food when compared to fasting in a statistically significant manner in all the three groups as shown in [ table 1 ] .
fasting and 2 hour post - prandial values ( meanstandard deviation ) of free t4 and tsh among the three groups
in our study we addressed a clinically relevant question : whether thyroid function tests ( free t4 and tsh ) should be estimated in fasting state or not ?
we observed that tsh values get lowered if estimated postprandially irrespective of the fasting levels .
but due to its low pulse amplitude and long half - life the circulating variations is only modest .
previous studies by scobbo et al . , kamat et al . and bandhopadhyay et al .
tsh secretion is heavily dependent on two factors namely thyrotropin releasing hormone ( trh ) and somatostatin ; the former stimulating and the latter inhibiting tsh .
a possible explanation for the acute postprandial decline of serum tsh is food induced elevation of circulating somatostatin and consequent suppression of tsh .
microparticle enzyme immuno assay ( second generation ) , radioimmunoassay , immunofluorescence assay but observed results similar to our study . in a recent study by sarkar comparing two third generation tsh assay methods , [ chemiluminescence ( architect ) vs electrochemiluminescence ( cobas ) ] ,
timing of sampling was considered as one of the factors which might have influenced the decline in tsh in the previous studies .
hence , whether the tsh suppression in our study was due to food related alteration in blood chemistry or timing of sample or both could not be clarified .
clinical guidelines for thyroid function testing or laboratory guidelines for free t4 and tsh estimation do not emphasize the time of phlebotomy or the fasting / non - fasting status of the patient .
clinically , in our study , the lowering of tsh postprandially led to reclassification of 15 out of 20 subjects ( 75% ) as euthyroid who would have otherwise been labelled as sch based on fasting tsh alone .
this may have a significant impact not only on the diagnosis but also monitoring of hypothyroidism , especially in situations where even marginal variations in tsh may be important like in pregnancy or sub - fertility . with recent guidelines for management of hypothyroidism during pregnancy stressing a target tsh of 2.5 miu / l or less
, the findings of our study may have more 0 . further , lack of uniformity in the time of sampling for tsh may lead to unnecessary repetition of tests especially in a resource limited setting . with the above observations
, we propose that a fasting tsh sample may be preferred to random or postprandial estimations as normal fasting values would obviate the need for retesting .
factors other than food or timing of sample have not been addressed in our study .
this may have clinical implications in the diagnosis and management of hypothyroidism , especially sch . | background : thyroid stimulating hormone ( tsh ) levels vary with the time of the day and probably in relation to food . in this study , we addressed the question of whether a fasting or non - fasting sample would make a clinically significant difference in the interpretation of thyroid function tests.materials and methods : fifty seven adult ambulatory patients were selected from our laboratory database and were divided into group a [ normal free thyroxine ( t4 ) and tsh ] , group b ( subclinical hypothyroid with increased tsh and normal free t4 ) and group c ( overt hypothyroid with low free t4 and high tsh ) .
thyroid functions ( free t4 and tsh ) were done in fasting state and 2 hours postprandially.results:tsh was suppressed in all subjects after food irrespective of the fasting levels .
free t4 values did not change significantly .
this resulted in reclassification of 15 out of 20 ( 75% ) subjects as subclinical hypothyroidism ( sch ) based on fasting values whose tsh values were otherwise within range in the postprandial sample .
this may have an impact on the diagnosis and management of hypothyroidism especially where even marginal changes in tsh may be clinically relevant as in sch and in pregnancy.conclusion:tsh levels showed a statistically significant decline postprandially in comparison to fasting values .
this may have clinical implications in the diagnosis and management of hypothyroidism , especially sch . | I
M
Statistical methods
R
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C |
traditionally , implant treatment is based on a 2-stage protocol with a healing period of 3 - 6 months during which the implants are submerged to achieve osseointegration.1 recently , this clinical suggestion has been challenged .
numerous practitioners now advocate immediate or early loading of implants.2 the advantages of immediately loaded implants are clear : they require shorter treatment periods and allow immediate recovery of function and esthetics.3 high success rate of immediately loaded implants in humans was first documented in the middle 1980s .
the 88% cumulative success rate on 1739 immediately loading implants was suggested.4 the clinical performance and prognosis of the single - stage surgical protocol are known to be comparable to the traditional 2-stage method.5 there are some articles reporting a cumulative survival rate of 95% , which investigated immediately loaded single implants.6,7 results from these studies suggest that immediate loading could achieve equal success rates as those found in delayed loading .
it is also known as a common claim that treatment with immediate loading improved patient satisfaction and was cost effective although no scientific evidence was presented to support.8 however , advantages of early or immediate loading as mentioned may be offset by an increased risk of implant failure .
it was reported that immediately loaded implants were approximately 3 times more likely to fail within 1 year of placement.3 furthermore , there have been few clinical studies investigating the success or failure rates of immediate loading based on korean implant systems .
the aim of this preliminary prospective study was to evaluate the outcome of immediate functional loading in partial edentulism , using sinusquick eb ( neobiotech co. , seoul , korea ) implant system .
four subjects ( 2 smokers and 2 non - smokers ) recruited from a population of patients under routine care at seoul national bundang hospital were enrolled in the study .
the patients were selected according to the following inclusion criteria : they were in normal general health with sufficient bone to allow the placement of implants at least 7 mm length .
the mean age of the subjects was 50.3 years ( range from 39 to 65 years ) , with a gender distribution of 100% men .
informed written consent was obtained from all subjects following approved institutional review board guidelines for clinical research .
surgery was performed under local anesthesia ( 1 : 100,000 epinephrine ) or conscious intravenous sedation with 1% propofol solution and midazolam .
implants were inserted according to the procedures recommended by manufacturers . during the period from april to october 2009 ( range from 2 to 6 months )
immediate loading was applied to the implants showing implant stability quotient ( isq ) and insertion torque values that were more than 60 and 35 ncm , respectively .
immediate loading is defined as provisional or final implant - supported restoration delivered within 2 weeks.9 therefore , provisional implant - supported fixed partial dentures were delivered within 2 weeks ( fig .
periapical radiographs were taken using commercially available film holders and a paralleling imaging technique during the investigating period . in each patient ,
peri - implant marginal bone level was evaluated by impax ( agfa co. , mortsel , belgium ) system of periapical radiographs .
measurements were recorded at the time of surgery , immediate loading , after 3-months of continued loading , and at the last follow - up ( fig .
marginal bone height was determined on these images by measuring the distance from a reference point , defined as the platform of the implant ( fig .
2 ) , to the most coronal point of bone - to - implant contact on both the mesial and distal sides of the implant . a single value for marginal bone height was then calculated by obtaining the mean of these two measurements for each implant .
the definition of implant success was based on the following clinical and radiologic criteria : 1 ) absence of clinically detectable implant mobility , 2 ) absence of pain or any subjective sensation , 3 ) absence of recurrent peri - implant infection , and 4 ) absence of continuous radiolucency around the implant.10
marked variability was noted in the implant sizes selected for placement , although implants 11.5 mm length and 5.0 mm diameter were most commonly used .
the mean follow - up period was 4.8 months ( range , 2 to 6 months ) .
mean marginal bone loss from implant surgery to immediate loading , 3-months follow - up and last follow - up was found to be 0.03 mm , 0.16 mm and 0.29 mm respectively ( table ii ) .
no implant failed up to 6 months after insertion , resulting in a 100% survival rate .
all the inserted implants showed successful integration and stable peri - implant condition up to six months .
primary stability was reported to be the most important determining factor on immediate implant loading.6 micromovements of more than 100 m were sufficient to jeopardize healing with direct bone - to - implant contact.6 szmukler - moncler et al . indicated that micromotions at the bone - implant interface beyond 150 m resulted in fibrous encapsulation instead of osseointegration.11
if the primary implant stability could not be achieved or was questionable , it was strongly recommended to follow a conventional treatment protocol.6 most agreed that an insertion torque of at least 32 nm and a resonance frequency analysis of at least 60 isq was required to achieve a high level of stability.12 in this study , mean isq of 15 early loaded implants was 64.9 4.9 .
generally , clinicians agreed that the quality of bone was significant for success in immediate loading .
the initial stability of the implant reduces in the first 3 - 6 weeks after placement due to remodeling and an increased ratio of woven to lamellar bone.12 barewal et al .
indicated that implants placed in areas of high bone quality are relatively stable over the early healing periods.13 however , we reported that both maxillary and mandibular arches showed no failure of implants although the sample size was too small to analyze the data .
horiuchi et al . also reported about no difference in the success rate between arches in immediate loading.14 further studies are required about the relationship between bone quality and the success rate of immediate loading .
it has been established that there are no absolute contraindications to implant placement although a number of conditions exist , which are associated with an increased risk of failure.12 tobacco was reported to be only a risk factor for the implant failure.3 however , the results of this investigation showed there was no implant failure in the participating patients who were smokers .
further controlled clinical studies are needed to evaluate the long - term success of early loaded implants .
within the limitation of this clinical study the preliminary results indicate that immediate loading of the implants in partial edentulism , based on sinusquick eb implant system , may be successful for short period up to six months . | statement of problemmany dental clinicians are concerned about immediate loading of inserted implants .
however , there have been few clinical studies surveying the success rates of immediate loading , based on korean implant systems.purposethe aim of this study was to evaluate the outcome of immediate functional loading of the implant ( sinusquick eb , neobiotech co. , seoul , korea ) in partially edentulous maxilla or mandible.material and methodstotal 15 implants were placed . within 2 weeks after implant insertion
, provisional implant - supported fixed partial dentures were delivered to the patients .
quantitatively , marginal bone loss was measured at the time of immediate loading , after 3-months of continued loading and at the last follow - up .
the mean follow - up period was 4.8 months.resultsmean marginal bone loss from implant surgery to early loading , 3-months follow - up and last follow - up was 0.03 0.07 mm , 0.16 0.17 mm and 0.29 0.19 mm .
no implant failed up to 6 months after insertion , resulting in a 100% survival rate.conclusionimmediate loading exhibited high success rate in partial edentulism for up to 6 months .
well - controlled long term clinical studies with large sample size are necessary to confirm this finding . | INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
patients with absence epilepsy or other idiopathic generalized epilepsy ( ige ) syndromes manifest 2.5 hz to 3.5 hz ( 3 hz on average ) bisynchronous typical spike - and - wave activity ( tswa ) in the interictal electroencephalogram ( eeg ) and during typical absence seizures.1
nonconvulsive status epilepticus ( ncse ) consists of sustained rhythmic / periodic epileptiforma activity in the eeg , along with alterations in mental status and/or behavior , in the absence of convulsive movements .
there are other definitions of ncse , but no definition ( including ours ) is universally accepted .
a wide range of cognitive - behavioral changes occurs in ncse , including mild inattention , confusion , stupor , and coma .
an even wider range of eeg patterns is seen in ncse , but many of these patterns are not conclusively epileptiform.2 ncse is traditionally divided into complex partial status epilepticus ( cpse)3 and absence status epilepticus ( ase).4 diagnosing ncse can be difficult in the presence of coma.5,6 first , the eeg in coma may show a typical cpse / ase pattern even though the clinical course and response to treatment are not compatible with cpse / ase .
second , the eeg in coma may show epileptiform abnormalities that defy classification into cpse or ase .
third , the eeg in coma may show abnormalities that can not be easily labeled as epileptiform .
ige - related ncse usually takes the form of absence status epilepticus.4 some adults with no history of seizure / epilepsy develop ase
de novo in the context of an acute brain disorder.4 the acute brain disorder in de novo ase is often an encephalopathy from drug overdose / withdrawal , fluid / electrolyte imbalance , or systemic infections.7 however , de novo ase is not routinely reported in hypoxic - ischemic encephalopathyb ; we are aware of only one previous case of de novo ase with tswa in hypoxic - ischemic brain injury.8 we present an unusual case of hypoxic - ischemic brain injury in which the patient s eeg showed benzodiazepine - resistant tswa even though the patient had no past or family history of seizure or epilepsy . after presenting the case ,
we examine the pathophysiology of post - hypoxic tswac and its implications for classifying and managing ncse .
the patient , a 65-year - old man with congestive heart failure ( left ventricular ejection fraction of 20% ) suffered cardiorespiratory arrest resulting in hypoxic - ischemic brain injury .
the history is also significant for hypertension , kidney disease , hepatitis c , and substance abuse with cocaine and marijuana .
home medications include digoxin , furosemide , spironolactone , carvedilol , lisinopril , aspirin , potassium chloride , and folic acid .
the patient was found unconscious , apneic , asystolic , and pulseless on a street in his neighborhood .
emergency responders arrived within 15 minutes and performed cardiopulmonary resuscitation ( cpr ) , including defibrillation , intubation , and artificial ventilation .
the patient arrived in the emergency room with a glascow coma scale of 3 ; dopamine was started .
on admission to the intensive care unit ( icu ) , the patient was in coma with intact brainstem function and stable vital signs ( rr = 14 , hr = 73 , bp = 100/70 , t = 36 c ) .
brain ct scan showed mild diffuse atrophy , chronic microvascular changes , and no acute lesions .
blood tests showed normal white cell count , low hemoglobin ( 11.5 g / dl ) , elevated serum glucose ( 255 mg / dl ) , normal blood urea , nitrogen and creatinine , and normal serum sodium , potassium , and calcium .
ventricular fibrillation occurred 21 hours after the first hypoxic - ischemic event ; cpr was performed for 15 minutes .
post - resuscitation , the patient manifested eyelid myoclonus occurring spontaneously every 3 to 12 seconds without any clear relationship to external stimulation ; this is most likely a form of acute posthypoxic myoclonus .
intravenous lorazepam was given : 7-mg over 7 hours ( first 4-mg , then 2-mg , then 1-mg ) resulting in cessation of eyelid myoclonus .
more lorazepam ( 10-mg ) was infused over the next 7 hours ( 0.01 mg / kg / hour over 4 hours , then 0.02 mg / kg / hour over 3 hours ) .
serum phenytoin was 18.6 g / ml 10 hours after the loading dose and 18.0 g / ml 38 hours later even though fosphenytoin was discontinued before the patient received any maintenance dose .
eeg monitoringd was initiated 23 hours after administering fosphenytoin and discontinuing lorazepam ; at that time the patient was in deep coma with no involuntary movements .
the eeg showed sustained typical spike - and - wave activity ( tswa ) confirming ncse and indicating typical absence status epilepticus ( fig .
intravenous midazolam was started : 0.2 mg / kg bolus and 0.05 mg / kg / hour infusion .
tswa persisted so another 0.2 mg / kg bolus was given 2.5 hours after the first bolus .
the eeg continued to show tswa , albeit with somewhat lower discharge frequency ( fig .
2 ) . the infusion rate was increased and at 0.5 mg / kg / hour the eeg showed intermittent suppression of spike - and - wave discharges ( fig .
3 ) . the midazolam infusion rate was further increased to achieve eeg burst - suppression . at a rate of 1.2 mg / kg / hour , tswa disappeared and
stable burst - suppression ( 612 bursts / minute ) was achieved with an infusion rate of 1.4 to 1.6 mg / kg / hour .
the patient s family requested withdrawal of life support 72 hours after admission and the patient died the following day .
eeg showed typical spike - and - wave activity ( tswa ) even though he had no history of seizure or epilepsy .
non - convulsive status epilepticus ( ncse ) was diagnosed and intravenous lorazepam and midazolam was administered .
the tswa proved to be relatively resistant to benzodiazepines , raising some important issues regarding the pathophysiology of post - hypoxic tswa and providing some insight into how ncse should be classified and managed .
typical spike - and - wave activity ( tswa ) is the eeg hallmark of absence seizure .
it is also a routine interictal eeg finding in absence epilepsy and other ige syndromes.1 for twsa to occur , the thalamocortical - corticothalamic network must be functionally intact and in a state where intrinsically bursting cortical pyramidal and thalamic neurons are mildly to moderately hyperpolarized and susceptible to producing high - frequency bursts of action potentials.9 meeren and colleagues published a review of the theories on the pathophysiology of tswa.9 this can be summarized as follows : ( 1 ) the centrencephalic theory posits a diffusely projecting pacemaker in the midline thalamus as the origin of tswa . ( 2 )
the thalamic clock theory is a refinement of the centrencephalic theory ; it localizes the abnormal pacemaker to the reticular thalamic nucleus .
( 3 ) the corticoreticular theory claims that tswa is generated by the abnormal interaction of the cortex and the reticular system of the thalamus and brainstem . ( 4 ) the cortical theory considers cortical hyperexcitability as the primary defect ; a hyperexcitable cortex transforms the normal rhythms of the thalamus into tswa .
( 5 ) the cortical focus theory is a synthesis of the cortical and corticoreticular theories ; it asserts that a cortical focus ( the perioral region of the somatosensory cortex is a prime suspect ) initiates the epileptic activity , spreads to other cortical areas , and entrains the thalamus producing a resonant circuitry for expressing tswa .
to these five theories , we add blumenfeld s comprehensive theory which argues that tswa can be provoked by different insults acting on different excitatory / inhibitory circuits.10 it is likely that absence status epilepticus ( ase ) shares the basic mechanisms of absence seizure . whether ictogenesis results in a self - limited absence seizure or in ase depends on the brain s seizure terminating mechanisms ;
such mechanisms are likely impaired in ase.11 patients with no history of ige can develop ase
de novo in the context of an acute brain disorder.4 pro and colleagues reported a patient with late - onset de novo ase whose eeg consistently showed interictal tswa over a 14-year period.12 their case indicates that , in some individuals with latent ige , exposure of the brain to toxic or metabolic factors can trigger a seizure later in life .
the fact that post - hypoxic tswa can look exactly like ige - related tswa implies that different insults acting on different brain circuits can turn on the basic mechanisms that lead to tswa ( see blumenfeld s theory above).10 this case also implies that extensive hypoxic - ischemic brain injury can spare the thalamocortical - corticothalamic network responsible for generating tswa / absence seizure.9 as stated above , a genetic predisposition to develop tswa / ase may be the reason our patient manifested tswa.12 it is also possible that all individuals have an inherent ability to produce tswa and that , in our patient , the pattern of cerebral hypoxic - ischemic injury has released
such ability.10 the patient s eye - lid movements occurred repeatedly at irregular intervals within a 7-hour period and stopped after a total of 7-mg of lorazepam was given .
these movements are consistent with post - hypoxic myoclonus and not with the more periodic eye - blink automatism seen in absence seizure .
it should also be emphasized that eyelid movements were not present when tswa was detected and that tswa is not a known eeg correlate of post - hypoxic myoclonic status epilepticus ; hence myoclonic status epilepticus would not be an appropriate diagnosis for our patient .
the patient has a history of substance abuse and he tested positive for cocaine and marijuana .
however , most drug - related de novo ase cases in the literature were linked to withdrawal of benzodiazepine ( bdz ) or to intoxication with alcohol or psychotropic drugs.4,7 alcohol was not detected in the patient s blood and we are not aware of any reports in which cocaine / marijuana was linked to de novo ase . still , this explanation can not be ruled out .
the patient received a 1440-mg loading dose of fosphenytoin ( a phenytoin prodrug ) 23 hours prior to the onset of eeg monitoring . in the setting of ige , phenytoin ( and other antiepileptic drugs )
can aggravate seizures13 and can even result in refractory ase.14 however , we are not aware of any report of phenytoin / fosphenytoin causing ase in patients with no ige .
the patient also received 17-mg of lorazepam ( a bdz and gaba - a receptor agonist ) 23 to 37 hours before the onset of eeg recording .
however , the absence of a history of chronic bdz use makes it unlikely that the patient went through a bdz withdrawal state .
enhanced gaba - a inhibition in the thalamus can trigger absence seizures in genetic and pharmacological models of absence epilepsy.15 however , we are not aware of any report of lorazepam causing ase in patients with no ige
. what is the significance of benzodiazepine - resistance in tswa / ase ? as a rule , ige - related ase responds rapidly and completely to bdz therapy.4,11 this is in line with the ability of gaba - a receptors to mediate the inhibition of the thalamic
pacemaker of tswa.15 a few cases of refractory ase were described in the literature but all of these patients had ige and were receiving phenytoin or carbamazepine.13 the rarity of post - hypoxic tswa makes it much more difficult to find any report of bdz - resistant post - hypoxic tswa .
we initially hypothesized that resistance indicates a difference in the mechanisms of post - hypoxic tswa and ige - related tswa.16 this is not necessarily the case and the basic mechanism of tswa genesis could still be the same , only that cerebral hypoxia - ischemia resulted in impairment of neural circuits where bdz act to suppress tswa .
for example , impairment of gaba inhibitory circuits in the cerebral cortex can increase the chance of cortical ictogenesis , thalamic entrainment , and tswa generation ( see cortical focus theory).9 in classifying seizures , the international league against epilepsy ( ilae ) made no formal reference to non - convulsive status epilepticus ( ncse).1719 the 1981 ilae seizure classification contains three diagnostic entities that qualify as ncse aura continua , complex partial status epilepticus , and absence status epilepticus ( ase).17 subtle status epilepticus was added in the 2006 report of the ilae classification core group bringing the number of ncse seizures to four.18 in our patient , the finding of tswa indicated ase ( more specifically typical ase ) but treatment response ( bdz - resistance ) and clinical outcome ( the patient died ) are diametrically opposite to that of ase ( especially typical ase ) .
such an electroclinical paradox is not uncommon in critically ill patients with ncse .
unfortunately , the 1981 and 2006 seizure classifications ignore this issue by omitting the many variations of ncse , especially ncse in coma.5,6 the 2010 ilae seizure classification also fails to address this issue.19 what s more , the ilae has completely excluded status epilepticus and focal seizures from the 2010 classification.20 the fact that ncse is more common than previously thought underscores the urgency to create a better classification system for status epilepticus and ncse .
recent studies have shown that ncse constitutes 25%50% of all cases of status epilepticus and the incidence of ncse in the critically ill can be as high as 50%.6 excluding some ncse subtypes from the seizure classification system on philosophical grounds is disadvantageous to the patient , the physician , and the researcher.19 while a seizure classification system can not be perfect at the outset , it should be heuristic and include all phenomena considered as ncse , including the ncse variations in comatose patients .
only a heuristic seizure classification system will result in better understanding and rational treatment of ncse . according to bauer and trinka , the confusion vis - - vis ncse in coma
can be overcome by having a separate ncse categorycomatose ncse or coma - ncse.5 the authors classified cpse and ase as ncse proper .
ncse proper has a good prognosis and , in most cases , the treatment of choice is turning off the seizure without aggressively anesthetizing the patient .
coma - ncse is more ominous and its prognosis depends almost entirely on the underlying brain disorder.5 while there is no firm evidence that suppressing the seizure improves prognosis in coma - ncse , anesthetizing the patient with midazolam or propofol may not be unreasonable if the patient is already on mechanical ventilation and is being monitored in the icu .
maganti and colleagues proposed an etiology - based classification for ncse , including ncse in coma , ncse in acute cerebral lesions , ncse in metabolic disorders , and ncse in those with pre - existing epilepsy.6 this more detailed approach may lend itself to better treatment recommendation , but most importantly better evaluation and testing of treatment recommendations to determine their value based on outcome .
we agree with bauer and trinka that it is important to differentiate coma - ncse from ncse proper5 and with maganti and colleagues that it is critical to account for etiology in classifying ncse.6 by combining the bauer / trinka and maganti proposals and adding some minor refinements , we were able to create a practical decision tree for ncse ( fig .
4 ) . as shown in this decision tree , as soon as ncse is diagnosed ( based on electroclinical data ) , the clinician should separate
ncse with significant irreversible brain injury ( sibi ) and ncse with no evidence of sibi .
most coma ncse with sibi is due to hypoxic - ischemic , traumatic , ischemic , infectious , or inflammatory insults to the brain .
most coma ncse with no sibi is due to epileptic , toxic , or metabolic factors , often with the confounding effects of anesthetics or sedative drugs that are regularly employed in the icu .
most non - coma ncse is due to purely epileptic , toxic , or metabolic factors , the majority of which are reversible .
occasionally , sibi of lesser severity may give rise to non - coma ncse .
such a heuristic approach can facilitate our understanding of ncse , especially ncse in coma . while the incidence depends on how one defines ncse , even more conservative estimates of the incidence of ncse ( eg , 25% )
warrant a detailed analysis of ncse cases with a goal toward increased understanding and better classification of ncse .
further work is needed to better define ncse , to determine which eeg patterns indicate ncse , and to establish treatment paradigms for the different types of ncse .
a more collaborative approach across hospitals / universities is warranted given the sample size that would be needed to determine treatment efficacy in so many ncse categories .
typical spike - and - wave activity ( tswa ) is the eeg hallmark of absence seizure .
it is also a routine interictal eeg finding in absence epilepsy and other ige syndromes.1 for twsa to occur , the thalamocortical - corticothalamic network must be functionally intact and in a state where intrinsically bursting cortical pyramidal and thalamic neurons are mildly to moderately hyperpolarized and susceptible to producing high - frequency bursts of action potentials.9 meeren and colleagues published a review of the theories on the pathophysiology of tswa.9 this can be summarized as follows : ( 1 ) the centrencephalic theory posits a diffusely projecting pacemaker in the midline thalamus as the origin of tswa . ( 2 )
the thalamic clock theory is a refinement of the centrencephalic theory ; it localizes the abnormal pacemaker to the reticular thalamic nucleus .
( 3 ) the corticoreticular theory claims that tswa is generated by the abnormal interaction of the cortex and the reticular system of the thalamus and brainstem . ( 4 ) the cortical theory considers cortical hyperexcitability as the primary defect ; a hyperexcitable cortex transforms the normal rhythms of the thalamus into tswa .
( 5 ) the cortical focus theory is a synthesis of the cortical and corticoreticular theories ; it asserts that a cortical focus ( the perioral region of the somatosensory cortex is a prime suspect ) initiates the epileptic activity , spreads to other cortical areas , and entrains the thalamus producing a resonant circuitry for expressing tswa .
to these five theories , we add blumenfeld s comprehensive theory which argues that tswa can be provoked by different insults acting on different excitatory / inhibitory circuits.10 it is likely that absence status epilepticus ( ase ) shares the basic mechanisms of absence seizure . whether ictogenesis results in a self - limited absence seizure or in ase depends on the brain s seizure terminating mechanisms ;
such mechanisms are likely impaired in ase.11 patients with no history of ige can develop ase
de novo in the context of an acute brain disorder.4 pro and colleagues reported a patient with late - onset de novo ase whose eeg consistently showed interictal tswa over a 14-year period.12 their case indicates that , in some individuals with latent ige , exposure of the brain to toxic or metabolic factors can trigger a seizure later in life .
the fact that post - hypoxic tswa can look exactly like ige - related tswa implies that different insults acting on different brain circuits can turn on the basic mechanisms that lead to tswa ( see blumenfeld s theory above).10 this case also implies that extensive hypoxic - ischemic brain injury can spare the thalamocortical - corticothalamic network responsible for generating tswa / absence seizure.9 as stated above , a genetic predisposition to develop tswa / ase may be the reason our patient manifested tswa.12 it is also possible that all individuals have an inherent ability to produce tswa and that , in our patient , the pattern of cerebral hypoxic - ischemic injury has released
such ability.10 the patient s eye - lid movements occurred repeatedly at irregular intervals within a 7-hour period and stopped after a total of 7-mg of lorazepam was given .
these movements are consistent with post - hypoxic myoclonus and not with the more periodic eye - blink automatism seen in absence seizure .
it should also be emphasized that eyelid movements were not present when tswa was detected and that tswa is not a known eeg correlate of post - hypoxic myoclonic status epilepticus ; hence myoclonic status epilepticus would not be an appropriate diagnosis for our patient .
the patient has a history of substance abuse and he tested positive for cocaine and marijuana .
however , most drug - related de novo ase cases in the literature were linked to withdrawal of benzodiazepine ( bdz ) or to intoxication with alcohol or psychotropic drugs.4,7 alcohol was not detected in the patient s blood and we are not aware of any reports in which cocaine / marijuana was linked to de novo ase . still , this explanation can not be ruled out .
the patient received a 1440-mg loading dose of fosphenytoin ( a phenytoin prodrug ) 23 hours prior to the onset of eeg monitoring . in the setting of ige , phenytoin ( and other antiepileptic drugs ) can aggravate seizures13 and can even result in refractory ase.14 however , we are not aware of any report of phenytoin / fosphenytoin causing ase in patients with no ige .
the patient also received 17-mg of lorazepam ( a bdz and gaba - a receptor agonist ) 23 to 37 hours before the onset of eeg recording .
however , the absence of a history of chronic bdz use makes it unlikely that the patient went through a bdz withdrawal state .
enhanced gaba - a inhibition in the thalamus can trigger absence seizures in genetic and pharmacological models of absence epilepsy.15 however , we are not aware of any report of lorazepam causing ase in patients with no ige
. what is the significance of benzodiazepine - resistance in tswa / ase ? as a rule , ige - related ase responds rapidly and completely to bdz therapy.4,11 this is in line with the ability of gaba - a receptors to mediate the inhibition of the thalamic
pacemaker of tswa.15 a few cases of refractory ase were described in the literature but all of these patients had ige and were receiving phenytoin or carbamazepine.13 the rarity of post - hypoxic tswa makes it much more difficult to find any report of bdz - resistant post - hypoxic tswa .
we initially hypothesized that resistance indicates a difference in the mechanisms of post - hypoxic tswa and ige - related tswa.16 this is not necessarily the case and the basic mechanism of tswa genesis could still be the same , only that cerebral hypoxia - ischemia resulted in impairment of neural circuits where bdz act to suppress tswa .
for example , impairment of gaba inhibitory circuits in the cerebral cortex can increase the chance of cortical ictogenesis , thalamic entrainment , and tswa generation ( see cortical focus theory).9
in classifying seizures , the international league against epilepsy ( ilae ) made no formal reference to non - convulsive status epilepticus ( ncse).1719 the 1981 ilae seizure classification contains three diagnostic entities that qualify as ncse aura continua , complex partial status epilepticus , and absence status epilepticus ( ase).17 subtle status epilepticus was added in the 2006 report of the ilae classification core group bringing the number of ncse seizures to four.18 in our patient , the finding of tswa indicated ase ( more specifically typical ase ) but treatment response ( bdz - resistance ) and clinical outcome ( the patient died ) are diametrically opposite to that of ase ( especially typical ase ) .
such an electroclinical paradox is not uncommon in critically ill patients with ncse .
unfortunately , the 1981 and 2006 seizure classifications ignore this issue by omitting the many variations of ncse , especially ncse in coma.5,6 the 2010 ilae seizure classification also fails to address this issue.19 what s more , the ilae has completely excluded status epilepticus and focal seizures from the 2010 classification.20 the fact that ncse is more common than previously thought underscores the urgency to create a better classification system for status epilepticus and ncse .
recent studies have shown that ncse constitutes 25%50% of all cases of status epilepticus and the incidence of ncse in the critically ill can be as high as 50%.6 excluding some ncse subtypes from the seizure classification system on philosophical grounds is disadvantageous to the patient , the physician , and the researcher.19 while a seizure classification system can not be perfect at the outset , it should be heuristic and include all phenomena considered as ncse , including the ncse variations in comatose patients .
only a heuristic seizure classification system will result in better understanding and rational treatment of ncse . according to bauer and trinka , the confusion vis - - vis ncse in coma
can be overcome by having a separate ncse categorycomatose ncse or coma - ncse.5 the authors classified cpse and ase as ncse proper .
ncse proper has a good prognosis and , in most cases , the treatment of choice is turning off the seizure without aggressively anesthetizing the patient .
coma - ncse is more ominous and its prognosis depends almost entirely on the underlying brain disorder.5 while there is no firm evidence that suppressing the seizure improves prognosis in coma - ncse , anesthetizing the patient with midazolam or propofol may not be unreasonable if the patient is already on mechanical ventilation and is being monitored in the icu .
maganti and colleagues proposed an etiology - based classification for ncse , including ncse in coma , ncse in acute cerebral lesions , ncse in metabolic disorders , and ncse in those with pre - existing epilepsy.6 this more detailed approach may lend itself to better treatment recommendation , but most importantly better evaluation and testing of treatment recommendations to determine their value based on outcome .
we agree with bauer and trinka that it is important to differentiate coma - ncse from ncse proper5 and with maganti and colleagues that it is critical to account for etiology in classifying ncse.6 by combining the bauer / trinka and maganti proposals and adding some minor refinements , we were able to create a practical decision tree for ncse ( fig .
4 ) . as shown in this decision tree , as soon as ncse is diagnosed ( based on electroclinical data ) , the clinician should separate
the next step is to differentiate between ncse with significant irreversible brain injury ( sibi ) and ncse with no evidence of sibi
. most coma ncse with sibi is due to hypoxic - ischemic , traumatic , ischemic , infectious , or inflammatory insults to the brain .
most coma ncse with no sibi is due to epileptic , toxic , or metabolic factors , often with the confounding effects of anesthetics or sedative drugs that are regularly employed in the icu .
most non - coma ncse is due to purely epileptic , toxic , or metabolic factors , the majority of which are reversible .
occasionally , sibi of lesser severity may give rise to non - coma ncse .
such a heuristic approach can facilitate our understanding of ncse , especially ncse in coma .
while the incidence depends on how one defines ncse , even more conservative estimates of the incidence of ncse ( eg , 25% ) warrant a detailed analysis of ncse cases with a goal toward increased understanding and better classification of ncse .
further work is needed to better define ncse , to determine which eeg patterns indicate ncse , and to establish treatment paradigms for the different types of ncse .
a more collaborative approach across hospitals / universities is warranted given the sample size that would be needed to determine treatment efficacy in so many ncse categories .
this is an unusual presentation of hypoxic - ischemic brain injury in that typical spike - and - wave activity ( tswa ) appeared in the eeg even though the patient never had seizure / epilepsy .
the tswa was relatively resistant to benzodiazepines suggesting that cerebral hypoxia - ischemia spared the thalamocortical apparatus generating tswa , but destroyed the inhibitory circuits where benzodiazepines act to suppress tswa .
post - hypoxic tswa is rare ; nevertheless , it highlights two important principles : first , the eeg allows us to diagnose ncse but does not always tell us how to treat , or whether we should even treat , a particular ncse pattern .
second , it is only by separating ncse proper from comatose ncse and taking into account the overall picture , including the presence of structural lesions , reversible causes , history of epilepsy , and prognosis that we can tailor ncse treatment to the best interest of the patient . | introductiontypical spike - and - wave activity ( tswa ) in the electroencephalogram ( eeg ) indicates idiopathic generalized epilepsy ( ige ) .
ige - related nonconvulsive status epilepticus ( ncse ) is typically an absence status epilepticus ( ase ) .
ase and tswa respond dramatically to benzodiazepines .
patients with no history of seizure / epilepsy may develop ase
de novo in the context of an acute brain disorder . however , we are aware of only one previous case of de novo ase with tswa in hypoxic - ischemic brain injury.case presentationa 65-year - old man , with congestive heart failure and history of substance abuse , survived cardiorespiratory arrest after 18 minutes of cardiopulmonary resuscitation .
post - resuscitation , the patient was in coma with intact brainstem function .
toxicology was positive for cocaine and marijuana .
eyelid myoclonus suggested ncse , which was initially treated with lorazepam and fosphenytoin .
eeg monitoring showed sustained tswa confirming ncse and demonstrating de novo ase ( the patient and his family never had seizure / epilepsy ) .
the tswa was resistant to lorazepam , levetiracetam , and low - dose midazolam ; it was eliminated only with midazolam at a dose that resulted in burst - suppression ( 1.2 mg / kg / hour).conclusionthis is an unusual case of tswa and hypoxic - ischemic brain injury in a patient with no history of seizure / epilepsy .
the tswa was relatively resistant to benzodiazepines suggesting that cerebral hypoxia - ischemia spared the thalamocortical apparatus generating tswa but impaired the cortical / thalamic inhibitory circuits where benzodiazepines act to suppress tswa .
albeit rare , post - hypoxic tswa offers us some valuable insights for classifying and managing nonconvulsive status epilepticus . | Introduction
Case Presentation
Discussion
Pathophysiology of TSWA
Classification of NCSE
Conclusion |
thirty - two eyes of 16 patients who underwent unilateral ptosis surgery were enrolled in the study .
the patients with other ocular disease , dry eye , history of a systemic disease that would affect ocular surface , history of contact lens use , and prior conjunctival surgery were excluded from the study . all patients underwent ptosis repair by an mcr with a technique similar to the described by putterman .
a subjective dry eye questionnaire was given to the patients questioning various dry eye symptoms , frequency of dryness , grittiness , burning , redness , crust formation on lashes and stickiness of eyelids in the morning .
responses were graded as follows : 0 - never ; 1 - rare ; 2 - sometimes ; 3 - frequently ; or 4 - always .
schirmer and tbut tests were performed after an application of a drop of proparacaine hydrochloride 0.5% .
fluorescein staining was recorded for 5 regions of the cornea ( superior , inferior , nasal , temporal , central ) and 4 regions of the conjunctiva ( superior , inferior , nasal , temporal ) .
the total staining score was the sum of the scores of all regions . rose - bengal staining ( rbs ) of the conjunctiva was performed by using a contacare rose - bengal ophthalmic strip ( contacare , baroda , india ) ; initially wetted with non - preserved buffered saline and then instilled on the inferior bulbar conjunctiva .
the eyelid margin was checked for meibomian gland disease ( mgd ) with slit lamp examination .
grade-2 indicated 3 to 4 blocked glands with thick secretions but without expression of glands .
impression cytology specimens were collected from the temporal and nasal conjunctiva of each eye using nitrocellulose ( sartorius , gttingen , germany ) filter paper .
the quantitative studies of conjunctival goblet cells and squamous metaplasia of conjunctival epithelial cells were conducted by taking photographs with a calibrated grid under a light microscope .
five non - overlapping areas of each randomly - selected sample were photographed and averaged for a single score .
the specimens were also assigned a grade of conjunctival epithelial squamous metaplasia according to nelson 's grading scheme .
the results were analyzed by using paired t - test and wilcoxon matched pair test .
patients were followed - up for mean 18.5 10.2 ( 4 - 40 ) months .
the mean subjective dry eye questionnaire score was 2.68 3.98 points in operated eyes and 2.00 2.63 points in control eyes .
there was no difference in the st and tbut scores before and after surgery in both operated and in the control eyes ( p = 0.79 , p = 1.00 , respectively ) .
the mean fluorescein and rbs scores , meibomian gland disease score and squamous metaplasia grading score of operated and non - operated eyes of each patient also showed no significant differences [ table 1 ] .
tear function and ocular surface examinations an impression cytology analysis showed no statistically significant change in goblet cell densities between operated and the control eyes ( > 0.05 ) .
conjunctival epithelium contains goblet cells that secrete mucin and the glands of krause and wolfring , which are responsible for basal tear secretion .
any damage in tear film leads to epithelial defects in the cornea , and these defects are stained with fluorescein .
this stain is blocked by tear proteins such as albumin and mucin , which cover the ocular surface . decreased mucin production plays a major role in ocular surface staining with rose - bengal . in comparison of operated and control eyes in terms of fluorescein and rbs , no significant difference was found between the eyes .
the pathological examination of samples taken from upper and lower bulbar conjunctiva of patients revealed no statistically significant difference in goblet cell density and grade of conjunctival metaplasia between operated and control eyes .
conjunctival forniceal shortening due to conjunctival resection has more impact on dry eye states with conjunctival deficiency such as , anophthalmic socket and conjunctival cicatrizing diseases .
the suture used in mcr may abrade into the filtration area and may be problematic in patients with trabeculectomy .
some investigators believe that an excision of conjunctiva may damage the accessory lacrimal glands of wolfring , located in the upper tarsal border , which may aggravate dry eye states .
we have incorporated all the frequently applied clinical and laboratory tests to evaluate the effects of mcr surgery on tear film stability .
our results conclude that an mcr surgery does not have any adverse effects tear film production and stability .
further studies with a larger patient population might shed light on the effect of an mcr on tear film and ocular surface . | muller muscle - conjunctival resection ( mcr ) is a surgical technique to correct mild and moderate ptosis . in this study ,
tear function tests and ocular surface are evaluated in patients who underwent unilateral surgery .
sixteen patients with normal preoperative tear function who underwent unilateral mcr were evaluated prospectively .
the fellow eyes of the patients were taken as the control group .
a dry eye assessment questionnaire , schirmer testing , tear film break - up time , fluorescein stain , rose - bengal stain , and conjunctival impression cytology were used to assess the tear film functions and ocular surface changes in the operated and non - operated eyes .
there was no statistically significant difference in the tear function tests and goblet cell densities between the operated and non - operated eyes .
the results indicate that an mcr procedure has no apparent effect on tear function tests and goblet cell density in patients with normal preoperative tear function . | Materials and Methods
Results
Discussion |
postoperative cognitive dysfunction ( pocd ) is a major complication characterized by disordered thinking and impaired consciousness following surgeries and anesthesia , especially in elderly individuals .
early studies focused on pocd after cardiac surgery , but a recent study conducted by evered et al . showed that it was associated with non - cardiac surgery and even with minor non - invasive procedures under sedation , such as coronary angiography .
a large multi - center study reported that the incidence of pocd was 25% 1 week after non - cardiac surgery and about 10% 3 months after non - cardiac surgery .
cognitive changes after surgery prolong hospital stay , elevate perioperative medical cost , decrease postoperative quality of life , and increase surgical morbidity and mortality
clinical studies have consistently suggested that the main causes of this injury include an exaggerated systemic inflammation , endothelial dysfunction , cerebral hypoperfusion , and microembolism .
others have specifically reported that pocd is indeed correlated with the serum protein levels of il-6 , tnf- , s100 , neuron - specific enolase ( nse ) , and malonaldehyde ( mda ) [ 911 ] .
in addition , animal studies have also shown that neuroinflammation and brain cell death after surgery and anesthesia may contribute to the brain functional changes [ 1214 ] .
although many drugs have been studied as neuroprotective during surgery and anesthesia , such as thiopental , propofol , ulinastatin , sevoflurane , and morphine , there is no agreement on the efficiency of prophylactic neuroprotectants in cardiac or non - cardiac surgery .
lidocaine , an inexpensive , widely available , and relatively safe compound , is a local anesthetic and class ib antiarrhythmic that readily crosses the blood - brain barrier .
initially demonstrated cerebral protection of lidocaine in a feline model of cerebral arterial gas embolism .
later clinical studies have also demonstrated the effects of lidocaine on perioperative neuroprotection [ 1921 ] .
however , the mechanisms underlying lidocaine treatment - induced neuroprotection remain incompletely understood . therefore , in the present study , we hypothesized that lidocaine ameliorated early cognitive damage in patients undergoing spine surgery .
also , we determined the effects of lidocaine treatment on the levels of il-6 , tnf- , s100 , nse , and mda in serum .
1 people s hospital and written informed consent was obtained from all patients before the study .
the study included 87 american society of anesthesiologists ( asa ) physical status i or ii patients aged greater than 65 years scheduled for a spine surgery from september 2013 to february 2015 .
exclusion criteria included : mini - mental state examination ( mmse ) score < 23 before surgery ; history of neurological diseases ( including alzheimer s disease and stroke history ) , psychological disorder , and drug or alcohol abuse ; history of diabetes mellitus , severe hypertension , severe anemia , hepatic or renal dysfunction ; unwillingness to comply with the protocol or procedures ; inability to speak and read chinese .
patients were randomly allocated to 2 treatment groups : ( 1 ) lidocaine treatment group ( n=40 ) , a bolus of 1 mg / kg of lidocaine over 5 minutes administered after induction of anesthesia and followed by a continuous infusion at 1.5 mg / kg / h until the end of the surgery ; or ( 2 ) control group ( n=40 ) , normal saline administered as a bolus and an infusion with the same volume and rate changes as the lidocaine group .
anesthesia was induced with midazolam ( 0.03~0.05 mg / kg ) , sufentanil ( 0.2~0.3 g / kg ) , cisatracurium ( 0.15~0.2 mg / kg ) and propofol ( 2 mg / kg ) .
anesthesia was maintained by intravenous injection of propofol ( 4~12 mg / kg / h ) and remifentanil ( 0.1~0.15 g / kg / min ) .
electrocardiogram , respiratory rate , pulse oximetry , petco2 and hemoglobin oxygen saturation ( spo2 ) were continuously monitored during surgery .
the blood samples were collected to observe changes in the levels of il-6 , tnf- , s100 , nse and mda before inducing anesthesia ( t1 ) , at the end of surgery ( t2 ) and three days after the end of surgery ( t3 ) .
blood samples ( 5 ml ) were allowed to clot for 2 h at room temperature and were then centrifuged for 15 min at 2000g at 4c .
the levels of il-6 , tnf- , s100 and nse were measured using an enzyme - linked immunosorbent assay ( elisa ) kit ( neobioscience technology company , beijing , china ) according to the manufacturer s protocol .
mda concentrations were determined using enzymatic methods following the manufacturer s instructions ( jiancheng biologic project company , nanjing , china ) .
cognitive function was evaluated pre - operatively and three days post - operatively in a quiet room with only the patient and the experienced psychometrician .
intergroup numerical data , including il-6 , tnf- , mda , s100 and nse concentrations were analyzed with the student s t - test ; intragroup numerical data were analyzed with repeated measures anova .
the differences between the values were considered to be significant at p<0.05 . all statistical tests and graphs
were performed using statistical program for social sciences 20.0 software ( spss , inc . ,
the study included 87 american society of anesthesiologists ( asa ) physical status i or ii patients aged greater than 65 years scheduled for a spine surgery from september 2013 to february 2015 .
exclusion criteria included : mini - mental state examination ( mmse ) score < 23 before surgery ; history of neurological diseases ( including alzheimer s disease and stroke history ) , psychological disorder , and drug or alcohol abuse ; history of diabetes mellitus , severe hypertension , severe anemia , hepatic or renal dysfunction ; unwillingness to comply with the protocol or procedures ; inability to speak and read chinese .
patients were randomly allocated to 2 treatment groups : ( 1 ) lidocaine treatment group ( n=40 ) , a bolus of 1 mg / kg of lidocaine over 5 minutes administered after induction of anesthesia and followed by a continuous infusion at 1.5 mg / kg / h until the end of the surgery ; or ( 2 ) control group ( n=40 ) , normal saline administered as a bolus and an infusion with the same volume and rate changes as the lidocaine group .
anesthesia was induced with midazolam ( 0.03~0.05 mg / kg ) , sufentanil ( 0.2~0.3 g / kg ) , cisatracurium ( 0.15~0.2 mg / kg ) and propofol ( 2 mg / kg ) .
anesthesia was maintained by intravenous injection of propofol ( 4~12 mg / kg / h ) and remifentanil ( 0.1~0.15 g / kg / min ) .
electrocardiogram , respiratory rate , pulse oximetry , petco2 and hemoglobin oxygen saturation ( spo2 ) were continuously monitored during surgery .
the blood samples were collected to observe changes in the levels of il-6 , tnf- , s100 , nse and mda before inducing anesthesia ( t1 ) , at the end of surgery ( t2 ) and three days after the end of surgery ( t3 ) .
blood samples ( 5 ml ) were allowed to clot for 2 h at room temperature and were then centrifuged for 15 min at 2000g at 4c .
the levels of il-6 , tnf- , s100 and nse were measured using an enzyme - linked immunosorbent assay ( elisa ) kit ( neobioscience technology company , beijing , china ) according to the manufacturer s protocol .
mda concentrations were determined using enzymatic methods following the manufacturer s instructions ( jiancheng biologic project company , nanjing , china ) .
cognitive function was evaluated pre - operatively and three days post - operatively in a quiet room with only the patient and the experienced psychometrician .
intergroup numerical data , including il-6 , tnf- , mda , s100 and nse concentrations were analyzed with the student s t - test ; intragroup numerical data were analyzed with repeated measures anova .
the differences between the values were considered to be significant at p<0.05 . all statistical tests and graphs
were performed using statistical program for social sciences 20.0 software ( spss , inc . ,
seven patients were excluded because of refusal to neuropsychological evaluation after operation , thus leaving eighty patients who completed the blood sample collection and neurocognitive tests .
there were no significant differences in age , sex , body weight , asa classification , operation time , and hospital stay between groups ( p>0.05 ) .
compared with the preoperative mmse scores , those on three days after surgery were significantly decreased in the control group . however , the mmse scores in the lidocaine group were markedly higher than those in the control group at t3 ( figure 1 , p<0.05 ) .
serum assays were performed at 3 time points : preoperation ( t1 ) , at the end of surgery ( t2 ) , and 3 days after the end of surgery ( t3 ) . in the control group ,
however , the lidocaine group had lower serum il-6 concentration than that in the control group ( figure 2a , p<0.05 ) .
there were no significant differences between groups in tnf- concentration at 3 time points ( figure 2b , p>0.05 ) .
serum levels of mda were higher at 3 days after surgery when compared with preoperative levels in the control group .
however , the elevated levels were dramatically attenuated by lidocaine treatment ( figure 3 , p<0.05 ) . obviously , no significant differences in the level of mda were observed between groups at t2 ( p>0.05 ) . in the control group
, there was a marked increase in the serum concentrations of s100 and nse at t2 and t3 compared with those at t1 .
however , lidocaine treatment clearly inhibited the up - regulation of s100 and nse in serum ( figure 4 , p<0.05 ) .
the present study revealed that lidocaine can improve cognitive performance in elderly patients undergoing spine surgery .
in addition , we reported for the first time that lidocaine can effectively decrease serum levels of il-6 , mda , s100 , and nse after non - cardiac surgery
. therefore , a potential mechanism underlying lidocaine treatment - induced neuroprotection may be to inhibit the release of il-6 , mda , s100 , and nse .
these findings indicate that lidocaine may be a promising therapeutic approach for the treatment of pocd .
lidocaine confers cerebral protection via many mechanisms , including reducing the cerebral metabolic rate , reducing the ischemic excitotoxin release , and decelerating the ischemic transmembrane ion shift .
additionally , recent evidence has suggested that the neuroprotective effects of lidocaine may be associated with its anti - inflammatory and anti - apoptotic properties .
however , previous clinical trials on the effects of lidocaine on pocd are contradictory , with improvements and no effect .
this variability may arise from different timing and dose of lidocaine administered and different patient selection . in our study ,
the infusion protocol was designed to deliver a 1 mg / kg bolus over 5 minutes after induction of anesthesia , followed by a continuous infusion at 1.5 mg / kg / h until the end of the surgery .
our results suggested that the mmse scores of patients in the lidocaine group were markedly higher than those in the control group 3 days after surgery , implying that lidocaine may attenuate cognitive function in patients undergoing spine surgery .
studies suggest that the magnitude of inflammatory response is a risk factor for cognitive dysfunction after major surgery .
surgical trauma activates the peripheral innate immune system , resulting in the release of inflammatory mediators , which impairs cognitive function .
xu and colleagues found that serum levels of il-6 increased after general anesthesia in abdominal surgery and the increase may be associated with the occurrence of pocd .
their results showed that il-6 might serve an indicator to guide the prevention and treatment of pocd .
serum tnf- level is also suggested to be a critical mediator of inflammation - induced neuronal dysfunction .
it is the first cytokine to be released following surgery and its peripheral blockade could limit the release of neuroinflammation and cognitive impairment in a mouse model of surgery - induced cognitive dysfunction .
however , the studies mentioned above did not demonstrate the relationship between pocd and the levels of serum tnf-. in agreement , our results showed that concentration of serum il-6 increased at the end of surgery and 3 days after the end of surgery .
there were no significant differences in tnf- level between the 2 groups at the 3 time points studied .
our results and those of others indicate that inflammatory response may be responsible for pocd and that lidocaine may prevent or reverse the cognitive deficits by inhibiting the response .
numerous studies support that oxidative stress precedes the development of neurodegenerative diseases such as pocd , mild cognitive impairment ( mci ) , parkinson s disease , and alzheimer s disease ( ad ) .
lipid peroxidation - induced oxidative dna damage is believed to contribute to neuronal death and neurological dysfunction .
previous studies have provided experimental evidence that increased levels of mda in peripheral blood are associated with development of ad and mci .
also , recent evidence suggests that elevated mda levels are a risk factor for cognitive decline in aged patients after orthopedic surgery and total hip - replacement surgery .
therefore , intervention using serum mda could be a primary prevention strategy for pocd . in the present study
we have shown a steady increase in serum mda levels in the control group but not in the lidocaine group .
s100 and nse are serum markers of neuronal injury and are increasingly used in diagnosis and prognosis of cognitive impairment after different kinds of surgery .
s100 , in astrocytes , is a calcium - binding protein that has been regarded as highly brain - specific .
normally , s100 can not pass the blood - brain barrier to enter the blood stream . however , serum concentrations are increased after damage to central nervous cells as well as blood - brain barrier dysfunction .
studies have confirmed the value of s100 in assessment of cognitive deficits after various surgeries .
therefore , s100 protein release is a plausible candidate in assessment of incidence , course , and outcome of pocd .
nse is an isoenzyme of the glycolytic enzyme enolase , usually found in the cytoplasm of neurons and cells of neuro - endocrine differentiation .
our study has demonstrated that both the serum s100 protein and nse levels significantly increased at the end of the surgery and on postoperative day 3 , suggesting that spine surgery may cause damage to brain tissue .
interestingly , s100 protein and nse levels were reduced after the administration of lidocaine , suggesting the neuroprotective effect of lidocaine .
a limitation of this study is that we did not measure the serum concentration of lidocaine during or after surgery .
an accepted therapeutic range is 2 to 5 g / ml , and detrimental effects , including arrhythmia , tremors , confusion , visual disturbances , impaired concentration , or even seizures and coma , usually occur at levels above 6 to 10
. however , we did not find any adverse effects caused by lidocaine in our study , which could be attributed to the relatively low lidocaine dose .
our results indicate that lidocaine treatment attenuated cognitive impairment in elderly patients undergoing spine surgery . furthermore , serum il-6 , s100 , nse , and mda were involved in the mechanism underlying the therapeutic effect of lidocaine on cognitive function .
therefore , the results indicated that lidocaine may be an effective neuroprotective agent for the treatment of pocd .
further clinical investigation is warranted to determine its potential on the subsequent development and progression of pocd . | backgroundthis study aimed to evaluate the effects of lidocaine treatment on cognitive impairment in aged patients undergoing spine surgery and to explore the underlying mechanism.material/methodspatients were randomly divided into 2 treatment groups : ( 1 ) saline ( control ) and ( 2 ) lidocaine .
after induction of anesthesia , the lidocaine group received lidocaine as a bolus of 1 mg / kg over 5 minutes , followed by a continuous infusion at 1.5 mg / kg / h until the end of the surgery .
we examined the effects of lidocaine treatment on the improvement of cognitive function using the mini - mental state examination ( mmse ) at preoperation and 3 days postoperation .
serum samples were collected to assess the levels of il-6 , tnf- , mda , s100 , and nse before inducing anesthesia , at the end of surgery , and 3 days after the end of surgery.resultswe found that the mmse scores in the lidocaine group were markedly higher than those in the control group at 3 days after surgery . moreover , lidocaine treatment markedly suppressed the release of il-6 , s100 , and nse into the serum at the end of surgery and 3 days after the end of surgery . in the control group , serum mda levels increased by 3 days after the end of surgery .
the lidocaine group had lower serum mda levels than those in the control group.conclusionslidocaine may be an effective neuroprotective agent in treating early postoperative cognitive dysfunction in elderly patients undergoing spine surgery . | Background
Material and Methods
Participants
Protocol and general anesthesia
Serum samples
Measurement of neurocognitive function
Statistical analysis
Results
Discussions
Conclusions |
physicians treat diseases . for this reason when increasing blood pressure was recognized as a risk factor for coronary artery disease and stroke ,
this cut point has generally become lower over time as evidence of benefit in treating blood pressure levels lower than the contemporary accepted cut points accumulated .
recognition of other risk factors such as dyslipidemia , higher risk groups such as the aged [ 1 , 2 ] , and those with comorbid conditions such as diabetes has led to differential treatment thresholds and target blood pressures resulting in confusing or conflicting guideline recommendations , depending on which peak body produced them .
is there a simpler way to identify those most likely to have a major adverse cardiovascular event who do not have overt disease , and therefore who needs rigorous therapeutic intervention for their blood pressure and other cvd risk factors ?
increasing blood pressure has a log - linear relationship with adverse risk for cardiovascular events . using this figure alone in clinical decision making risks overtreatment ( medicalization where medication adverse events likely to exceed benefit , and adverse cost - effectiveness ) and undertreatment ( failure to act where medication benefit is likely to exceed adverse events and be cost effective ) . estimating absolute risk
, the risk of having an adverse cardiovascular event over a specified period of time ( usually 5 or 10 years ) , is a superior method to measurement of blood pressure alone to identify who is most at risk of having a cardiovascular event and therefore who would most likely benefit from intervention [ 5 , 6 ] .
it does this because the figure derived is more holistic , incorporating other cvd risk factors that explain almost all risk including blood pressure , and directly estimates why we treat blood pressure in any population without cvd , to prevent its onset and complications .
guidelines are increasingly recognizing the benefit of utilizing absolute risk scores rather than blood pressure measurements alone [ 712 ] .
cvd risk calculators are based on algorithms derived from observational prospective cohort studies such as the framingham study [ 13 , 14 ] .
cvd risk calculators must be used to estimate absolute risk in those without prior cvd events as physician estimation is unreliable .
physicians can reliably estimate relative risk , the risk of an individual having a myocardial infarction or stroke relative to others of the same age and gender .
the problem with relative risk is that by its very nature it excludes the two most important drivers of cvd risk , age and gender .
there are validated algorithms specifically derived from hypertensive populations available but such scores would tend to fragment absolute risk scores back into their constituent risk factor classification rather than unify and simplify the process .
risk factors used in the framingham risk score ( age , gender , blood pressure , serum cholesterol , smoking status , and presence of diabetes ) but the benefits of this increase in complexity is marginal . absolute risk scores ( ars ) derived from these algorithms have proven to have good discrimination in their source population ( e.g. , framingham risk score c statistic 0.763 ( males ) , 0.793 ( females ) ) but do have problems in subgroups not adequately represented in the study population . for example , calculators derived from the framingham risk score have an upper age limit because no persons over 74 years were included in the study population . obviously , from an international perspective , not only subgroups but whole populations were missing .
for example , australian aborigines , an important high adverse cvd risk ethnic minority in australia , have resisted all attempts to be reliably and validly incorporated into australian algorithms derived from the framingham risk score [ 11 , 18 ] .
they are by definition at high risk and should be aggressively managed . those with target organ damage due to elevated blood pressure such as left ventricular hypertrophy and hypertensive retinopathy or nephropathy .
those with blood pressure 180/100 mm hg . those from a non - caucasian population unless the risk score utilized has been derived from and/or validated in this population or has been recalibrated .
once identified and
the decision has been made to treat based on absolute risk score , then management reverts to that of the individual risk factor .
the concept of treating absolute risk per se is supported by clinical trials such as the hope study , but the hypothesis needs to be formally tested in studies such as ongoing and planned polypill trials .
this management strategy promises simpler treatment regimens , lower direct costs , and superior clinical outcomes . outside of the polypill
if adverse effects prevent drug therapy of a particular risk factor then intervene on another .
management of persons with elevated blood pressure is best done through an absolute risk approach including the use of absolute risk scores .
this promises better targeted therapy , simpler more flexible management regimens , and superior clinical outcomes . | increasing blood pressure has a continuum of adverse risk for cardiovascular events .
traditionally this single measure was used to determine who to treat and how vigorously . however , estimating absolute risk rather than measurement of a single risk factor such as blood pressure is a superior method to identify who is most at risk of having an adverse cardiovascular event such as stroke or myocardial infarction , and therefore who would most likely benefit from therapeutic intervention . cardiovascular disease ( cvd ) risk calculators
must be used to estimate absolute risk in those without overt cvd as physician estimation is unreliable .
incorporation into usual practice and limitations of the strategy are discussed . | 1. Introduction
2. Absolute Cardiovascular Risk
3. Cardiovascular Disease Risk Calculators
4. Conclusions |
the medical records of 83 patients initially diagnosed as lca before 6 months of age , in the department of ophthalmology of 2 tertiary referral centers , were evaluated .
all patients fulfilled the diagnostic criteria currently proposed by de laey and a search for metabolic or degenerative central nervous system disorders was performed in all patients .
patients initially diagnosed with alleged neurologic or systemic abnormalities , or patients whom revealed any other hereditary retinal disease during follow - up were excluded .
brain mri was performed in 31 patients who consented to the examination ( 37.3% ) .
the described research adhered to the tenets of the declaration of helsinki and approval to conduct this study was obtained from the institutional review board .
out of 31 patients , 6 patients ( 19.4% ) had radiologically documented brain abnormalities .
other alterations on mri with unknown clinical significance were found in 3 subjects , which included a small cerebellum and 2 subjects with mild external hydrocephalus .
the following cases depict the clinical characteristics of lca patients associated with specific radiological brain abnormalities , including absent septum pellucidum and cerebellar vermis hypoplasia .
she was born from healthy non - consanguineous parents and had an unremarkable family history .
cycloplegic refractive errors were -1.50 d sph = -3.00 d cyl 180 od , -3.50 d sph = -3.00 d cyl 180 os .
erg was extinguished in both eyes and she was diagnosed as lca . systemic evaluation for metabolic abnormalities , as well as of the kidney , liver , and skin were unremarkable .
genetic analysis of mutations in crx , crb1 , gucy2d , aipl1 , rdh12 , rpe65 , lrat , rpgrip1 , and tulp1 were unremarkable .
development was within the normal limit and neurologic evaluation was unremarkable at the time of diagnosis .
however , brain mri showed a prominent enlargement in the subarachnoid space of both frontal convexities , and the septum pellucidum was absent .
no focal lesions were present in the brain and the size of the corpus callosum and white matter myelination was normal . at the age of 15 months ,
a follow - up brain mri was taken , and external hydrocephalus with absent septum pellucidum was stationary ( fig .
a diagnosis of septo - optic dysplasia was considered , however , normal appearance of the optic nerve and normal pituitary function left the diagnosis doubtful .
strabismus surgery was performed for an esotropia of 40 prism diopters ( pd ) . at the age of 3 years
, the patient could still not fixate with the eyes and nystagmus was present , while ocular alignment was orthotropic .
cognitive and motor development was moderately delayed approximately 10 months , yet was slowly improving .
he was born after an uneventful pregnancy , and his medical history was unremarkable in the perinatal period .
development was within normal limits and neurologic evaluation was unremarkable at the initial presentation . however , brain mri revealed hypoplasia of the cerebellar vermis with enlarged 4th ventricles .
systemic evaluation for metabolic abnormalities , as well as of the kidney , liver , and skin were unremarkable . genetic analysis of mutations in crx , crb1 , gucy2d , aipl1 , rdh12 , rpe65 , lrat , rpgrip1 , and tulp1 were negative . at 8 years of age , development was within normal limit .
his visual acuity was at the level of light perception in both eyes , and nystagmus had diminished ; however , erg was still extinguished .
out of 31 patients , 6 patients ( 19.4% ) had radiologically documented brain abnormalities .
other alterations on mri with unknown clinical significance were found in 3 subjects , which included a small cerebellum and 2 subjects with mild external hydrocephalus .
the following cases depict the clinical characteristics of lca patients associated with specific radiological brain abnormalities , including absent septum pellucidum and cerebellar vermis hypoplasia .
she was born from healthy non - consanguineous parents and had an unremarkable family history .
cycloplegic refractive errors were -1.50 d sph = -3.00 d cyl 180 od , -3.50 d sph = -3.00 d cyl 180 os .
erg was extinguished in both eyes and she was diagnosed as lca . systemic evaluation for metabolic abnormalities , as well as of the kidney , liver , and skin were unremarkable .
genetic analysis of mutations in crx , crb1 , gucy2d , aipl1 , rdh12 , rpe65 , lrat , rpgrip1 , and tulp1 were unremarkable .
development was within the normal limit and neurologic evaluation was unremarkable at the time of diagnosis .
however , brain mri showed a prominent enlargement in the subarachnoid space of both frontal convexities , and the septum pellucidum was absent .
no focal lesions were present in the brain and the size of the corpus callosum and white matter myelination was normal . at the age of 15 months ,
a follow - up brain mri was taken , and external hydrocephalus with absent septum pellucidum was stationary ( fig .
a diagnosis of septo - optic dysplasia was considered , however , normal appearance of the optic nerve and normal pituitary function left the diagnosis doubtful .
strabismus surgery was performed for an esotropia of 40 prism diopters ( pd ) . at the age of 3 years , the patient could still not fixate with the eyes and nystagmus was present , while ocular alignment was orthotropic .
cognitive and motor development was moderately delayed approximately 10 months , yet was slowly improving .
he was born after an uneventful pregnancy , and his medical history was unremarkable in the perinatal period .
development was within normal limits and neurologic evaluation was unremarkable at the initial presentation . however , brain mri revealed hypoplasia of the cerebellar vermis with enlarged 4th ventricles .
systemic evaluation for metabolic abnormalities , as well as of the kidney , liver , and skin were unremarkable .
genetic analysis of mutations in crx , crb1 , gucy2d , aipl1 , rdh12 , rpe65 , lrat , rpgrip1 , and tulp1 were negative . at 8 years of age , development was within normal limit .
his visual acuity was at the level of light perception in both eyes , and nystagmus had diminished ; however , erg was still extinguished .
pathologic findings in brain mri of lca subjects with systemic or neurologic abnormalities are documented in up to 33% of patients [ 3,5 - 7 ] .
while most of the findings are mild and nonspecific alterations on mri , such as slight reduction of the dimensions of the chiasm and/or optic nerve or white matter alterations , 2 to 10% of patients reveal pathologic findings of hypoplasia of the cerebellar vermis . without consideration of mild nonspecific alterations in brain mri ,
the present study showed similar results to former reports , as 7% of primary lca subjects were found to have pathologic brain imaging ; half of the patients showing cerebellar vermis hypoplasia and the other patients revealing mild external hydrocephalus or absence of septum pellucidum , of which the latter is a novel finding in the present study .
notably , the perinatal history or neurologic examinations of the patients were unremarkable at the time of presentation before 6 months of age . moreover , as the brain mri was performed in only 37% of all patients who consented to the examination , the true incidence of abnormal brain structures may be larger .
. found that a significant amount of brain pathology occurs in children with congenital disorders of the peripheral visual system and the number of lesions varies directly with the degree of visual impairment and both correlate with developmental outcome .
therefore , brain imaging studies in patients initially diagnosed as lca subjects should be mandatory , even if systemic or neurologic abnormalities are not obvious .
case 1 and 2 showed mri findings known to be a part of syndromic associations , namely septo - optic dysplasia with absent septum pellucidum and joubert syndrome with cerebellar vermis hypoplasia .
however , the cases differed in many aspects from the typical syndromic characteristic of septo - optic dysplasia or joubert syndrome . in case 1 ,
absence of the septum pellucidum without any other radiologic abnormalities lead to a differential diagnosis of septo - optic dysplasia .
the clinical picture of absent septum pellucidum and congenital nystagmus overlapped with the clinical and radiological features of septo - optic dysplasia . on the other hand ,
the present case differed from typical septo - optic dysplasia in a number of specific aspects , i.e. , the absence of hypoplasia of the optic disc , the absence of pituitary abnormalities and the absence of typical neurological impairment ( e.g. , myoclonic seizure was not present in our lca subject ) .
the absence of septum pellucidum may be an isolated finding and , although a characteristic sign of septo - optic dysplasia , it is not pathognomonic .
congenital absence of the septum pellucidum is not associated with significant intellectual , behavioral , or neurological deficits in the majority of patients with septo - optic dysplasia .
thus , the clinical significance of an absent septum pellucidum in case 1 with mild developmental delay is unknown .
case 1 may be considered as a type of complex lca associated with the absence of septum pellucidum and mild developmental delay . in case 2 ,
cerebellar vermis hypoplasia is found in up to 20% of brain imaging findings in lca subjects .
the " molar tooth " sign is now recognized as present not only in joubert syndrome but also in various cerebellooculo - renal syndromes ( arima , coach , and senior - loken ) .
the joubert syndrome and related disorders are frequently associated with an lca - like ocular phenotype .
therefore , looking for systemic involvement in lca patients with cerebellar vermis hypoplasia , including the kidney , liver and skin , to allow the delineation of lca subtypes , is crucial .
however , the two lca patients in the present study with cerebellar vermis hypoplasia differed significantly from the typical characteristics of joubert syndrome - related disorders in a number of specific aspects , i.e. , the absence of episodes of apnea - tachypnea , the absence of oculomotor apraxia - like head thrust , less severe neurological involvement ( e.g. , minimal hypotonia ) , and the absence of systemic involvement .
thus , the subjects ( including case 2 ) could be considered as a type of complex lca associated with cerebellar vermis hypoplasia or another entity of joubert syndrome - related disorders with only the ocular lca phenotype . in a previous report
, mutations in cep290 were found in lca patients associated with mental retardation or neurodevelopmental degeneration in the absence of the other systemic features of joubert syndrome .
these individuals may represent a genetic subtype of lca or an undiagnosed systemic disorder which remains to be elucidated .
unfortunately , the genetic analysis of the cep290 mutation was not available in the present study .
further studies and genetic evaluation are required to define this heterogeneous disease . in conclusion , lca has complex phenotypic features , and an absence of septum pellucidum can be associated with lca , which has not been documented in the literature .
neurologic abnormalities may not be definite in a blind subject , especially under 6 months of age .
thus , brain imaging is mandatory in patients primarily diagnosed with lca in the absence of definite neurologic abnormalities . | purposeto report the incidence and new findings of abnormal brain imaging studies associated with patients initially diagnosed with leber 's congenital amaurosis ( lca ) without definite systemic abnormalities and to determine the need for brain imaging studies in these patients.methodsa retrospective review of medical records was performed in 83 patients initially diagnosed as lca and without definite systemic abnormalities before the age of 6 months in 2 tertiary referral centers .
brain magnetic resonance imaging was performed in 31 of 83 patients ( 37.3%).resultssix of 31 patients ( 19% ) had radiologically documented brain abnormalities .
two patients had cerebellar vermis hypoplasia , 1 patient showed an absence of septum pellucidum , 2 subjects showed mild external hydrocephalus , and 1 patient was found to have a small cerebellum.conclusionsapproximately one fifth of the lca patients in whom brain imaging was performed were associated with brain abnormalities , including the absence of septum pellucidum , which has not been documented in the literature .
brain imaging is mandatory in patients primarily diagnosed with lca , even without definite neurologic or systemic abnormalities . | Materials and Methods
Results
Incidence of radiological brain abnormalities
Case 1. Absent septum pellucidum
Case 2. Cerebellar vermis hypoplasia
Discussion |
first , we attempted to find representatives of all disciplines involved in the surgical management of dfo , including podiatry , orthopedic surgery , plastic surgery , vascular surgery , and general surgery .
second , we sought experts with extensive and direct clinical experience in the management of dfo ( table 1 ) . finally , we preferentially chose panelists with an appointment at an academic health care institution , involvement in clinical training programs , or experience of publishing original manuscripts in peer - reviewed journals on the topic of diabetic foot complications or foot reconstruction .
our intent was to select surgeons who had read and thought critically about the surgical management of dfo .
members of the expert panel , listed in alphabetical order the authors first constructed an initial open - ended questionnaire seeking questions that would elicit opinions on clinical practices regarding the management of dfo .
all questions and subsequent statements assumed forefoot osteomyelitis without significant peripheral artery disease or orthopedic / podiatric hardware .
we then used the responses to create statements that we asked the panelists to rate using a nine - point likert scale : 1=completely disagree , 2=strongly disagree ; 3=disagree , 4=somewhat disagree , 5=neutral , 6=somewhat agree , 7=agree , 8=strongly agree , 9=completely agree .
they were also provided space for additional comments or explanations of their scoring , identification of exceptions , or any other feedback .
the first round of the questionnaire involved a total of 63 items in the following four sections : 1 ) initial diagnosis and selection of patients for operative management ( statements 118 ) ; 2 ) operative management ( statements 1941 ) ; 3 ) postoperative care ( statements 4256 ) ; and 4 ) general principles ( statements 5763 ) .
we then conducted a second round of scoring on those items with a mean likert score between 6.40 and 7.0 .
this follow - up modified scoring survey included survey items based on panelist feedback , along with the group average for the item and the panelist 's previous personal score for that item .
first , we attempted to find representatives of all disciplines involved in the surgical management of dfo , including podiatry , orthopedic surgery , plastic surgery , vascular surgery , and general surgery .
second , we sought experts with extensive and direct clinical experience in the management of dfo ( table 1 ) . finally , we preferentially chose panelists with an appointment at an academic health care institution , involvement in clinical training programs , or experience of publishing original manuscripts in peer - reviewed journals on the topic of diabetic foot complications or foot reconstruction .
our intent was to select surgeons who had read and thought critically about the surgical management of dfo .
the authors first constructed an initial open - ended questionnaire seeking questions that would elicit opinions on clinical practices regarding the management of dfo .
all questions and subsequent statements assumed forefoot osteomyelitis without significant peripheral artery disease or orthopedic / podiatric hardware .
we then used the responses to create statements that we asked the panelists to rate using a nine - point likert scale : 1=completely disagree , 2=strongly disagree ; 3=disagree , 4=somewhat disagree , 5=neutral , 6=somewhat agree , 7=agree , 8=strongly agree , 9=completely agree .
they were also provided space for additional comments or explanations of their scoring , identification of exceptions , or any other feedback .
the first round of the questionnaire involved a total of 63 items in the following four sections : 1 ) initial diagnosis and selection of patients for operative management ( statements 118 ) ; 2 ) operative management ( statements 1941 ) ; 3 ) postoperative care ( statements 4256 ) ; and 4 ) general principles ( statements 5763 ) .
. we then conducted a second round of scoring on those items with a mean likert score between 6.40 and 7.0 .
this follow - up modified scoring survey included survey items based on panelist feedback , along with the group average for the item and the panelist 's previous personal score for that item .
thirteen panelists participated in the initial round . an additional panelist was added after the initial round to represent vascular surgery .
the overall response rate of selected panelists was 95% ( 39 of 41 possible responses ) . at the end of the first round ,
a total of 33 items reached our defined consensus cut - off : 19 items had a mean score of 6.50 . among the items , 16 scored a weighted mean between 6.50 and 6.99 , and we then did an additional round of scoring on these .
after this additional scoring round , we achieved consensus ( mean likert score 7 ) on five more statements , for a total of 38 consensus statements .
these consensus statements included 7 statements concerning the initial diagnosis and selection of patients for operative management , 15 statements about intraoperative management , 10 statements on postoperative management , and 6 general statements on general strategies and principles relevant to surgical management of dfo ( tables 25 ) .
consensus statements for initial diagnosis and selection of patients for operative management of diabetic forefoot osteomyelitis dfo , diabetic foot osteomyelitis .
consensus statements on general strategies and principles relevant to the surgical management of diabetic forefoot osteomyelitis dfo , diabetic foot osteomyelitis .
the consensus panel initially agreed on seven statements relevant to the initial diagnosis of dfo and patient selection ( table 2 ) .
the presence of visible , chronically exposed trabecular bone within a forefoot ulcer was deemed sufficient to establish the diagnosis of dfo . a magnetic resonance imaging ( mri ) or biopsy of a specimen of bone ( for culture or histopathology )
were identified as second - line diagnostic modalities of choice for confirmation of dfo when the diagnosis was otherwise uncertain .
surgical resection of affected bone was recommended when any one of the following is present : systemic toxicity ; an open or infected joint space ; the presence of a prosthetic heart valve ; clinical or imaging evidence of substantial cortical destruction , osteolysis , macroscopic bone fragmentation , or necrotic bone ; or visible , chronically exposed trabecular bone .
no consensus was reached on whether or not the presence of visible cortical bone is sufficient for the diagnosis of dfo or if this should just represent an indication for surgical resection ( see supplementary file ) .
the panel reached consensus on 15 statements regarding specific operative details ( table 3 ) .
the majority agreed that any concomitant deep soft tissue infection should be addressed first , and definitive ( final ) resection of bone should be undertaken 37 days after addressing the soft tissue infection .
there was agreement that negative pressure wound therapy dressings with instillation should be a consideration but is not required for treatment of deep soft tissue infection ( table 3 , also supplementary file ) .
panelists reached consensus that any bone resection should be done with a power saw ( e.g. micro - sagittal or micro - oscillating saw ) ; this should include all areas of necrotic , fragmented bone or areas of significant cortical destruction seen on x - ray , and continue until grossly healthy - appearing bone is seen at the resection margin .
the surgeon should collect a total of four specimens between the initial and final operations : grossly abnormal bone sent for 1 ) histopathology and 2 ) microbiology ( gram - stained smear , aerobic and anaerobic cultures ) and the proximal - most bone resected ( i.e. the bone margin ) for 3 ) histopathology and 4 ) microbiology .
the panel thought that metatarsal head / proximal phalangeal ostectomy was an acceptable alternative to toe / ray amputation for the surgical management of dfo of these bones .
finally , the panelists agreed that a surgeon should consider adjunctive podiatric / orthopedic procedures including adjunctive tendo - achilles lengthening for any patient with a major biomechanical abnormality of the affected forefoot , particularly when these pose increased risk of reulceration or transfer ulcers .
the panelists reached consensus for a total of 10 statements on postoperative management ( table 4 ) .
when delayed primary closure is not possible , the surgeon should use negative pressure wound therapy dressings to develop a wound bed prior to autogenous skin grafting or for delayed secondary wound healing .
absorbent , non - adherent dressings are the preferred alternative when negative pressure dressings can not be used .
offloading was identified as an important component of postoperative management and clinicians should ensure this is provided to all patients who have undergone surgery for dfo . a removable cast walker or posterior splint
was considered the preferred offloading modality , but open - toed shoes with multidensity inserts were considered acceptable for patients with a single toe or dorsal foot wound .
health care providers should make rollator walkers , crutches , or canes available to patients for additional support during ambulation .
when the bone margin specimen shows either histopathological findings compatible with osteomyelitis or pathogenic microorganisms in microbial culture , clinicians should prescribe postoperative antibiotic treatment .
there was a high level of heterogeneity with no consensus on statements describing the timing for the removal of the dressing placed at the time of the initial operation .
similarly , there was no consensus on recommendations on the use of total contact casts or non - removable cast walkers during the postoperative period ( see supplementary file ) .
the panelists reached consensus approval on six statements regarding overall strategies or principles ( table 5 ) .
multidisciplinary / interdisciplinary teams were identified as important in improving treatment outcomes and reducing leg amputation rates .
the importance of establishing a biomechanically stable foot not simply a healed wound was an important goal in the management of forefoot osteomyelitis .
there was agreement that well - established definitions of dfo treatment success and failure are lacking , and that there are no widely accepted guidelines currently available for monitoring postoperative treatment response .
finally , the panelists did not reach consensus on initial statements estimating the major amputation rate associated with dfo in the setting of adequate arterial perfusion and no orthopedic / podiatric hardware when options of < 5% , 510% , and 1020% were provided ; consensus was reached when statements were modified to estimate a major amputation rate of < 10% .
thirteen panelists participated in the initial round . an additional panelist was added after the initial round to represent vascular surgery .
the overall response rate of selected panelists was 95% ( 39 of 41 possible responses ) . at the end of the first round ,
a total of 33 items reached our defined consensus cut - off : 19 items had a mean score of 6.50 . among the items , 16 scored a weighted mean between 6.50 and 6.99 , and we then did an additional round of scoring on these .
after this additional scoring round , we achieved consensus ( mean likert score 7 ) on five more statements , for a total of 38 consensus statements .
these consensus statements included 7 statements concerning the initial diagnosis and selection of patients for operative management , 15 statements about intraoperative management , 10 statements on postoperative management , and 6 general statements on general strategies and principles relevant to surgical management of dfo ( tables 25 ) .
consensus statements for initial diagnosis and selection of patients for operative management of diabetic forefoot osteomyelitis dfo , diabetic foot osteomyelitis .
consensus statements on general strategies and principles relevant to the surgical management of diabetic forefoot osteomyelitis dfo , diabetic foot osteomyelitis .
the consensus panel initially agreed on seven statements relevant to the initial diagnosis of dfo and patient selection ( table 2 ) .
the presence of visible , chronically exposed trabecular bone within a forefoot ulcer was deemed sufficient to establish the diagnosis of dfo . a magnetic resonance imaging ( mri ) or biopsy of a specimen of bone ( for culture or histopathology )
were identified as second - line diagnostic modalities of choice for confirmation of dfo when the diagnosis was otherwise uncertain .
surgical resection of affected bone was recommended when any one of the following is present : systemic toxicity ; an open or infected joint space ; the presence of a prosthetic heart valve ; clinical or imaging evidence of substantial cortical destruction , osteolysis , macroscopic bone fragmentation , or necrotic bone ; or visible , chronically exposed trabecular bone .
no consensus was reached on whether or not the presence of visible cortical bone is sufficient for the diagnosis of dfo or if this should just represent an indication for surgical resection ( see supplementary file ) .
the panel reached consensus on 15 statements regarding specific operative details ( table 3 ) .
the majority agreed that any concomitant deep soft tissue infection should be addressed first , and definitive ( final ) resection of bone should be undertaken 37 days after addressing the soft tissue infection .
there was agreement that negative pressure wound therapy dressings with instillation should be a consideration but is not required for treatment of deep soft tissue infection ( table 3 , also supplementary file ) .
panelists reached consensus that any bone resection should be done with a power saw ( e.g. micro - sagittal or micro - oscillating saw ) ; this should include all areas of necrotic , fragmented bone or areas of significant cortical destruction seen on x - ray , and continue until grossly healthy - appearing bone is seen at the resection margin .
the surgeon should collect a total of four specimens between the initial and final operations : grossly abnormal bone sent for 1 ) histopathology and 2 ) microbiology ( gram - stained smear , aerobic and anaerobic cultures ) and the proximal - most bone resected ( i.e. the bone margin ) for 3 ) histopathology and 4 ) microbiology .
the panel thought that metatarsal head / proximal phalangeal ostectomy was an acceptable alternative to toe / ray amputation for the surgical management of dfo of these bones .
finally , the panelists agreed that a surgeon should consider adjunctive podiatric / orthopedic procedures including adjunctive tendo - achilles lengthening for any patient with a major biomechanical abnormality of the affected forefoot , particularly when these pose increased risk of reulceration or transfer ulcers .
the panelists reached consensus for a total of 10 statements on postoperative management ( table 4 ) .
when delayed primary closure is not possible , the surgeon should use negative pressure wound therapy dressings to develop a wound bed prior to autogenous skin grafting or for delayed secondary wound healing .
absorbent , non - adherent dressings are the preferred alternative when negative pressure dressings can not be used .
offloading was identified as an important component of postoperative management and clinicians should ensure this is provided to all patients who have undergone surgery for dfo . a removable cast walker or posterior splint
was considered the preferred offloading modality , but open - toed shoes with multidensity inserts were considered acceptable for patients with a single toe or dorsal foot wound .
health care providers should make rollator walkers , crutches , or canes available to patients for additional support during ambulation .
when the bone margin specimen shows either histopathological findings compatible with osteomyelitis or pathogenic microorganisms in microbial culture , clinicians should prescribe postoperative antibiotic treatment .
there was a high level of heterogeneity with no consensus on statements describing the timing for the removal of the dressing placed at the time of the initial operation .
similarly , there was no consensus on recommendations on the use of total contact casts or non - removable cast walkers during the postoperative period ( see supplementary file ) .
the panelists reached consensus approval on six statements regarding overall strategies or principles ( table 5 ) .
multidisciplinary / interdisciplinary teams were identified as important in improving treatment outcomes and reducing leg amputation rates .
the importance of establishing a biomechanically stable foot not simply a healed wound was an important goal in the management of forefoot osteomyelitis .
there was agreement that well - established definitions of dfo treatment success and failure are lacking , and that there are no widely accepted guidelines currently available for monitoring postoperative treatment response .
finally , the panelists did not reach consensus on initial statements estimating the major amputation rate associated with dfo in the setting of adequate arterial perfusion and no orthopedic / podiatric hardware when options of < 5% , 510% , and 1020% were provided ; consensus was reached when statements were modified to estimate a major amputation rate of < 10% .
diagnosing and treating dfo is perhaps the most contentious area regarding management of diabetic foot complications ( 8) .
thus , it is not surprising , for example , that the reported percentage of patients with dfo who undergo a surgical procedure ranges from 5% at centers that utilize primary medical management ( 9 ) , to 2040% in centers utilizing a selective surgical approach ( 10 , 11 ) , to 100% in centers that adopt a primarily surgical approach ( 12 ) . while the selection of a primary treatment strategy should continue to be a major focus for clinicians , areas of practice heterogeneity exist throughout all aspects of managing dfo , and
the fact that a wide variety of medical and surgical specialties are involved in its management may contribute to such practice heterogeneity .
a dearth of high - quality evidence assessing various aspects of dfo management may be largely responsible for this practice heterogeneity and uncertainty .
one randomized 52 patients between primary antibiotic therapy versus primary surgical therapy , and found no significant differences in outcomes ( 13 ) .
while it is remarkable that the authors were able to undertake such a trial , the many limitations ( 14 ) of this small trial make it difficult to generalize the results . in another study ,
40 patients treated without surgery randomized to receive either 6 or 12 weeks of antibiotic therapy again had similar outcomes ( 15 ) .
the four published studies that have described the outcomes of primarily surgical therapy were all retrospective single - center studies and enrolled a total of 424 patients ( 4 , 12 , 16 , 17 ) .
good - quality evidence is available to support practices relevant , although not exclusive , to dfo management , such as the randomized trials on various offloading modalities for foot ulcers ( 1821 ) .
furthermore , several authors have published a case series describing good outcomes with surgical techniques that can be applied to appropriately selected patients with dfo ( 2225 ) .
unfortunately , there is very little evidence to support many of the most basic aspects of surgical management , such as how to determine the extent of bone that should be resected determined , whether surgical wounds should be closed or left open , and whether is it useful to obtain a bone margin specimen for culture or histology to guide postoperative management .
we undertook the current study with the aim of providing some starting points from which further discussion and investigation on the optimal management of dfo can proceed .
the expert panel reached high levels of agreement on many statements , particularly in operative and postoperative management of dfo .
in particular , they agreed on several absolute indications for performing surgical resection of bone .
the panel also agreed on the importance of obtaining a total of four bone specimens for histopathological and microbiological processing two from grossly abnormal bone and two from the proximal - most bone margin .
they agreed that delayed primary closure is the preferred wound healing method , in conjunction with procedures to address biomechanical deformities .
if delayed primary closure is not possible , the panelists preferred negative pressure wound therapy or absorbent , non - adherent dressings , followed by autogenous skin grafting .
finally , the panelists reached high levels of consensus on various modalities of offloading footwear .
there was a wide distribution of ratings on several items with ultimately did reach consensus as well as many items which did not reach a consensus . among these areas of uncertainty are some rather fundamental topics .
one was the optimal timing for when to remove the dressing placed at the time of the initial operation .
some surgeons felt strongly that the dressing should be removed within 24 hours , while others felt that it should be left in place for several days after the initial operation .
another was the rate of major ( above - ankle ) amputation that would be expected among patients with dfo and adequate arterial perfusion ; panelists initially split between the original statements that provided the options of < 5% and 510% .
panelists could not reach consensus on the use of total contact casts or non - removable cast walkers .
although these offloading options have been shown to provide significantly better wound healing rates than offloading shoes and removable cast walkers ( 19 , 26 , 27 ) , there was no consensus about their use in the early postoperative period .
our panel agreed that there is no well - established standard definition of treatment success or failure after dfo surgery .
areas of uncertainty include utilization of biomarkers such as ( esr ) erythrocyte sedimentation rate ; and ( crp ) c - reactive protein in establishing diagnosis of dfo ( 28 ) , the value of various methods of establishing the diagnosis of dfo , and additional relative or absolute indications for surgical resection of bone in dfo .
first , not all of the expert members endorse all of the statements that reached consensus . nevertheless , reaching consensus was difficult or impossible unless at least 11 of the 13 members had some level of agreement with any given statement .
conventional delphi methods were employed in this study , with a mean composite score of at least 7.0 ( agree ) required to reach consensus .
furthermore , the statements reaching consensus represent no more than statements of opinion on which there were high levels of agreement among surgeons with experience and expertise in the field .
of course , these statements are by no means definitive , and in some cases are not even supported by previously reported data .
second , it is our intention that these statements provide guidance on the management of most cases of forefoot dfo not associated with peripheral arterial disease or orthopedic / podiatric hardware .
not all statements can be followed in all patients , and clinical judgment should be combined with a deep understanding of a specific patient 's dfo episode when managing individual cases .
the management of peripheral arterial disease requires consultation with specialty providers ( vascular surgeons , angiologists , interventional cardiologists , or radiologists ) in adherence to societal guidelines where appropriate ( 5 ) .
the management of midfoot and heel osteomyelitis , as well as the management of osteomyelitis associated with orthopedic / podiatric hardware , can be very challenging and should involve orthopedic or podiatric surgeons with specific expertise in these areas . in summary , we identified statements on the perioperative and postoperative management of dfo on which a panel of expert surgeons from various disciplines reached consensus . we hope that these statements may help share or even establish
best practices that can be applied in appropriate clinical situations pending the publication of more high - quality data .
we will be waiting with interest to see if those data provide evidence to support or refute our consensus statements on various practices within dfo management . | backgroundthe aim of this study was to develop consensus statements that may help share or even establish
best practices in the surgical aspects of managing diabetic foot osteomyelitis ( dfo ) that can be applied in appropriate clinical situations pending the publication of more high - quality data.methodswe asked 14 panelists with expertise in dfo management to participate .
delphi methodology was used to develop consensus statements .
first , a questionnaire elicited practices and beliefs concerning various aspects of the surgical management of dfo .
thereafter , we constructed 63 statements for analysis and , using a nine - point likert scale , asked the panelists to indicate the extent to which they agreed or disagreed with the statements .
we defined consensus as a mean score of greater than 7.0.resultsthe panelists reached consensus on 38 items after three rounds . among these , seven provide guidance on initial diagnosis of dfo and selection of patients for surgical management .
another 15 statements provide guidance on specific aspects of operative management , including the timing of operations and the type of specimens to be obtained .
ten statements provide guidance on postoperative management , including wound closure and offloading , and six statements summarize the panelists agreement on general principles for surgical management of dfo.conclusionsconsensus statement on the perioperative management of dfo were formed with an expert panel comprised of a variety of surgical specialties .
we believe these statements may serve as
best practice guidelines until properly performed studies provide more robust evidence to support or refute specific surgical management steps in dfo . | Methods
Selection of topics and participants
Delphi technique
Results
Response rate and number of rounds
Summary of statements reaching consensus
Discussion
Supplementary Material |
enterococcus has emerged as a nosocomial pathogen in the last two decades , causing urinary tract infections , genital tract infections , and endocarditis due to its colonizing capacity and multidrug resistance [ 1 , 2 ] . the emergence of multidrug resistant enterococci to commonly used antibiotics , for example , aminoglycosides and cephalosporins , is because of their ability to attain and transfer the drug resistance gene , giving rise to enterococci with high level aminoglycoside ( hlar ) and glycopeptide resistance .
however , combinations of penicillins with aminoglycosides are synergistically bactericidal against enterococci in vitro and are effective in treating severe enterococcal endocarditis .
the mechanism of this synergy has been explained by the enhanced uptake of aminoglycosides in the presence of penicillins or other agents which inhibit cell wall synthesis .
an increased frequency of high level resistance to aminoglycoside antibiotics ( mic > 8,000 g / ml ) in clinical isolates of enterococci has been reported which were also resistant to synergism with the penicillins . this study was aimed at determining the antibiotic susceptibility of enterococci isolated from various clinical samples with reference to aminoglycoside and vancomycin .
so if the knowledge of hlar prevalence is available , clinicians can prescribe the various drug combination ( cell wall inhibitor + aminoglycosides ) at the very beginning of treatment avoiding the unnecessary usage of other antimicrobials .
this study was conducted on 100 isolates of enterococcus spp . from various clinical specimens including urine , pus , blood , semen , vaginal swab , and throat swab during a period of six months from july
the samples were processed immediately after collection and enterococcus isolates were identified by standard protocols based on gram 's staining , colony morphology , catalase test , bile solubility , growth in sodium chloride , bile esculin test , and sugar fermentation tests .
antibiotic sensitivity testing of enterococci was performed using kirby - bauer disc diffusion method and mueller - hinton agar supplemented with 5% sheep blood was used as per clsi guidelines .
the antibiotics discs used were penicillin ( 10 u ) , gentamicin ( 10 g ) , ciprofloxacin ( 5 g ) , linezolid ( 30 g ) , vancomycin ( 30 g ) , erythromycin ( 15 g ) , and doxycycline ( 30 g ) , and nitrofurantoin ( 300 g ) was also added in urinary isolates .
, high level ( 120 g ) gentamicin and streptomycin ( 300 g ) discs were placed on the agar medium .
plates were incubated at 37c for 24 hours , and diameter of zone of inhibition was measured .
resistance was indicated by no zone and susceptibility by a zone of diameter 10 mm .
the test was quality controlled using e. faecalis atcc 29212 ( susceptible ) and e. faecalis atcc 51299 ( resistant ) .
vancomycin resistance was determined by using e - strip ( hi - media ) on blood agar for those isolates which were resistant by disc diffusion test .
out of a total of 100 isolates , 41% from urine and semen and 25% from blood followed by 19% from pus and 8% from body fluid drains were included in the study .
the majority of clinical samples from which enterococcus spp . were recovered were from indoor ( 60% ) in comparison to outdoor ( 40% ) patients .
enterococcus isolates from blood samples were resistant to penicillin ( 64% ) , and urinary isolates were resistant to fluoroquinolones ( 53.6% ) .
commonly used antimicrobials were found to be sensitive in enterococcus spp . recovered from vaginal swab samples ( table 1 ) .
high level gentamicin resistance ( hlgr ) was more common in urine samples ( 41.5% ) followed by blood ( 36% ) samples .
high level streptomycin resistance ( hlsr ) was more common in pus samples ( 52.6% ) followed by blood samples ( 36% ) ( table 2 ) . in table 3 , more resistance to nitrofurantoin in enterococcus isolates from urine samples was not noticed in indoor ( 17% ) patients in comparison to opd ( 7.3% ) .
similarly resistant to glycopeptides ( vancomycin ) , fluoroquinolones were more common in hospitalized patients . also , hlar was more common in indoor ( 39% ) versus outdoor patients ( 25% ) ( table 3 ) .
it was more common in ipd patients ( 28% ) as compared to opd patients ( 4% ) .
high level aminoglycoside resistance was more common in vancomycin resistant enterococci ( vre ) isolates than vancomycin sensitive enterococci ( vse ) isolates .
five out of nine of the enterococcus isolates resistant to vancomycin by disk diffusion test were found resistant to these glycopeptides by e - strip test ( table 4 ) .
over the last few years , they have become important nosocomial pathogens probably due to inherent resistance to antibiotics ( cephalosporins ) , ability to adhere to indwelling medical devices , and ability to survive in adverse environmental conditions .
antimicrobial resistance in enterococcus has been increasing mainly in hospitalized patients [ 911 ] . in our study , out of the total 100 enterococcus isolates from different clinical specimens , the majority were recovered from hospitalized ( 60% ) patients in comparison to outdoor ( 40% ) patients , similar to other studies .
a combination of penicillin and aminoglycosides is considered as treatment of choice for enterococcal infections ; therefore , resistance against these antibiotics has important clinical results effecting therapeutic prognosis . in the present study
, enterococcus isolates from blood samples were found to be penicillin resistant in 64% strains ( 16/25 = 64% ) ( mic ranges from 16 to 32 g / ml ) , which could be due to resistance mechanism involving low affinity penicillin binding proteins or production of lactamases . in this study ,
prevalence of drug resistance to various antibiotics was as follows : ciprofloxacin ( 25% ) , penicillin ( 66.67% ) , and nitrofurantoin in urinary isolates ( 24.3% ) .
one isolate of enterococcus spp . was found to be resistant to linezolid having inhibitory zone of diameter less than 15 mm .
this type of antibiogram has been documented in earlier studies also [ 1214 ] . here a low prevalence of fluoroquinolone ( 25% ) and other antibiotic resistance was found in comparison to other studies , 72% and 62% , respectively , which could be due to very precise and judicious use of this antimicrobial in our institute .
the present study demonstrated high prevalence of hlar ( gentamicin and streptomycin ) 29% and 35% , respectively .
hlgr was more common in urine samples ( 41.5% ) followed by blood ( 36% ) samples .
these findings are also reported in some studies . however , a higher and lower prevalence level of hlgr and hlsr have been reported in few studies , respectively [ 13 , 14 , 16 ] . in our study ,
hlar to both gentamicin and streptomycin was found in 22% isolates , specifically more in blood isolates .
hlar in these enterococcal strains nullify the efficacy of beta lactam and aminoglycoside combination therapy .
therefore , differentiation of hlar from simple intrinsic resistance is important and should be adopted as a part of routine microbiology laboratories . in this study
, it was found that hlar was more common in ipd ( 28% ) as compared to opd patients ( 4% ) , similar to other studies .
vancomycin resistance was found in nine isolates of enterococcus by disc diffusion method ; out of nine isolates five isolates were confirmed as vre on e - strip test having mic ( > 64 gm / ml ) . in india , the prevalence of vre has been reported to be between 0 and 30 per cent [ 1722 ] . in the present study , resistance to vancomycin was maximum in blood isolates , that is , 16.25% ( 4/25 ) , with more prevalence in indoor patients ( table 4 ) .
it could be explained by the facts that in hospitalized patients use of broad spectrum antimicrobials is common practice and it leads to colonization pressure for selection of vancomycin resistance strains .
it may increase the risk of cross - infection among hospitalized patients via staff members and environmental contamination with vre strains .
out of four vre strains , three were found to be sensitive to higher concentration of either of or both the aminoglycosides ( table 5 ) .
so the combination of the higher level aminoglycoside with cell wall inhibitor can be considered for treatment of vre infection after antibiotic susceptibility testing .
control efforts . due to lack of effective therapy for multiple antibiotic resistant enterococcal infection , prevention of the dissemination of these strains
. a very precise use of antimicrobials , for example , cephalosporins , and anti aerobic drugs should be in practice .
there are certain recommendations to reduce the cross - contamination by these organisms which include surveillance for colonization , identification of colonized and infected patients with their isolation , the use of gowns and gloves by health staff ( barrier method ) , hand washing with an antiseptic after gloves removal , and avoiding contact with environmental surfaces after gloves removal .
medical equipment , for example , stethoscopes and blood pressure cuffs , must be dedicated to hlr patients .
environmental decontamination is also required with effective disinfectants ( isopropyl alcohol , hypochlorite , and phenolic and quaternary ammonium salts ) [ 2629 ] .
deficiency of effective antimicrobial therapy and control measure for prevention of dissemination for multiple drug resistant enterococci are among the major factors for increasing prevalence of vre and hlar .
thus , it becomes important for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated .
it becomes equivalently important that clinicians who are in direct contact with patients should go primarily for first / low generation of antibiotics for simple infections , for example , sore throat , rather than switching to higher class of antimicrobials . |
aims . this study was aimed at knowing the prevalence of vancomycin and high level aminoglycoside resistance in enterococcal strains among clinical samples .
study design .
it was an investigational study .
place and duration of study .
it was conducted on 100 enterococcus isolates , in the department of microbiology , pt .
bds pgims , rohtak , over a period of six months from july to december 2014 . methodology .
clinical specimens including urine , pus , blood , semen , vaginal swab , and throat swab were processed and enterococcus isolates were identified by standard protocols .
antibiotic sensitivity testing of enterococci was performed using kirby - bauer disc diffusion method .
results . high level gentamicin resistance ( hlgr ) was more common in urine samples ( 41.5% ) followed by blood ( 36% ) samples .
high level streptomycin resistance ( hlsr ) was more common in pus samples ( 52.6% ) followed by blood samples ( 36% ) .
resistance to vancomycin was maximum in blood isolates .
conclusion .
enterococci resistant to multiple antimicrobial agents have been recognized .
thus , it is crucial for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion |
when cryosurgery has to be done for deeply embedded lesions , the freezing time has to be increased proportionately .
this freezes an area beyond the lesion creating a large halo of frozen tissue around the lesion , which results in painful hemorrhagic blistering and longer healing time . in case of verrucae
, there may be spread of the warts to the periphery and normal tissue after the healing of the blister .
various methods of targeted cryotherapy are available such as the cotton tip application , use of needles or smaller nozzles to create a small jet of spray and use of plastic cone ( otoscope specula ) to restrict the lateral spread of cryogen as it is being sprayed
this innovation describes the use of the simple disposable biopsy punch to deliver targeted cryotherapy .
what is unique about the use of the biopsy punch is that it not only prevents lateral spread but as the cryogen passes through the punch , the punch starts to sink into the lesion delivering cryotherapy to deeper parts of the lesion , which is not possible with any of the above methods .
punches of different sizes , according to the size of the lesion [ figure 1a]test tube holder [ figure 1b]cryocan and liquid nitrogen [ figure 1c]gauzesurgical blade no . 15 with blade holder for paring in case of warts .
punches of different sizes , according to the size of the lesion [ figure 1a ] test tube holder [ figure 1b ] cryocan and liquid nitrogen [ figure 1c ] surgical blade no . 15 with blade holder for paring in case of warts .
different sized punches are cut to half for treating plantar warts , at first paring is done to reduce the thickness of the lesion and determine the size of the wart .
the punch is cut to half the length , and the plastic part is covered with gauze . for smaller punches ,
the punch is kept on the lesion with slight pressure so that there is no gap between the punch metal tip and the skin .
note that when the punch is cut , the edge should be smooth and flat so that the nozzle sits perfectly on it .
the liquid nitrogen is passed , and as the liquid nitrogen spray touches the lesion through the punch , the metal tip of the punch also gets cooled , which then slowly starts sinking into the lesion [ figure 2 ] . for the treatment of warts ,
freezing is continued till the bevelled edge of the punch enters the skin . when deeper cryotherapy is required , the punch is driven further inside the lesion .
this helps in deeper delivery of the cryogen and increases efficacy , with least damage to the surrounding tissue .
the punch need not be rotated to cut into the lesion ; rather the effect of cooling itself makes the punch cold cut into the lesion [ video 1 online at www.jcasonline.com ] .
the nozzle of cryocan kept onto the top of the punch the punches remain embedded till they are frozen [ figure 3 ] .
the punches fall off on their own in 10 - 20 seconds depending on the depth reached .
the next punch can be placed adjacent to the previous punch and the process is repeated .
the procedure can be performed two or three times on the same lesion depending on its size . in keloids ,
this technique is useful even in larger lesions like keloid where it can be sprayed into multiple areas with less pain and damage to the tissues . following the procedure ,
blister occurs , which is very rare , the fluid collected can be expressed out with a needle or syringe .
two punches are placed one beside the other this technique can be used for the treatment of verrucae , particularly plantar warts [ figure 4a and b ] , dermatofibroma , pyogenic granuloma , keloids and hypertrophic scars , cherry angioma and nevi .
( a ) before cryo paring is done to determine the size of punches , ( b ) after two weeks the paring is done to determine the reduction in the warts . in this case complete recovery
is noted there is targeted damage to the unwanted tissue , sparing collateral damage to the surrounding skin .
deeper delivery of the cryogen is possible by use of punches that slide deeper into the lesions as the cryosurgery is being performed .
uniform temperature at the depth can be maintained , unlike other methods , where optimal temperature is reduced with increasing depth .
punches of different sizes , according to the size of the lesion [ figure 1a]test tube holder [ figure 1b]cryocan and liquid nitrogen [ figure 1c]gauzesurgical blade no . 15 with blade holder for paring in case of warts .
punches of different sizes , according to the size of the lesion [ figure 1a ] test tube holder [ figure 1b ] cryocan and liquid nitrogen [ figure 1c ] surgical blade no . 15 with blade holder for paring in case of warts .
for treating plantar warts , at first paring is done to reduce the thickness of the lesion and determine the size of the wart .
the punch is cut to half the length , and the plastic part is covered with gauze . for smaller punches ,
the punch is kept on the lesion with slight pressure so that there is no gap between the punch metal tip and the skin .
note that when the punch is cut , the edge should be smooth and flat so that the nozzle sits perfectly on it .
the liquid nitrogen is passed , and as the liquid nitrogen spray touches the lesion through the punch , the metal tip of the punch also gets cooled , which then slowly starts sinking into the lesion [ figure 2 ] . for the treatment of warts ,
freezing is continued till the bevelled edge of the punch enters the skin . when deeper cryotherapy is required , the punch is driven further inside the lesion .
this helps in deeper delivery of the cryogen and increases efficacy , with least damage to the surrounding tissue .
the punch need not be rotated to cut into the lesion ; rather the effect of cooling itself makes the punch cold cut into the lesion [ video 1 online at www.jcasonline.com ] .
the nozzle of cryocan kept onto the top of the punch the punches remain embedded till they are frozen [ figure 3 ] .
the punches fall off on their own in 10 - 20 seconds depending on the depth reached .
the next punch can be placed adjacent to the previous punch and the process is repeated .
the procedure can be performed two or three times on the same lesion depending on its size . in keloids ,
this technique is useful even in larger lesions like keloid where it can be sprayed into multiple areas with less pain and damage to the tissues . following the procedure ,
blister occurs , which is very rare , the fluid collected can be expressed out with a needle or syringe .
this technique can be used for the treatment of verrucae , particularly plantar warts [ figure 4a and b ] , dermatofibroma , pyogenic granuloma , keloids and hypertrophic scars , cherry angioma and nevi .
( a ) before cryo paring is done to determine the size of punches , ( b ) after two weeks the paring is done to determine the reduction in the warts . in this case
there is targeted damage to the unwanted tissue , sparing collateral damage to the surrounding skin . pain and blistering reaction
deeper delivery of the cryogen is possible by use of punches that slide deeper into the lesions as the cryosurgery is being performed .
uniform temperature at the depth can be maintained , unlike other methods , where optimal temperature is reduced with increasing depth .
. the biopsy punch should be in complete contact with the skin to avoid leakage from the edges that this can lead to unwanted side effects like blisters in the surrounding normal skin .
using the simple disposable biopsy punch for targeted cryotherapy , reduces side effects and delivers optimum temperatures in the depth of the lesion . | cryotherapy is a commonly used office procedure that causes destruction of tissue by cryonecrosis due to rapid freezing and thawing of cells .
the limitation in treating plantar warts and deeper dermal lesions is that the freeze time should be longer to penetrate deeper , which results in collateral damage to normal skin surrounding the lesion .
this results in unwanted side effects of prolonged pain , blistering and haemorrhage and increased healing time .
the cone spray technique was used to reduce collateral damage , but deeper penetration is difficult to achieve .
an innovative technique using disposable biopsy punches is described that ensures deeper freezing as compared to the plastic cone .
the metal cutting edge of the punch enters deeper into the lesions as the liquid nitrogen is passed , sparing damage to surrounding skin . | BACKGROUND
INNOVATION
Instruments
Technique
Indications
Advantages of targeted cryotherapy
LIMITATIONS
CONCLUSION |
taiwan had 468 hospitals in 2003 , 393 ( 83.9% ) private and 75 ( 16.1% ) public .
data were collected from emergency departments in which the number of patients exceeded 500 per month .
the data collected included the following : accreditation of the hospitals , the average monthly volume of emergency department patients from march to may 2003 , the effects of the sars epidemic on emergency department workers , types of ppe supplied , kinds of icms implemented , as well as timing and origin of instruction to use ppe and icms during the sars epidemic . before data analyses , hospitals were classified into 3 levels , medical center ( level a ) , regional hospital ( level b ) , or local hospital ( level c ) .
in general , the number of hospital beds at these hospitals , based on reference data , was > 500 in level a , 200500 in level b , and < 200 in level c. the average monthly volume of emergency department patients was an average of the monthly emergency department volumes during the 3-month period .
the effects on emergency department staff were measured according to a 4-point scale , as follows : 1 ) had fever and needed to stay at home , 2 ) quarantined at home with fever or no fever , 3 ) quarantined at hospital , and 4 ) probably had sars .
the basic ppe recommended by the doh included head and shoe covers , goggles , face shield , gloves , apron , disposable gown , surgical mask , and n95 respiratory mask .
the high - level protective respirators were defined as p100/ n100/ ffp3 ( approved by the national institute for occupational safety and health [ niosh ] ) and powered air - purifying respirators with full - body isolation suit .
icms included the guidelines from the doh ( defined as basic icms ) as well as additional measures , such as having a fever triage ward or referral to a sars screening team , implemented by emergency departments during the sars outbreak .
the timing of complete ppe implementation or having icms completely in place was classified into 2 stages : 1 ) early stage , from march to late april , 2 ) late stage , from late april to mid - june .
the order to use ppe or icms came from 3 sources : 1 ) emergency department workers themselves , 2 ) emergency department administrators , and 3 ) hospital administrators .
the difficulty of implementing or instituting icms was rated on a scale from 1 ( mildly difficult ) to 5 ( very difficult ) .
the effectiveness of implementing icms was rated on a scale from 1 ( less effective ) to 5 ( very effective ) .
one - way analysis of variance ( anova ) was used to test the differences in the average monthly volumes of patients among different levels and types of emergency departments .
the associations between categorical variables were analyzed by chi - square and fisher exact test .
the rating scale of difficulty and effectiveness of icms was represented by median and interquartile range ( iqr ) .
the correlation between difficulty and effectiveness was analyzed by spearman rank correlation ; p values < 0.05 were considered significant .
a total of 213 emergency departments were initially included in this study ; 152 ( 71.4% ) were private hospitals and 61 ( 28.6% ) were public hospitals .
one hundred emergency departments responded to the questionnaire ( respondent rate = 46.9% ) . among these ,
15 emergency departments were medical centers ( respondent rate = 65.2% , including 6 public and 9 private emergency departments ) , 28 emergency departments were regional hospitals ( respondent rate = 38.9% , including 10 public and 18 private ) , and 57 emergency departments ( respondent rate = 44.9% , including 14 public and 43 private ) were local hospitals .
the emergency department volumes and assessment of the effects of the sars outbreak on emergency department workers are shown in table 1 . from march to may 2003 , the average monthly volume of emergency department patients in level a hospitals was 6,200 ( range 3,42911,080 ) and 3,828 ( range 1,8645,770 ) in level b hospitals , both of which were significantly larger than the average number of patients in level c hospitals ( average 2,246 , range 7293,236 ) ( p = 0.001 ) .
no significant differences in emergency department volume were found between public ( average 2,642 , range 1,3646,258 ) and private hospitals ( average 3,398 , range 72911,080 ) .
the most frequent effect of the sars outbreak on emergency department workers was " fever and needed to stay at home . "
emergency department workers in level a and b hospitals had a higher probability of being affected during the sars outbreak , regardless of job type .
when the effects of the sars outbreak on public and private hospitals were compared , significant differences were found between the type of quarantine at hospitals .
represents the number of eds that responded " yes " to at least 1 ed staff member in a given category of possible sars impact .
the number in parentheses is the percentage of the total eds in a particular hospital level or type .
the use of basic ppe did not differ significantly among emergency departments at different hospital levels
. however , level a emergency departments used high grade ppe ( p100/n100/ffp3 or powered air - purifying respirator ) more often than emergency departments at level b and c hospitals .
however , in terms of additional icms , emergency departments of level c hospitals used more restrictive measures when transferring patients in and out .
the use of icms in public and private hospital was significantly different in patients who were transferred out .
eighty percent ( 80/100 ) of hospitals completely implemented use of ppe , and 66% ( 66/100 ) of hospitals implemented their icms at the late stage of the sars outbreak .
the instruction to use ppe originated from emergency department managers in 60% of level a , 46% of level b , and 23% of level c hospitals .
the order to implement icms came from hospital managers in 33% of level a , 50% of level b , and 62% of level c hospitals . *
ed , emergency department ; sars , severe acute respiratory syndrome . * sars , severe acute respiratory syndrome ; ppe , personal protective equipment ; icm , infection control measures ; ed , emergency department .
table 5 shows the rating scales and correlations of difficulty and effectiveness of icms in emergency departments . of the basic icms , instituting isolation at emergency departments was thought to be the most difficult ( median = 3 score ) .
the effectiveness of additional icms was also thought to be less than that of basic icms .
significant negative correlations were seen between perceived difficulty and effectiveness of implemented icms , except for body temperature at admission , institution of a fever screening station , and closure of the emergency department .
* sars , severe acute respiratory syndrome ; iqr , interquartile range ; ed , emergency department .
in our study , emergency department workers in larger hospitals were more severely affected by the sars outbreak than staff at smaller hospitals .
first , as our data showed , emergency departments at larger hospitals tend to have more patients requiring emergency services than those at smaller emergency departments .
thus , overcrowding and more frequent contact with patients would increase the incidence of person - to - person transmission .
emergency department workers may have become infected even without contact with a hospitalized sars patient ( 14 ) .
persons living in urban areas may be more likely to travel overseas , which would increase their chances of contracting an infectious disease .
a similar phenomenon was reported at hospitals in cities with a high population density , such as beijing , hong kong , singapore , and toronto ( 1419 ) .
third , fever patients tended to visit larger hospitals in the belief that they would be able to see a specialist who could identify the fever source . finally , emergency departments at larger hospitals had more patients transferring in , so some patients with fever of unknown origin may have been transferred from lower level hospitals . in taiwan ,
emergency department volume does not always correlate well with either hospital bed number or with hospital location ( such as urban versus .
this lack of correlation may explain , in part , the large variation in numbers of emergency department patients in level a hospitals . during the sars outbreak ,
therefore , preventing outbreaks at larger hospitals during an epidemic of an emerging disease is essential . in our analysis
, most of the emergency departments in taiwan followed the guidelines for basic ppe provided by the doh .
some hospitals did not use these basic recommended ppe because they already had other ppe that performed the same function .
in fact , a substantial problem for hospitals during the sars epidemic was the cost of basic ppe , such as surgical masks and n95 respiratory masks , which increased costs markedly during this period .
the quantity of ppe required by larger hospitals was very large , which placed a financial hardship on these hospitals , even though many of them did not encounter any sars cases .
some hospitals were so anxious to acquire sufficient basic ppe that they even requested recycled ppe if it was available .
the supply of higher grade respirators was greater at larger hospitals than at smaller hospitals .
this finding may have been because hospital outbreaks were generally more common at larger hospitals and more patients who needed emergency resuscitation were transferred to larger hospitals .
transfers inevitably increased the risk for transmission to emergency workers ( 14 ) . in our analysis , most hospitals implemented basic icms during the sars epidemic , but smaller emergency departments more frequently used more restrictive icms . paradoxically , smaller hospital appeared to be more alert to the emerging disease , although the number of emergency department patients was lower than in larger emergency departments .
this may have been because smaller hospitals were aware that they lacked the ability and capacity to treat sars patients and therefore implemented additional icms to prevent an outbreak .
smaller hospitals were more likely than larger hospitals to restrict the patients from being transferred in than to transfer out suspected case - patients during the epidemic . placing the suspected sars patients in an isolation room
however , most hospitals , both public and private , found this a considerable challenge ( 2022 ) .
lack of isolation rooms became the key reason for transferring patients out and restricting the transfer of patients in . because of the difficulty of isolating all suspected patients , implementing additional icms became the best strategy for most emergency departments .
this strategy may have resulted in more patients with fever being transferred to a large hospital , thereby exposing these hospitals to a high risk of an outbreak .
an inadequate number of isolation rooms will still be a problem in the next large - scale epidemic . because the effects of sars on the healthcare system were unknown in early stages , most hospitals had no clearly defined response plan and were unsure when to implement icms . in our analysis ,
use of ppe or icms in the emergency department usually began at the outset of the epidemic ( outbreak at hospital a ) .
the attitude of most hospital administrators was to keep an eye on the situation , especially in private hospitals .
this attitude was similar to that of health policymakers in the initial stage who were concerned that the sars epidemic would have a devastating effect on the nation 's economy and would cause widespread panic .
few hospitals actually prepared an infection response plan in the early stage that included the preparation of ppe and design of icms .
some hospitals did not even begin to consider how to implement these measures until they were directly facing the sars epidemic .
ppe and icms were a financial hardship for private hospitals ( > 80% of hospitals in taiwan ) .
the reimbursement for private hospitals comes from the bureau of national health insurance , depending on what services are provided . during the sars epidemic ,
the overall volume of patients decreased , which affected the income of emergency departments and hospitals ( 23 ) . in a future epidemic , without immediate government assistance at the crucial early stage , the effectiveness of hospitals ' response will be reduced . implementing basic icms was easier and more effective than implementing additional icms .
most of the basic icms were directly ordered by the doh , so hospitals were required to fully support the emergency departments .
this fact may explain why these basic icms were rated low in difficulty to implement and thought to have high effectiveness . whether additional icms protected smaller hospitals is unclear , but
these measures did appear to decrease the risk of an outbreak in lower level hospitals .
this finding may have been because physicians were required to spend more time communicating with patients or with outside hospitals , which also had the effect of causing the number of complaints and disagreements between physicians and patients to rise .
some of these additional icms will place a great strain on the healthcare system and render it incapable of functioning normally .
early recognition and rapid initiation of infection control precautions are the most important strategies for controlling large - scale infectious disease outbreaks ( 24 ) . if recognizing a new or large - scale contagious infectious disease in the early stage is not possible , implementing additional icms in hospitals , especially smaller hospitals , may be unavoidable because the first priority for hospital managers is to prevent a hospital outbreak . to avoid disrupting the healthcare system when additional icms are implemented
, the doh should do its utmost to provide full financial support and other assistance .
if equal support from the doh for all hospitals is not practical , a centralized system for suspected patients may be considered as a strategy to reduce the severity and extent of an epidemic .
this strategy may decrease the high incidence of person - to - person transmission in larger hospitals and may enhance the ability of smaller hospitals to treat patients with suspected cases . implementing a centralized system of quarantine is controversial , however , because ethical issues are involved ( 25 ) .
the response rate of the present study was low , so results may have been affected by nonresponse bias .
the nonresponding emergency departments may have had less effective systems in place when they responded to the sars epidemic .
in addition , little seasonal variation in emergency department volume occurs in taiwan , and any seasonal variation in the 3-month period was likely to be negligible .
the findings of this study suggest that policymakers should understand the different abilities of hospitals to respond to an epidemic .
in addition , support and control measures should be implemented more effectively and made immediately available to all hospitals , whether public or private .
understanding the ability and capacity of different hospitals to respond to a contagious disease will enable policymakers to design effective infection control measures to safeguard the health of the nation .
in our study , emergency department workers in larger hospitals were more severely affected by the sars outbreak than staff at smaller hospitals .
first , as our data showed , emergency departments at larger hospitals tend to have more patients requiring emergency services than those at smaller emergency departments .
thus , overcrowding and more frequent contact with patients would increase the incidence of person - to - person transmission .
emergency department workers may have become infected even without contact with a hospitalized sars patient ( 14 ) .
persons living in urban areas may be more likely to travel overseas , which would increase their chances of contracting an infectious disease .
a similar phenomenon was reported at hospitals in cities with a high population density , such as beijing , hong kong , singapore , and toronto ( 1419 ) .
third , fever patients tended to visit larger hospitals in the belief that they would be able to see a specialist who could identify the fever source . finally , emergency departments at larger hospitals had more patients transferring in , so some patients with fever of unknown origin may have been transferred from lower level hospitals . in taiwan ,
emergency department volume does not always correlate well with either hospital bed number or with hospital location ( such as urban versus .
this lack of correlation may explain , in part , the large variation in numbers of emergency department patients in level a hospitals . during the sars outbreak ,
therefore , preventing outbreaks at larger hospitals during an epidemic of an emerging disease is essential .
in our analysis , most of the emergency departments in taiwan followed the guidelines for basic ppe provided by the doh .
some hospitals did not use these basic recommended ppe because they already had other ppe that performed the same function .
in fact , a substantial problem for hospitals during the sars epidemic was the cost of basic ppe , such as surgical masks and n95 respiratory masks , which increased costs markedly during this period .
the quantity of ppe required by larger hospitals was very large , which placed a financial hardship on these hospitals , even though many of them did not encounter any sars cases .
some hospitals were so anxious to acquire sufficient basic ppe that they even requested recycled ppe if it was available .
the supply of higher grade respirators was greater at larger hospitals than at smaller hospitals .
this finding may have been because hospital outbreaks were generally more common at larger hospitals and more patients who needed emergency resuscitation were transferred to larger hospitals .
transfers inevitably increased the risk for transmission to emergency workers ( 14 ) . in our analysis , most hospitals implemented basic icms during the sars epidemic , but smaller emergency departments more frequently used more restrictive icms . paradoxically , smaller hospital appeared to be more alert to the emerging disease , although the number of emergency department patients was lower than in larger emergency departments .
this may have been because smaller hospitals were aware that they lacked the ability and capacity to treat sars patients and therefore implemented additional icms to prevent an outbreak .
smaller hospitals were more likely than larger hospitals to restrict the patients from being transferred in than to transfer out suspected case - patients during the epidemic . placing the suspected sars patients in an isolation room
was recommended . however , most hospitals , both public and private , found this a considerable challenge ( 2022 ) .
lack of isolation rooms became the key reason for transferring patients out and restricting the transfer of patients in .
because of the difficulty of isolating all suspected patients , implementing additional icms became the best strategy for most emergency departments .
this strategy may have resulted in more patients with fever being transferred to a large hospital , thereby exposing these hospitals to a high risk of an outbreak .
an inadequate number of isolation rooms will still be a problem in the next large - scale epidemic .
because the effects of sars on the healthcare system were unknown in early stages , most hospitals had no clearly defined response plan and were unsure when to implement icms . in our analysis ,
use of ppe or icms in the emergency department usually began at the outset of the epidemic ( outbreak at hospital a ) .
the attitude of most hospital administrators was to keep an eye on the situation , especially in private hospitals .
this attitude was similar to that of health policymakers in the initial stage who were concerned that the sars epidemic would have a devastating effect on the nation 's economy and would cause widespread panic .
few hospitals actually prepared an infection response plan in the early stage that included the preparation of ppe and design of icms .
some hospitals did not even begin to consider how to implement these measures until they were directly facing the sars epidemic .
ppe and icms were a financial hardship for private hospitals ( > 80% of hospitals in taiwan ) .
the reimbursement for private hospitals comes from the bureau of national health insurance , depending on what services are provided . during the sars epidemic ,
the overall volume of patients decreased , which affected the income of emergency departments and hospitals ( 23 ) . in a future epidemic , without immediate government assistance at the crucial early stage , the effectiveness of hospitals ' response will be reduced .
most of the basic icms were directly ordered by the doh , so hospitals were required to fully support the emergency departments .
this fact may explain why these basic icms were rated low in difficulty to implement and thought to have high effectiveness .
whether additional icms protected smaller hospitals is unclear , but these measures did appear to decrease the risk of an outbreak in lower level hospitals . however , difficulty and effectiveness of icms had a significant negative correlation .
this finding may have been because physicians were required to spend more time communicating with patients or with outside hospitals , which also had the effect of causing the number of complaints and disagreements between physicians and patients to rise .
some of these additional icms will place a great strain on the healthcare system and render it incapable of functioning normally . early recognition and rapid initiation of infection control precautions are the most important strategies for controlling large - scale infectious disease outbreaks ( 24 ) . if recognizing a new or large - scale contagious infectious disease in the early stage is not possible , implementing additional icms in hospitals , especially smaller hospitals , may be unavoidable because the first priority for hospital managers is to prevent a hospital outbreak . to avoid disrupting the healthcare system when additional icms are implemented
, the doh should do its utmost to provide full financial support and other assistance .
if equal support from the doh for all hospitals is not practical , a centralized system for suspected patients may be considered as a strategy to reduce the severity and extent of an epidemic .
this strategy may decrease the high incidence of person - to - person transmission in larger hospitals and may enhance the ability of smaller hospitals to treat patients with suspected cases . implementing a centralized system of quarantine is controversial , however , because ethical issues are involved ( 25 ) .
the response rate of the present study was low , so results may have been affected by nonresponse bias .
the nonresponding emergency departments may have had less effective systems in place when they responded to the sars epidemic .
in addition , little seasonal variation in emergency department volume occurs in taiwan , and any seasonal variation in the 3-month period was likely to be negligible .
the findings of this study suggest that policymakers should understand the different abilities of hospitals to respond to an epidemic .
in addition , support and control measures should be implemented more effectively and made immediately available to all hospitals , whether public or private .
understanding the ability and capacity of different hospitals to respond to a contagious disease will enable policymakers to design effective infection control measures to safeguard the health of the nation . | how emergency departments of different levels and types cope with a large - scale contagious infectious disease is unclear .
we retrospectively analyzed the response of 100 emergency departments regarding use of personal protective equipment ( ppe ) and implementation of infection control measures ( icms ) during the severe acute respiratory syndrome outbreak in taiwan .
emergency department workers in large hospitals were more severely affected by the epidemic .
large hospitals or public hospitals were more likely to use respirators .
small hospitals implemented more restrictive icms .
most emergency departments provided ppe ( 80% ) and implemented icms ( 66% ) at late stages of the outbreak .
instructions to use ppe or icms more frequently originated by emergency department administrators .
the difficulty of implementing icms was significantly negatively correlated with their effectiveness .
because ability to prepare for and respond to emerging infectious diseases varies among hospitals , grouping infectious patients in a centralized location in an early stage of infection may reduce the extent of epidemics . | Materials and Methods
Results
Discussion
Impact of SARS on Emergency Departments in Larger Hospitals
Use of High-Grade PPE and Additional ICMs
Use of PPE and ICMs in Late Stage of Epidemic
Implementing Additional ICMs |
aortic dissection is a life - threatening condition affecting up to 30 people per million each year .
it remains a devastating disease with one of the highest mortality rates of cardiovascular diseases and multiple unanswered questions regarding treatment .
endovascular repair of uncomplicated acute aortic type b dissections ( aad - b ) is especially controversial and its associated post - implantation syndrome ( pis ) vague .
we present a case of a 57-year - old man with an uncomplicated aad - b managed with tevar ; and the subsequent refractory pains , only later managed with steroids as atypical pis .
although most definitions do not include local features of inflammation ( including pain ) as part of pis ; its place in systemic inflammatory response syndrome ( sirs ) and pis is highlighted .
a 57-year - old man of african descent known to have hypertension ; presented with acute onset , progressively worsening chest pain radiating to the back ( severity > 8 ; numeric pain rating scale ) .
he had no other comorbidities , was a non - smoker and did not volunteer any history of alcohol use .
he was on metoprolol , irbesartan and hydrochlorothiazide for his hypertension ; which he admitted to using only when he felt unwell . on examination
, he had a blood pressure of 172/119 ( right arm ) and 137/92 ( left arm ) .
high - sensitivity troponin t was elevated ( 19.29 ng / ml ) , but not rising .
an electrocardiogram ( ecg ) and a transthoracic 2d - echocardiogram showed left ventricular hypertrophy and no wall motion abnormalities .
a computed tomography aortogram ( cta ) was done showing an aortic dissection involving the descending aorta extending to the left common iliac , with iliac , superior mesenteric and both renal arteries arising from true lumen ( fig .
he was admitted to the intensive care unit and started on morphine and labetalol infusion at 2 mg / h .
figure 1:(a ) ct aortogram showing the dissection , 3d reconstruction ; ( b ) immediate post - tevar showing contrast only in true lumen .
( a ) ct aortogram showing the dissection , 3d reconstruction ; ( b ) immediate post - tevar showing contrast only in true lumen .
the patient was selected for tevar according to cooper 's proposed algorithm and the decision reinforced by the history of non - compliance to medical therapy . a valiant captiva ( medtronic ) 38 200 mm stent was deployed through the right femoral artery into the aorta , landing proximally just distal to the left subclavian artery and distally just distal to the celiac artery with an overlap of 110 mm and effective occlusion of the false lumen ( fig . 1b ) . of note ,
12 h post - tevar however ; the patient started complaining of colicky , non - specific abdominal pains ; associated with a leukocytosis , decreased platelet counts , the absence of fever and rising c - reactive protein ( crp ) ( fig .
he was managed conservatively with non - steroidal anti - inflammatory agents ( nsaids ) , antispasmodics and proton pump inhibitors ( ppis ) .
five days later , pains persisted , increasing in severity ; with minimal relief with nsaids and antispasmodics .
an array of tests were done including procalcitonin ( normal ) ; blood cultures ( negative ) ; liver function tests ( normal ) , serum amylase / lipase ( normal ) ; abdominal ultrasound ( minimal gall bladder sludge ) ; repeat ecgs ( no changes ) ; oesophagoscopy ( normal ) and magnetic resonance imaging of the thoracic and lumbar spine ( normal ) .
a repeat cta showed the graft in place without endoleaks . in total , 16 days post procedure ; he was started on intravenous steroids ( for possible pis ) with dramatic clinical improvement and subsequently discharged home after 48 h in a stable condition .
note the initial attenuation of crp , with normalization of wbc / plt on fifth day post - tevar and the drastic drop in crp / plt with initiation of steroids on day 14 .
note the initial attenuation of crp , with normalization of wbc / plt on fifth day post - tevar and the drastic drop in crp / plt with initiation of steroids on day 14 .
described over two centuries ago ; aortic dissections result from an intimal tear that leads blood into a false lumen between the intimal and medial layers of the aortic wall .
as classified by daily et al . in 1970 at stanford ; type a dissections involve the ascending aorta and type b involve only the descending aorta . the latter account for over a third of aortic dissections [ 1 , 5 ]
; a majority hypertension - related and are uncomplicated so assigned by the absence of malperfusion , hypotension , refractory hypertension or findings suggestive of impending rupture .
once a diagnosis of an uncomplicated aad - b is established , a majority of patients are started on medical therapy ; and this has been widely accepted as the standard treatment [ 2 , 3 ] .
this entails intensive anti - impulse therapy ; the cornerstone of which is reduction of pressure on the aortic wall and subsequent decreased propagation of the false lumen .
while a majority of patients so treated are discharged with no complications ; the long - term outcomes are perturbing with high mortality and complication rates .
subsequently , questions abound about the possibility of multimodal therapy with adjunctive tevar in a bid to improve the outcomes .
this stent graft placement occludes the primary intimal tear promoting false lumen thrombosis and subsequent aortic remodeling ; with resultant reduction of disease progression , late adverse events and aorta specific mortality [ 7 , 8 ] . despite this
, however , the use of tevar in uncomplicated aad - b remains controversial ; and is not without risks , one of which is pis . first described in 1999 , pis , is a vaguely defined sirs following endovascular repair that presents with a noninfectious fever , leukocytosis ( > 12 000/l ) , elevated crp ( > 10 mg / l ) and coagulation disturbances including thrombocytopenia in the context of negative blood culture results ; and occurs in up to 60% patients .
though the exact pathophysiology remains an enigma , it is believed to result from complex endothelial - stent fabric interactions where tissue injury activates complement and induces production of various pro - inflammatory mediators including tumor necrosis factor- , interleukin ( il)-1 , il-6 ; with local and systemic effects . while the systemic effects are well appreciated , and define pis
; there is hardly any mention of its local effects , including local pain . in kenya ,
compliance to antihypertensives has been shown to be low , and thus an added benefit with tevar in management of this case postulated . while the patient developed evident sirs with tachycardia , leukocytosis , thrombocytopenia and high serum crp post - tevar ; it is his refractory pain that was worrying .
evaluation for the etiology of this , from endoleaks to cord ischemia yielded no positive result and thus the eventual management as an atypically presenting pis , refractory to nsaids . although pis is a largely systemic inflammatory response ; perigraft inflammation has been shown in experimental studies and thus features of local inflammation ( like pain in our case ) should be borne in mind to avoid delayed treatment with associated lengthy hospital stays .
h.g . was the primary doctor in the care of this patient and was involved in providing data needed ( including images ) in preparation of this report .
he was also very resourceful in literature review especially on hypertension locally and internationally ; and in the preparation of the manuscript , including revising it critically for important intellectual content .
p.w . was the lead author with significant contribution in case report framework , literature review , draft revision and final submission .
patient consent was obtained ( available for review if needed ) for publication of this case report . | aortic dissection is a life - threatening condition and has one of the highest mortality rates of cardiovascular diseases .
it remains a devastating disease ; with multiple unanswered questions concerning treatment modalities .
the role of thoracic endovascular aortic repair ( tevar ) in these patients ; especially those with uncomplicated acute aortic type b dissections ( aad - b ) is especially controversial although it has been shown to have better long - term outcomes compared to medical therapy alone . for those who have tevar , up to 60% may develop an acute , transient systemic inflammatory response syndrome that remains vaguely defined .
the role of local inflammation in this post - implantation syndrome ( pis ) has not been highlighted .
we present a case of a 57-year - old male patient with an uncomplicated aad - b who developed an
atypical pis post - tevar with severe refractory abdominal pains ; leukocytosis and raised c - reactive protein .
the role of local inflammation in pis is highlighted . | Introduction
Case Report
Discussion
Authors contributions
Consent for publication
Conflict of interest statement
Funding |
barium as a positive radiographic contrast medium for evaluation of gastrointestinal disorders has historically been associated with the first application of x - rays in medicine .
it formed the mainstay as a primary diagnostic modality to investigate disorders of the gastrointestinal tract .
various improvisations in the composition of barium and its techniques ( high density , double contrast ) allowed accurate anatomical depiction .
technical innovations in the form of image intensification , automatic exposure , automatic film processor systems , etc . , provided ease in performing such tedious procedures .
there were , however , concerns regarding the excessive exposure of ionizing radiation to both , the radiologist and the patient . the advent of ultrasonography , endoscopy , capsule endoscopy , physiological monitoring , and axial imaging ( computed tomography [ ct ] and magnetic resonance [ mr ] scanning ) relegated the superiority of barium examinations . over the last 30 years , there has been a steady decline in requests for barium studies due to the above reasons .
now , barium meal evaluation of stomach and small bowel is performed only in teaching institutions and in mofussil areas , where there is dearth of modern imaging facilities .
this art comes with practice and with dwindling number of investigations asked for ; there is no time to master the same .
being operator depended , the clinicians would not trust the studies performed by juniors and would thus seek their answers from the gamut of other investigations available .
mastering the art of barium examination is time consuming and in the present economic trends , is not cost - effective .
with passage time , there will be nobody who would be able to perform an appropriate and optimal barium examination .
furthermore , the present generation of clinicians and radiologists rely more on endoscopy and axial imaging , and would not like to spend more time in the time - consuming barium examinations .
this decline of barium investigations has had negative repercussions on the residency training programs as they are not aware of the proper techniques , advantages , and limitations of the study . in odd cases where
one is forced to perform such an investigation , they would be on tender hooks to go about it and perform a shoddy study .
there are areas where endoscopy , axial imaging , and physiologic monitoring are inadequate ; they are invasive , time consuming , expensive , and not without their shortfallings , ambiguity , and complications .
it is primarily the financially driven initiatives that have propagated the use of other complementary modalities as a primary modality(s ) of choice over the conventional barium examination .
barium studies , till date , remains the safest , fastest , and cheapest diagnostic investigation to evaluate vague abdominal symptoms and the art has to be revived .
motility disorders of the gastrointestinal tract from pharynx to anus and submucosal lesions are best and quickly evaluated by barium studies .
no other modality can be as faster , safer , and more accurate than this age - old barium evaluation .
it is useful for both , diagnostic as well as for follow - up to evaluate response to therapy .
other specialties such as speech therapy and the ear - nose and throat , also require barium studies for accurate anatomic depiction by barium studies for the detection of vocal cord movements , artificial voice box evaluation , and other benign conditions such as diverticulum .
the real - time evaluation of the upper digestive tract by barium is a boon for them as it resolves many ambiguous disorders .
however , for staging purposes in esophageal cancers , almost all esophageal disorders can be diagnosed by barium swallow examination .
barium study is a quick investigation to evaluate response to treatment and to evaluate the postoperative status and their complications in gastroenterology , being a faster and cheaper investigation . in our institution
, we perform a large number of loopogram ( distal and proximal ) using barium before closure to external bowel diversion .
to perform even a simple and easy study such as loopogram , one needs to be aware of basics and basis of the use of barium studies in tubular structures such as mucosal coating , distension , the barium pool , projections , and compression to optimize visualization of abnormalities at single- and double - contrast studies .
at tata memorial hospital , we evaluated the audit data over 10 years from january 2006 to december 2015 .
the data were divided into three groups , namely , barium swallows , barium meals , and enemas and others ( enteroclysis , loopogram , and cologram ) .
being a tertiary cancer center and with the availability of most of the latest imaging technologies , we were seldom performing barium studies for the primary evaluation of the stomach and colon .
there has been a slow but steady decline in the number of barium studies performed in our hospital over the past 10 years . in our institute
, we found a trend of overall decrease in the number of barium studies from 2989 to 672 in 10 years with 76% regression . out of all the barium studies ,
the major decline was seen in the number of barium swallows which went down from 2801 to 471 in 10 years with an approximately 83% decrease over interval period [ figure 1 ] .
overall , 76% regression in the number of barium studies with a major decline seen in the number of barium swallows being performed having 83% decrease the decline is probably due to the fact that ours is a tertiary cancer center where most of the cases are already worked by basic imaging investigations outside .
similar trends are seen in the united states . in the data published by medicare , during a 3-year period ( 19992001 ) ,
there was approximately 33% decrease in the number of double - contrast barium enema ( dcbe ) for colorectal cancer screening , and in 2008 , the u.s .
preventive services task force recommendations for colorectal cancer screening guidelines the use of dcbe is omitted .
one of the many postulated reasons for the decline of use of dcbe , the most important are ( 1 ) wider availability and increasing use of cross - sectional imaging tests , such as abdominal ct and mr imaging ( mri ) , ( 2 ) higher sensitivity and specificity of ct and mri in detecting polyps , ( 3 ) alternatives such as ct- and mri - based virtual colonoscopy , and ( 4 ) very few trained radiologist who can perform and interpret this technique and teach radiology trainee for performing such procedures .
not surprisingly , there was a slight increase in the number of loopogram , reflecting the increase in surgical intervention .
the decline of barium evaluation of esophagus , stomach , and small bowel is attributed to diverse factors including the ever - increasing accessibility of endoscopy and sophisticated cross - sectional imaging modalities such as ct and mri . at our institution ,
barium studies are now used primarily to evaluate the pharynx , larynx , and cervical anastomosis and to evaluate the external bowel diversion loop before closure . for accurate anatomic delineation of the lesion
, our radiotherapy department still uses the lateral barium study for the neck at six feet distance , for the larynx and pharynx .
similarly , many centers across the world still use contrast swallow studies for the assessment of anastomotic leaks , and in a recent study by price et al . showed that barium swallow is a specific test for detecting anastomotic leaks after esophagectomy .
rarely , we have had to resort to enteroclysis ; for the odd cases , a routine barium follows through examination sufficed .
tata memorial centre ( tmc ) grant in aid under dae is one of the largest comprehensive cancer centers in asian subcontinent .
tmc rural outreach program ( tmcrop ) in southern maharashtra districts ( approximate population 3 million ) in the western part of india in 2002 . from 2008 ,
we introduced barium swallow examination , a faster and cheaper investigation for early esophageal cancer detection , as a primary tool for screening program in our tmcrop .
besides , being a primary diagnostic tool for many upper gastrointestinal disorders , these barium fluoroscopic examinations also have an important role in the evaluation of patients after gastrointestinal surgery ( including postesophagectomy and anastomosis surgery ) and in the diagnosis of a host of postoperative complications ( e.g. , perforation and obstruction ) .
hence , in the era of personalised cancer medicine and molecular functional imaging there is a need to revitalize barium studies that have been ignored for a long time .
importantly , there is a need to interact proactively with the clinicians who are our feeders .
joint discussions should be held , and it must be emphasized to them that other imaging modalities are complementary to barium studies and that there are many instances whereby , this cheaper and faster investigation would be beneficial to the patient . they must be coerced , and we should demonstrate a good study to encourage them to look our way . another problem that needs to be resolved is that of the available literature .
newer books on imaging do not lay sufficient stress on the various techniques of performing barium studies ; whatever little they may add , is insufficient .
it is not that barium examination can be mastered by reading books or through the internet .
it is the older editions on the subject that describe in depth the nuances in the performance of barium studies .
hence , it is the need to preserve older textbooks that describe these procedures in depth .
they also contain more illustrations than the newer ones , as other modalities were not in vogue then .
the institution also plays an important role in regard with sanctioning of grants for fluoroscopic equipment .
they too must be made aware of the fact that fluoroscopic equipment for barium studies is a must in the armamentarium of the radiodiagnosis department .
money should be spent not only on such equipment but also for the other modern and latest equipment . finally , to gather experience ,
junior doctors need to weed out frank cases to perform barium examination on diagnosed cases for applying and learning the art of barium studies , and importantly , the art of fluoroscopy . unlike vascular and interventional studies , fluoroscopy is an art in barium examination where one does not try to diagnose lesions on fluoroscopy .
fluoroscopy is performed to see adequacy of contrast coating , its passage , and to record images of lesions ( suspicious or obvious ) in orthogonal views .
our aim to preserve the age - old technique of barium studies not only to keep it going on but also for the betterment and appropriate management of the patient .
our goal is not to save barium studies simply to keep this technology alive , per se , but rather to preserve barium radiology for the quality in patient care . | with the advent of the modern imaging technologies , the present era of oncology is seeing steady decline in requests for barium studies due to the many reasons .
it is prudent to mention here , that , barium examinations can not be made obsolete !
our aim to preserve the age old technique of barium studies not only to keep it going on but also for the betterment and appropriate management of the patient .
our goal is not to save barium studies simply to keep this technology alive , per se , but rather to preserve barium radiology for the quality in patient care . | INTRODUCTION
IT IS PRUDENT TO MENTION HERE, THAT, BARIUM EXAMINATIONS CANNOT BE MADE OBSOLETE!
TATA MEMORIAL CENTER EXPERIENCE
TO RESURRECT BARIUM STUDIES
CONCLUSION
Financial support and sponsorship
Conflicts of interest |
informed consent to publish this case report was obtained from the patient . a 31-year - old woman ( gravida 1 , para 0 ) with a gestational age of 29 weeks
the echocardiography and ct angiography revealed an acute aortic dissection ( stanford type a , debakey type 1 ) with severe aortic regurgitation .
her nt - probnp ( 885.9 pg / ml ) and d - dimer ( 0.79 g / ml ) levels were elevated .
the patient had no history of vascular events , hypertension , or previous vascular surgery .
she presented a clinical feature of suspected marfan syndrome and her family history included the sudden death of her father .
she was admitted to the intensive care unit for close hemodynamic monitoring of both the mother and the fetus .
her vital signs were within the normal range at admission ( systolic blood pressure : 120150 mmhg ; heart rate : 7080 beats / min ) .
the pediatrician reported that the fetus ' vital signs were stable and would be viable for extra - uterine life under adequate intensive care with surfactant therapy .
a cardiac anesthesiologist , thoracic surgeon , obstetrician and pediatrician discussed the treatment plan and concluded that a cesarean section followed by an ascending aorta and aortic arch replacement should be performed .
the thoracic surgeon and anesthesiologist decided not to perform a sternotomy prior to the cesarean section after weighing the risks and benefits of a sternotomy before the cesarean section . because the chest pain had developed two days prior to her visit to our er and the patient 's hemodynamic status was stable , the risk of spontaneous rupture during the cesarean section was not considered to be high .
we also considered that the hemodynamic response and surgical incision for a sternotomy could only increase the risk of aortic rupture , and a delayed cesarean section due to a sternotomy could jeopardize the fetus ' life .
instead , the thoracic surgical team was prepared for cpb in the operating room during the cesarean section .
the right radial and right femoral arterial blood pressure and central venous pressure were monitored .
labetalol 15 mg / h and nitroprusside 1 g / kg / min were infused before surgery .
a total intravenous anesthesia with propofol and remifentanil was performed with monitoring by transesophageal echocardiography ( tee ) and cerebral oximeter monitoring ( inovos5100b , troy , usa ) . a large bore central venous line ( 9 fr .
advanced venous access catheter , edwards lifesciences llc , irvine , ca , usa ) was accessed and connected with a rapid volume infuser ( level 1 , level 1 technologies , rockland , ma , usa ) .
the intraoperative tee revealed severe aortic regurgitation and intimal flap in the ascending aorta ( fig .
1 ) . cardiac output monitoring by a swan - ganz catheter was not performed so as not to delay the cesarean section and because a possible arrhythmia during the catheter insertion could result in hemodynamic instability . instead
, the left ventricular systolic function , the cardiac output and the degree of aortic regurgitation were monitored by tee during the cesarean section .
the hemodynamic goal during the cesarean section was to avoid an abrupt increase in blood pressure and to maintain an adequate preload and cardiac output .
considering the severe aortic regurgitation and dissection , the heart rate was controlled between 60 and 80/min .
a 1280 gm male baby was delivered with a 1 min apgar score of 2 and resuscitated with an endotracheal intubation .
the baby was admitted to the neonatal intensive care unit ( icu ) and treated with a surfactant . during the cesarean section ,
the hemodynamic parameters were kept stable with the inotropic support of dopamine ( 510 g / kg / min ) and norepinephrine ( 0.10.4 g / kg / min ) and a transfusion of packed red blood cell 2 units .
colloid 500 ml ( volulyte , fresenius kabi , bad homburg , germany ) was administered during the cesarean section .
after the cesarean section , an ascending aorta and total arch replacement , valve sparing aortic root replacement , aortic valvuloplasty with commissuroplasty , and a free graft from the aorta to the right coronary artery were performed under cpb . weaning from cpb was successful with dopamine ( 3 g / kg / min ) , dobutamine ( 3 g / kg / min ) , and nitroglycerine ( 1 g / kg / min ) .
a transfusion of prbc 2 units , cryoprecipitate 10 units , and platelet concentrate 16 units was guided by thromboelastography .
the patient was transferred to the cardiac icu and discharged without any complications on postoperative day # 6 .
aortic dissection during pregnancy is a rare condition that is life - threatening for both the pregnant woman and the baby , with a survival rate of only 20%30% .
it is often associated with a genetic or anatomic predisposition including marfan syndrome or bicuspid aortic valve .
marfan syndrome has been reported in up to 50% of the aortic dissections during pregnancy , with emergency surgery frequently performed at diagnosis .
the incidence of dissection during pregnancy in marfan syndrome patients was reported to be 3% .
it mostly occurs during the third trimester of pregnancy due to the hyperdynamic state and hormonal effects on the vasculature .
eighty percent of the dissections during pregnancy are a type a dissection , and 87.5% of these cases occur in the pre - partum period .
this results in a connective tissue disorder which often affects the cardiovascular system and may cause aortic root dilatation and aortic dissection .
aortic root dilatation and the rapidity of this dilatation were reported to be critical risk factors for aortic dissection during pregnancy .
aortic dissection in the peripartum period mostly occurs during the third trimester , which is a period of maximal hemodynamic stress .
estrogen was reported to inhibit collagen and elastin deposition in the aorta while progesterone promotes the deposition of noncollagen proteins in the aorta .
when these hormonal effects occur in a patient with marfan syndrome , aortic dissection could be initiated during pregnancy .
previous case series have reported the successful management of aortic dissection in pregnant women through a cesarean section with subsequent emergency aortic surgery for a stanford type a dissection or by conservative management for a stanford type b dissection . in previous case series , the sternotomy was performed prior to the cesarean section to facilitate urgent cpb for possible hemodynamic instability during the cesarean section . however , in our case , the cesarean section was performed without a prior sternotomy . as mentioned previously , the onset of dissection was two days before the surgery , and the risk of rupture during the cesarean section was not considered to be high . in the case of an acute type b dissection , minimizing the surgical repair of the dissection
beta - blockers , calcium channel blockers and vasodilators such as sodium nitroprusside are commonly - used an anti - hypertensive agents .
vasodilators should not be used alone due to reflex tachycardia . for a type a dissection before the 30 week of gestation , aortic repair with the fetus in utero
although a high - flow , high pressure normothermic perfusion has been considered to be the safest for the fetus , there have been several case reports of successful hypothermia or deep hypothermic circulatory arrest during pregnancy before the 28 week of gestation . in our case ,
the cesarean section was performed before the aortic surgery , although before the 30 week of gestation . a decision based on the pediatrician 's opinion
was made that the gestational age was near 30 weeks , and the fetus was mature enough for extra - uterine life .
aortic dissection could occur during pregnancy in patients with marfan syndrome . to prevent this potential catastrophic event ,
preconception counseling should recommend prepregnancy elective aortic root replacement surgery in marfan syndrome patients with an aortic root enlargement .
elective replacement with valve - sparing procedures and a homograft can be performed with low morbidity and mortality in patients with marfan syndrome .
we performed general anesthesia for the cesarean section in our marfan syndrome patient with aortic dissection during pregnancy .
we chose general anesthesia because neuraxial anesthesia could produce a sympathetic block which would cause hemodynamic instability in this patient treated with a beta - blocker and vasodilator .
however , anesthesia for a cesarean section in marfan syndrome patients without aortic dissection would be different .
a previous article recommended performing a regional anesthesia for the cesarean section so as to avoid high blood pressure which could induce aortic dissection .
aortic dissection can occur during pregnancy in patients with marfan syndrome , mainly in the third trimester . if possible , surgical repair of the enlarged aortic root should be performed prepartum . however , if aortic dissection stanford type a occurs during pregnancy in a marfan syndrome patient , a cesarean section followed by aortic surgery is currently the best modality for treatment .
careful hemodynamic management during the cesarean section under general anesthesia with tee monitoring ensured the safety of the patient and baby without a prior sternotomy in our case . | aortic dissection during pregnancy is a devastating event for both the pregnant woman and the baby .
we report a case of acute aortic dissection ( stanford type a ) in a pregnant woman with marfan syndrome at the 29th week of gestation .
she underwent a cesarean section followed by an ascending aorta and total arch replacement with cardiopulmonary bypass , without a prior sternotomy .
the hemodynamic parameters were kept stable during the cesarean section by using inotropes and vasopressors under transesophageal echocardiography monitoring .
the newborn survived after endotracheal intubation and management in a neonatal intensive care unit . | Case Report
Discussion |
penile cancer is rare in developed countries , whereas in india and other asian countries the incidence is decreasing .
the incidence of squamous cell carcinoma ( scc ) of penis varies worldwide with age , circumcision , and hygiene practices .
phimosis , lack of neonatal circumcision , human papillomavirus ( hpv ) infection , penile lichen sclerosis , exposure to tobacco products , and psoralen ultraviolet a ( puva ) are the known risk factors for development of scc of penis . up to 50% of patients with scc of penis delay seeking medical attention by more than a year due to embarrassment , guilt , fear , ignorance , and personal neglect .
the level of denial is substantial , given that the penis is observed and handled on a daily basis .
a 60-year - old unmarried male , presented with a history of multiple ulcers over his right groin of 4 months duration .
it started as a painless swelling over the right groin , which evolved into multiple ulcers with foul swelling discharge .
. he does not give history of arthralgia , loss of weight , or loss of appetite .
the patient also gave history of multiple blisters over the trunk and extremities of 1 month duration .
there was no history of oral erosions or drug intake prior to the onset of lesions .
on general examination , the patient was moderately built and nourished , there was no pallor , icterus , pedal edema , or generalized lymphadenopathy .
examination of the right inguinal region showed three tender excavating ulcers of sizes 6 8 , 5 7 , and 3 5 cm , respectively , located adjacent to each other with rolled out , everted edges , and foul smelling purulent , blood - stained discharge .
enlarged , grouped , and matted tender inguinal lymphnodes of sizes 5 7 cm were felt below the ulcer .
examination of the left inguinal region also revealed enlarged , grouped , tender inguinal nodes of size 8 6 cm present both above and below the inguinal ligament giving the appearance of groove sign of greenblatt [ figure 1 ] .
right groin showing multiple excavating ulcers , left groin shows groove sign of greenblatt and multiple tense bullae with erosions over thighs and penile shaft genital examination revealed phimosis .
a firm , indurated , non tender mass of size 3 2 cm was felt through the prepucial skin on the glans penis , located on the 11o - clock to 2 o - clock position .
multiple tense bullae filled with clear fluid and erosions were present over upper limbs , lower limbs [ figure 1 ] , and trunk .
there were no oral erosions . with the above clinical findings , a differential diagnosis of lymphogranuloma venereum ( lgv ) or scc of penis with regional metastasis associated with bullous pemphigoid ( bp ) occurring as a para neoplastic phenomenon were considered .
igg antibody for chlamydia trachomatis ( serovars l1 , l2 , l3 ) was negative .
vdrl for syphilis and elisa for human immunodeficiency virus ( hiv ) 1 and 2 were nonreactive .
wedge biopsy of the ulcer in the right groin and from the penile growth showed ulcerated hyperplastic epidermis with severe dysplasia .
the dysplastic epithelial cells invade the basement membrane forming nests with keratin pearls , consistent with keratinizing scc of penis [ figure 2 ] .
skin biopsy taken from a tense bulla over the trunk , showed a sub epidermal bulla with mild lymphocytic infiltrate in the upper dermis consistent with bp .
the dysplastic epithelial cells are seen to invade the basement membrane hence , a diagnosis of keratinizing scc of penis with regional lymphnode metastasis and bp was confirmed .
scc of penis may present either as a flat growth , infiltrating , or papillary growth .
the lesion is not visible in most patients since the prepuce is non - retractile .
the inguinal nodes increase in size followed by erosion of the skin over the nodes .
it starts as a small transient , inconspicuous lesion on the genitalia and spreads to regional lymphnodes resulting in bubo and suppurative lymphadenopathy . in our patient ,
scc of penis was masquerading as lgv since he presented with history of inguinal bubo , which later ulcerated .
moreover , the primary scc was not noticed by the patient and was not visible during examination since the prepuce was non- retractile .
a biopsy from the groin ulcer and penile growth that was felt on palpation had confirmed the diagnosis .
the occurrence of bp in this patient can be attributed as a paraneoplastic phenomenon to scc of penis .
pemphigoid has been reported in numerous patients with malignancies , like breast cancer , b - cell lymphoma , lung cancer , gastric cancer , cancers of colon or rectum , endometrial cancer , and many others . in scc ,
this could lead to production of antibodies directed against these antigens that results in the development of bp in these patients . to the best of our knowledge
this is the first case of scc of penis with bp occurring as a paraneoplastic phenomenon .
we present this case to highlight that scc of penis can masquerade as lgv and a high index of suspicion is required for an early diagnosis of scc penis . | we report a case of a 60-year old unmarried male who presented with multiple ulcers and foul smelling discharge from the groin since 4 months and multiple tense bullae over the trunk of 1 month duration .
groove sign was present .
investigations for lymphogranuloma venereum ( lgv ) and other sexually transmitted diseases were negative .
histopathology from the ulcer in the groin and growth in the penis revealed squamous cell carcinoma ( scc ) .
skin biopsy of bulla was diagnostic of bullous pemphigoid ( bp ) .
we report a rare case of scc masquerading as lgv with bp occurring as a paraneoplastic phenomenon . | INTRODUCTION
CASE REPORT
DISCUSSION |
lactobacillus rhamnosus gg ( lgg ) is a gram positive lactic acid bacterium that is part of the commensal microflora in humans .
it is generally regarded as safe and has been used extensively in food products and health supplements .
meta - analysis of probiotic supplementation during pregnancy and early infancy indicates a reduced risk ratio of developing eczema in early infancy .
a meta - analysis of lgg supplementation showed increased treatment responders in subjects with abdominal pain related gastrointestinal disorders and irritable bowel syndrome .
ohashi et al . also found that long - term consumption of lactobacillus casei was associated with the reduced risk of bladder cancer .
lgg was also shown to possess antitumor effects in animal models of bladder cancer [ 6 , 7 ] .
the antitumor effects were comparable to that induced by mycobacterium bovis , bacillus calmette - gurin .
intravesical instillations of lgg resulted in an influx of dendritic cells ( dcs ) and neutrophils . despite bcg 's efficacy
it is associated with significant side - effects and less toxic therapies are needed ; thus this study further evaluates the immunotherapeutic potential of lgg .
dcs are antigen presenting cells that play an important role in cancer immunotherapy by stimulating cytotoxic t lymphocytes ( ctl ) and polarizing t helper cells towards a th1 profile .
dc maturation causes enhanced expression of surface costimulatory molecules and production of cytokines and chemokines .
however , extensive stimulation of dc can result in dc exhaustion that is characterized by diminished production of il-12 which is necessary for ctl induction and interferon gamma ( ifn ) production .
dc exhaustion can be the result of prolonged exposure to a stimulus or exposure to a very high dose of a stimulus either of which scenarios are possible when analyzing the interaction of microbes with immune cells .
miyazaki et al . showed that , upon inflammation , neutrophils migrate from the site of infection to neighboring lymph nodes where they undergo apoptosis and are taken up by dcs , thus ensuring that neutrophil derived antigens are presented to t cells .
neutrophils are also able to directly transfer antigens to dcs as was demonstrated by morel et al . , studying bcg infected neutrophils .
this work evaluates the impact of the dose of lgg and time of exposure on dc activation in the absence and presence of neutrophils and the consequent stimulation of t cells . the mouse orthotopic tumor model used to assess the intravesical instillation of lgg into the bladder followed the clinical protocol of bcg immunotherapy and was performed over a 2-hour time frame .
thus , this was the minimum time of interaction that was analyzed and 18 h was chosen as the maximum time of interaction as , beyond this time frame , dc viability was greatly reduced after exposure to a high dose of lgg .
the death induced by lgg on longer exposure may be a consequence of lactic acid production as observed with cancer cells exposed to lgg .
l. rhamnosus gg ( national collections of industrial and marine bacterial ltd . , uk ) was streaked onto deman rogosa sharpe ( mrs ) agar ( difco laboratories , usa ) and incubated at 37c in 5% co2 .
single colonies were used to produce seed cultures ( 9 h ) which were used to start 50 ml cultures .
bacteria were harvested at the late log phase by centrifugation ( 1699 g for 10 minutes at room temperature ) and washed twice with sterile saline ( 0.85% nacl ) .
the colony forming units ( cfu ) were determined by plating serial dilutions of the bacterial samples on mrs agar plates which were incubated at 37c in 5% co2 .
protocols were approved by the institutional animal care and use committee ( iacuc ) at the national university of singapore .
bone marrow derived neutrophils and dc were generated as previously described . in brief , neutrophils were derived by positive selection with anti - ly6 g microbead kit ( miltenyi biotec , germany ) and were at least 95% positive for ly6 g , by flow cytometry .
dcs were obtained from the bone marrow derived cells after 9 days of culture ( with fresh media replacement every other day ) in rpmi 1640 supplemented with 10% heat - inactivated fcs , 50 m 2-mercaptoethanol , 1% penicillin , streptomycin , glutamine , mem ( minimum essential medium ) , and 0.1% sodium pyruvate with 40 ng / ml of gm - csf ( bd bioscience , usa ) .
the media used for both neutrophil and dc experiments were dmem supplemented with 10% fetal bovine serum ( hyclone , usa ) , 2 mm l - glutamine ( gibco , japan ) , 50 g / ml penicillin g ( sigma aldrich , usa ) , and 50 m of 2-mercaptoethanol ( merck , germany ) with the addition of 20 ng / ml of gm - csf for the culture of dendritic cells .
t lymphocytes were isolated from spleens of naive c57bl/6 mice and enriched with the easyseptm t cell isolation kit ( stemcell technologies , vancouver , canada ) .
the lgg to cell ratios of 5 : 1 and 10 : 1 were defined as exposure to low dose lgg , while exposure to a ratio of 100 : 1 was defined as exposure to high dose lgg .
neutrophils ( 5 10 ) and dcs ( 2.5 10 ) were cocultured with lgg at bacteria to mammalian cell ratios of 5 : 1 , 10 : 1 , and 100 : 1 for 2 h in 24-well plates , before 200 g / ml of gentamicin was added for 2 h at 37c to kill extracellular lgg .
cells were washed thrice with pbs to remove extracellular bacteria and then neutrophils were incubated with dcs ( 2.5 10 ) for 18 h and dcs were incubated in fresh media for 18 h. untreated neutrophils and dcs were evaluated as controls .
for 2.5 10 dcs the bacteria cfu that corresponded to 5 : 1 , 10 : 1 , and 100 : 1 were 1.25 10 , 2.5 10 , and 25 10 cfu . the neutrophils ( 5 10 cells ) were treated with 2.5 10 , 5 10 , and 50 10 cfu of lgg that corresponded to 5 : 1 , 10 : 1 , and 100 : 1 , lgg to cells . for the dc 18
h experiment the dcs were exposed to lgg for 18 h. similarly cells were treated with bcg at a 5 : 1 ratio .
the supernatants were assayed for tnf- , il-12p70 , and il-10 ( ebioscience , san diego , usa ) and prostaglandin e2 ( pge2 ) ( cayman chemical , usa ) by elisa using a genios pro microplate reader ( tecan , switzerland ) .
the cells were harvested in pba ( pbs with 1% bovine serum albumin and 0.01% sodium azide ) for flow analysis of surface markers . il-10 and
cox-2 were inhibited by pretreatment with 400 ng / ml of anti - il-10 antibody ( biolegend , san diego , usa ) and 10 m of ns398 ( sapphire bioscience , australia ) , respectively , for 30 mins at 37c prior to addition of bacteria and
fixed dcs were double stained with anti - cd11c antibody and antibodies of the following surface markers : cd40 , cd80 , cd83 , cd86 , and mhc ii ( biolegend ) or the respective isotype controls in pba ( 1x pbs with 1% bsa , 0.05% sodium azide ) for 20 mins at 4c .
after that the cells were washed once and resuspended with pba before they were analyzed with bd facs canto using facs diva software ( becton dickinson , usa ) .
dcs or t cells were resuspended in lda medium ( nctc 109 and rpmi 1640 [ 1 : 1 ] ) , supplemented with 10% heat - inactivated fcs , 10 mm l - glutamine , 1 mm oxaloacetic acid , 0.2 u of bovine insulin per ml , and 50 m 2-mercaptoethanol .
naive t cells ( 1.0 10 cells / ml ) were cultured with untreated dcs ( 1.0 l0 cells / ml ) or dcs stimulated with lactobacilli for 2 h and 18 h ( treated as described above ) , in 200 l of lda medium in 96-well u - bottom plates at 37c under 5% co2 for 5 days .
one - way anova with post hoc bonferroni test was used to analyze all the data except for the comparison of cytokine profile of the treatment groups with anti - il-10 antibody or ns398 and their respective controls , which were analyzed with student 's t - test .
a short exposure ( 2 h ) to low dose lgg ( lgg to cell ratios of 10 : 1 and 5 : 1 ) reduced dc viability slightly ( 91.7 2.0% and 94.7 1.7% , resp . ) and there was little loss in viability even at 18 h. exposure to activated neutrophils had a similar effect .
however at a prolonged exposure of 18 h to high dose lgg ( 100 : 1 lgg to dc ratio ) , there was reduced dc viability ( 63.7 1.8% ) .
about 50% of neutrophils were dead ( apoptotic and necrotic death ) at 18 h after the initial 2 h exposure to lgg regardless of dose .
but in contrast there was increased lgg internalized with exposure to increased lgg dose . at a 5 : 1 ratio of lgg :
neutrophils there were 228 51 cfu internalized/5 10 neutrophils and this almost doubled after exposure to 10 : 1 and more than doubled again after exposure to 100 : 1 lgg to neutrophils .
activation markers on nave dc were examined and after exposure to lgg there was a significant increase in all markers with respect to nave dc , table 1 . as a further control dcs
after high dose lgg exposure for 2 h , there was significantly ( p < 0.05 ) higher expression of cd80 , cd86 , and cd40 compared to low dose lgg .
but at 18 h coincubation with lgg , expression of cd86 and cd40 was significantly reduced ( p < 0.05 ) after exposure to high dose lgg compared to low dose lgg .
dcs cocultured with neutrophils , activated with low doses of lgg for 2 h , showed higher expression of cd86 and reduced cd83 compared to dcs exposed directly to low dose lgg for 2 h. dc exposed to bcg at a dose of 5 : 1 , for 2 h or 18 h , or stimulated by neutrophils activated with bcg did not show a difference in surface marker expression .
in contrast lgg at the same dose showed changes in the expression of cd83 and cd86 .
neutrophils cultured with lgg showed decreased mhc class i expression , no increase in mhc class ii expression , and an increase in cd11b expression when placed directly in contact with lgg .
cd11b is an activation marker for neutrophils and has been shown to activate dc maturation via interaction with dc - sign .
mhc class ii mean fluorescence index ( mfi ) showed a doubling on exposure to low dose lgg or activated neutrophils , table 2 .
the mfi for mhc class ii , cd40 , and cd80 was decreased after exposure to dc activated for 18 h , with either high dose lgg or neutrophils activated with high dose lgg ( p < 0.05 ) .
but the reverse was true for cd83 when dcs were exposed to high dose lgg for 18 h ( p < 0.05 ) , table 2 .
more il-10 was produced after exposure to high dose lgg ( figure 1(a ) ) for 2 h and 18 h and via neutrophil mediated activation .
tnf- production was higher in dc exposed to high dose lgg ( figure 1(a ) ) for 2 h , but at 18 h , dc exposed to low dose lgg produced more tnf-. both indirect ( via neutrophils ) and direct dc activation for 18 h resulted in more il-12p70 production after low dose lgg exposure ( figure 1(a ) ) . with a short 2 h exposure to lgg ,
lgg activated neutrophils ( 2 h ) induced more il-10 , tnf- , and il-12p70 production in dc compared to dc exposed directly to low dose lgg for 2 h. at high dose lgg there was no significant difference in il-12p70 and tnf- production , whether the dc was stimulated directly with lgg or with activated neutrophils .
in contrast , when dc and neutrophils were exposed to bcg at a 5 : 1 ratio , there was comparable production of il-10 from all groups except dc exposed to bcg for 18 h. the amount of il-12p70 produced after bcg stimulation was at least 10-fold lower than that produced by lgg .
since , at high dose lgg , il-10 production is significantly higher in dcs , as well as dc treated with activated neutrophils , we determined if the dose dependent effects on il-12 production were due to il-10 levels .
pge2 is known to modulate il-10 expression ; induce indoleamine 2,3-dioxygenase ( a potent suppressor of dc function ) ; and modulate chemokine production and dc maturation and il-12p70 production [ 1820 ] .
therefore pge2 and il-10 production / function were inhibited individually and the impact on il-12p70 production was monitored , figure 1(b ) .
there was a significant increase in pge2 levels on dc stimulation with high rather than low dose lgg . at the concentration of ns398 that completely blocked the production of pge2 ( figure 1(b ) )
there was no significant effect on either il-10 or il-12p70 levels , figure 1(b ) . blocking il-10 with a neutralizing antibody
this corresponded to 2.1- and 4.4-fold increases , respectively , in dc stimulated with neutrophils activated with low and high dose lgg .
expression of surface markers on dendritic cells was not significantly affected by the presence of either the anti - il-10 antibody or ns398 ( data not shown ) .
neutrophils stimulated with lgg did not induce ifn production by t cells ( figure 2(a ) ) but induced il-2 production ( figure 2(b ) ) .
dcs stimulated with lgg activated neutrophils induced a significant increase ( p < 0.05 ) in ifn production ( figure 2(a ) ) and a slight increase in il-2 production by t cells ( figure 2(b ) ) similar to dc directly activated with low dose lgg .
the dc - neutrophil - t cell triple cell culture by itself induced il-2 production .
a dose dependent effect was clearly seen with ifn production ( p < 0.05 ) ( figure 2(a ) ) which was consistent with the decrease in il-12p70 production that was observed earlier .
direct or indirect dc activation with low dose lgg for 2 h induced more ifn and il-2 production by t cells ( p < 0.05 for ifn ) compared to high dose lgg , figures 2(a ) and 2(b ) . at 18 h
on activation by lgg , there was an increase in the percentage of dc expressing cd40 , cd80 , and cd86 with increasing dose .
but only cd40 had a significant increase in mfi ( p < 0.05 ) , indicating increased protein expression . cd86 and
cd80 interact with cd28 on t cells while cd40 binds to the cd40 ligand on t cells to induce t cell activation . on prolonged exposure
( 18 h ) to high dose lgg , the percentage of cells expressing cd86 and cd40 and the mfi of cd40 , cd86 , and cd80 were reduced .
thus , these dcs may not be as able to activate t cells as efficiently ; that is , there is a point beyond which lgg dose can be inhibitory to dc activation .
a similar effect was observed with l. casei which also has antitumor effects and other commensal lactobacilli such as l. gasseri , l. johnsonii , and l. reuteri .
different lactobacillus species induce variable levels of il-10 , il-12 , and tnf- via toll - like receptor ( tlr ) dependent activation of dc .
indirect dc stimulation via lgg activated neutrophils showed no difference in tnf- production with increasing dose .
lgg is known to adhere to epithelial cells with greater ability than other lactobacillus species .
such binding to dc may also increase the cellular signals triggered by direct interaction with dc .
found that lgg pili s spacba could interact with dc - sign and that blocking this interaction reduced dc cytokine production .
dc - sign also modulates tlr activation and it is possible that lgg pili interaction with dc - sign could have modulated tlr activated cytokine production .
an 18 h exposure to low dose lgg produced more tnf- and il-12 than exposure to high dose lgg .
it is likely that prolonged exposure led to increasing phagocytosis of lgg with time which resulted in triggering the downregulation of tnf- and il-12 production .
il-12 production is tlr2 independent but tlr9 , myd88 , and ros dependent .
however , with a short exposure to lgg there was a dose dependent effect only on tnf- production which might reflect tlr2 engagement .
as neutrophils are generally the first to encounter microbes and move to the lymph nodes to educate dc , we evaluated the dose effect of lgg on the ability of neutrophils to activate dc . stimulating dc with lgg treated neutrophils
exposed to low dose lgg induced higher cd86 than direct stimulation of dcs with lgg .
neutrophils cultured with high dose lgg induced a decrease in the mfi of cd40 , cd80 , and cd83 ( p < 0.05 ) on dcs . though the number of neutrophils undergoing apoptosis was similar at all doses of lgg the number of internalized lgg increased with dose of lgg .
the latter may have resulted in increased lgg or lgg components being transferred to dc [ 11 , 12 ] causing strong stimulation of dc and consequently dc exhaustion . in line with this hypothesis , il-12p70 and
tnf- production were much higher when the dcs were cultured with neutrophils activated with low dose lgg .
dcs are known to internalize apoptotic cells which like necrotic cells are able to stimulate dc .
phagocytosis of apoptotic cells induces anti - inflammatory signals such as the high levels of il-10 which was found in this study .
ifn production was higher in t cells cocultured with dc and neutrophils treated with low dose lgg for 2 h rather than high dose lgg .
similarly , when dcs were treated with lgg at 200 : 1 bacteria to cell ratio , phenotypic maturation and cytokine production but not th1 polarization were observed .
instead , the cd4 cells were converted to hyporesponsive t cells that secrete low ifn. thus , for optimal t cell activation , low dose lgg is overall the better therapeutic option .
prolonged stimulation of dcs ( for 24 h or longer ) can result in the loss of the ability to produce cytokines like il-12 , which is termed dc exhaustion [ 9 , 34 ] .
reported that the optimal temporal window to induce dc maturation in order to have sustained il-12 production for cancer immunotherapy is narrow , with a time frame of 1018 h , but our results indicate that a 2 h exposure is sufficient for dc maturation .
further lgg was much better at inducing il-12p70 production than bcg , the current standard therapy for bladder cancer .
il-10 is widely reported to downregulate dc maturation [ 35 , 36 ] and its ability to activate t cells as well as induce dc apoptosis .
pge2 , a potent inducer of il-10 , was also found to be produced in greater amounts when dcs were stimulated with neutrophils treated with high dose lgg .
however , it was still lower than the levels produced by dc coculture with neutrophil stimulated with low dose lgg , suggesting that there are other inhibitory factors aside from il-10 .
low dose lgg stimulates dc to induce greater th1 polarization in t cells compared to high dose lgg .
in mice lgg ( 1 10 cfu/100 l ) was effective at reducing tumor growth with comparable efficacy to bcg connaught ( 1 10 cfu / ml ) .
the former is roughly in the range of a 100 : 1 , lgg to cells for 2 h. thus future analysis should consider the effect of a 10-fold lower dose of lgg as an immunotherapeutic agent .
the dose response is an important consideration if lgg is to be used for human bladder cancer therapy . | this study evaluates the ability of lactobacillus rhamnosus gg ( lgg ) to activate dc and neutrophils and modulate t cell activation and the impact of bacterial dose on these responses .
murine bone marrow derived dc or neutrophils were stimulated with lgg at ratios of 5 : 1 , 10 : 1 , and 100 : 1 ( lgg : cells ) and dc maturation ( cd40 , cd80 , cd86 , cd83 , and mhc class ii ) and cytokine production ( il-10 , tnf- , and il-12p70 ) were examined after 2 h and 18 h coculture and compared to the ability of bcg ( the present immunotherapeutic agent for bladder cancer ) to stimulate these cells . a 2 h exposure to 100 : 1 ( high dose ) or an 18 h exposure to 5 : 1 or 10 : 1 ( low dose ) , lgg : cells , induced the highest production of il-12 and upregulation of cd40 , cd80 , cd86 , and mhc ii on dc . in dcs stimulated with lgg activated neutrophils il-12 production decreased with increasing dose .
lgg induced 10-fold greater il-12 production than bcg .
t cell ifn and il-2 production was significantly greater when stimulated with dc activated with low dose lgg . in conclusion , dc or dc activated with neutrophils exposed to low dose lgg induced greater th1 polarization in t cells and this could potentially exert stronger antitumor effects . thus the dose of lgg used for immunotherapy could determine treatment efficacy . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
congenital absence of the lumbosacral articular facet joint is an uncommon anomaly [ 112 ] ; it is usually asymptomatic and sometimes presents with a concomitant nerve root anomaly . to our knowledge ,
only three cases of congenital absence of the lumbosacral articular facet joint and a concomitant conjoined nerve root have been reported in the literature [ 9 , 12 ] .
a 39-year - old man presented with acute low back pain and right leg radiating pain .
his symptoms had rapidly worsened until he was not able to walk because of right foot drop and severe leg pain .
he was diagnosed a lumbar disc herniation at l5-s , and was referred to our hospital for surgical treatment .
neurological examinations showed a positive straight - leg raising test at 30 on the right side . on manual muscle test ( mmt ) ,
muscle weakness was found in the right leg : tibialis anterior was 1/5 , extensor hallucis longus was 0/5 , right flexor hallucis longus was 3/5 , and triceps surae was 3/5 .
the right patella tendon reflex was normal and the right ankle jerk reflex was diminished .
these findings were unusual symptoms of single - level lumbar disc herniation at l5-s1 . from plain anteroposterior ( ap ) and lateral radiographs of the lumbar spine , it was difficult to detect some spinal abnormalities . slight disc space narrowing at l5-s1 was seen on the lateral view ( fig . 1 ) .
myelographs showed incomplete blockade at the l5 pedicular level , especially on the right side ( fig . 2 ) .
computed tomography ( ct ) following myelography showed absence of the right l5-s1 facet joint ( fig .
multidetector three - dimensional computed tomography ( 3d - ct ) confirmed the absence of s1 superior articular process ( fig .
magnetic resonance ( mr ) images showed disc herniation on the right side at the l5-s1 intervertebral disc level ( fig .
axial mr t2-weighted images at the l5-s disc level showed disc herniation ( fig . 5b , left panel ) , which migrated cranially ( fig .
5b , right panel ) . based on the above neurological and radiological investigations , we diagnosed the patient as having a migrating mass that compressed the l5 nerve root and disc level lumbar disc herniation that compressed the s1 nerve root , causing these symptoms.fig .
1plain ap and lateral radiographs of the lumbar spine . despite slight disc space narrowing at l5-s1
2myelograms showing incomplete block at l5 pedicular level , especially on the right side ( arrows)fig .
43d - ct showing complete absence of the s1 superior articular process ( circle)fig .
b axial mr t2-weighted images showing the herniated nucleus pulposus ( hnp , left panel ) , which migrated cranially ( b , right panel ) plain ap and lateral radiographs of the lumbar spine . despite slight disc space narrowing at l5-s1 , there was no clear abnormality myelograms showing incomplete block at l5 pedicular level , especially on the right side ( arrows ) ct performed following myelography , showing absence of the right l5-s1 facet joint 3d - ct showing complete absence of the s1 superior articular process ( circle ) a mr sagittal images showing disc herniation at l5-s1 intervertebral disc level .
b axial mr t2-weighted images showing the herniated nucleus pulposus ( hnp , left panel ) , which migrated cranially ( b , right panel ) at first , to detect the l5 nerve root , we performed a fenestration at the right l4 - 5 using a microscope . at the l4 - 5 level
we found that the l5 nerve root originated from the caudal level of the l5 pedicle and conjoined with the s1 nerve root ( fig .
the large disc herniation at l5-s1 migrated cranially and was found beneath the abnormal conjoined nerve roots .
after removal of the disc herniation and unroofing of the nerve root , no obvious mobility of the conjoined nerve root was obtained.fig .
the absence of the s1 superior articular processes was obvious , and the l5 and s1 nerve roots were conjoined schema of anomalies in this case .
the absence of the s1 superior articular processes was obvious , and the l5 and s1 nerve roots were conjoined postoperatively , leg pain immediately disappeared and muscle weakness slightly improved 3 months later : mmt of the right tibialis anterior and extensor hallucis longus became 2/5 , and that of the right flexor hallucis longus and triceps surae became 5/5 . at follow - up
examination 1 month later , axial mr t2-weighted images from the s1 pedicle level showed the two confluent ( l5 and s1 ) rootlets ( fig .
the patient was informed that data concerning the case would be submitted for publication , and he consented.fig .
7sagittal mr images obtained 1 month after the surgery , showing two confluent ( l5 and s1 ) rootlets ( arrows ) sagittal mr images obtained 1 month after the surgery , showing two confluent ( l5 and s1 ) rootlets ( arrows )
congenital absence of the lumbosacral articular facet joint is rare , with only 25 cases reported in the english - language literature [ 112 ] . in the past cases ,
rowe et al . found only one case of congenital absence of the inferior articular process of l5 in 1,539 specimens .
the etiology of this anomaly is generally accepted as follows : there are three primary ossification centers in each vertebra : one center in the vertebral body and two in the neural arches [ 5 , 6 , 8 , 11 , 13 ] .
although congenital absence of the lumbosacral articular facet joint seems to cause spinal instability , in only 3 of 25 cases was spinal fusion performed [ 5 , 7 , 13 ] .
most of the symptomatic patients were treated with conservative therapy and became asymptomatic . only in severe cases
numerous cases have been reported in the literature [ 2 , 9 , 12 , 1426 ] .
reported that 0.25% of 1,200 patients who underwent ct or mri had conjoined nerve roots .
kadish et al . also reported that conjoined l5 and s1 roots are the most frequent ( 52.2% ) .
stated that lumbosacral nerve root anomalies were almost asymptomatic and sometimes presented with symptoms by disc herniation .
the present case showed both anomalies , i.e. , complete absence of the superior articular process of s1 and conjoined l5 and s1 nerve roots , which were symptomatically compressed by a large disc herniation .
osseous anomalies in the lumbosacral spine associated with conjoined nerve roots such as this case have been rarely reported .
however , rask stated that the presence of congenital osseous defects of the lumbosacral spine should indicate presence of nerve root anomalies . to our knowledge
, there have been 11 cases , including defect or anomaly of the vertebral arch , spondylosis , spondylolisthesis , and bifid sacrum , presenting with conjoined nerve roots [ 9 , 11 , 12 , 15 , 17 , 2426 ] .
only three previous cases were similar to the present case ( table 1 ) [ 9 , 12].table 1summary of previous cases of congenital absence of the lumbosacral articular facet joint associated with conjoined nerve rootcaseauthorssex , age ( years)symptomslocation of anomaliesconjoined nerve rootspresence of hnp1postacchini et al .
unknownlbpl5-s1l5 & s1present caseyoshioka et al.male , 39lbp , leg pain , palsyl5-s1l5 & s1+hnp herniated nucleus pulposus , lbp low back pain summary of previous cases of congenital absence of the lumbosacral articular facet joint associated with conjoined nerve root hnp herniated nucleus pulposus , lbp low back pain in summary , we report a quite rare case of congenital absence of lumbosacral articular facet and conjoined nerve root .
surgeons should be aware of this nerve root anomaly when a patient shows an unusual clinical presentation and/or congenital osseous anomaly . | we report a rare case of congenital absence of the l5-s1 facet joint , which was associated with a conjoined nerve root .
combination of these two anomalies has been quite rarely reported in the literature .
a 39-year - old man presented with acute low back pain and right leg radiating pain . muscle weakness and sensory disturbance of the right leg were also apparent in the region innervated by l5 and s1 nerve roots .
preoperative multidetector three - dimensional computed tomography ( 3d - ct ) showed complete absence of the right s1 superior articular process .
magnetic resonance ( mr ) images showed lumbar disc herniation at right l5-s1 level that migrated cranially .
intraoperative findings revealed that the right l5 nerve root and s1 nerve root were conjoined , and the conjoined nerve root was compressed by l5-s1 disc herniation , which led to impairment of the conjoined nerve root by a single - level lumbar disc herniation .
after removal of the disc herniation , his right leg pain immediately subsided , however muscle weakness and sensory disturbance persisted .
surgeons should be aware of this nerve root anomaly when examining a patient who shows an unusual clinical presentation and/or congenital osseous anomaly . | Introduction
Case report
Discussion |
optical rotations were
measured on a jasco p-2000 polarimeter ( c g/100 ml )
equipped with a halogen lamp ( 589 nm ) and a 1 ml microcell .
all nmr spectra were acquired with a varian mercuryplus 400 spectrometer
using solvent signals ( dmso - d6 : h , h 2.50 ; c , c 39.52 ;
cdcl3 : h , h 7.24 ppm ; c , c 77.23 ppm ) as references .
c correlations were determined
with gradient - enhanced inverse - detected hsqc and hmbc experiments ,
respectively .
hplc was performed on a shimadzu
lc-20at instrument with a shimadzu spd - m20a uv / vis photodiode detector
and a shimadzu elsd - ltii detector .
the leaves of cornus florida l. were collected from
a one - mile radius around timber lake , oxford ,
mississippi , during the spring and summer of 2011 by m.t.h .
voucher
specimens are kept in the hamann laboratory at the university of mississippi ,
school of pharmacy , oxford , ms ( cf2011 ) .
the leaves of c. florida ( 15.0
kg , dry weight ) were extracted with 90%
aqueous ethanol and dried under reduced pressure to give a crude extract
( 900 g ) .
a portion of this crude extract ( 400 g ) was separated on
a silica gel column ( 20 70 cm ) using a stepwise gradient of
hexanes / etoac ( 100:0 , 80:20 , 50:50 , and 0:100 , v / v , each 3 l ) and
etoac / meoh mixtures ( 80:20 , 60:40 , 50:50 , and 0:100 , v / v , each 3 l )
to afford eight fractions .
fraction e ( 220 g ) was then chromatographed
on hp-20 ( 8 50 cm ) using a stepwise gradient of acetone / water
( 10:90 , 20:80 , 50:50 , 60:40 , 80:20 , and 100:0 , v / v , each 2 l ) to give
six subfractions ( fr .
fraction
e2 ( 40 g ) was chromatographed on a preparative c18 reversed - phase mplc column ( 20 250 mm ; 2035% ch3oh / h2o over 40 min , 3565% ch3oh / h2o over 20 min ; flow rate : 12 ml / min ) to afford eight
subfractions ( fr . e2a
e2h ) . fraction
e2d ( 4.0 g ) was chromatographed on a preparative c18 reversed - phase hplc column ( shim - pak rp - c18 column ;
5 m ; 20 250 mm ; 845% ch3cn / h2o over 120 min , 7 ml / min ) to yield eight subfractions ( fr .
e2d-1e2d-8 ) .
fraction
e2d-4 ( 50 mg ) was purified by c8 reversed - phase
hplc ( polar - c8 ; 5 m ; 10 250 mm ; 1545%
ch3oh / h2o over 90 min , 5 ml / min ) to yield cornusoside
a ( 1 , 5.0 mg , tr 76.2 min ) .
fraction e2d-6 ( 832 mg ) was subjected to silica
gel column chromatography using ch2cl2/meoh
( 90:10 to 0:100 , v / v ) to afford nine fractions ( fr .
fraction e2d-6-a ( 320 mg ) was further purified by c8 reversed - phase hplc
( polar - c8 ; 5 m ; 10 250 mm ; 525% ch3cn / h2o over 70 min , 5 ml / min ) to yield two fractions .
the first fraction ( 60 mg )
was purified on a polymeric hplc column
( hamilton prp-1 ; 5 m ; 20 250 mm ; 1040% ch3cn / h2o over 50 min , 7 ml / min ) to yield cornolactone
c ( 4 , 8.0 mg , tr 41.5 min ) .
the second fraction ( 40 mg ) was chromatographed on a polymeric hplc
column ( hamilton prp-1 ; 5 m ; 20 250 mm ; 1545%
ch3cn / h2o over 70 min , 7 ml / min ) to yield cornolactone
b ( 3 , 4.0 mg , tr 37.9 min ) .
cornolactones a ( 2 , 15.0 mg ) and d ( 5 , 12
mg ) were purified from the remaining crude extract ( 500 g ) using a
similar procedure ( see supporting information ) . colorless gum ; [ ]d 13.8 ( c 0.01 , meoh ) ;
uv ( meoh ) max ( log ) 230 ( 3.4 ) nm ; ir ( kbr )
max 3355 , 2922 , 1730 , 1073 , 1020 cm ; h and c nmr ( 400 mhz , d6-dmso ) , see table 1 ; hresims m / z 413.1417 [ m + na ] ( calcd
for c17h26o10na , 413.1418 ) . colorless gum ; [ ]d + 3.3 ( c 0.2 , meoh ) ; uv ( meoh )
max ( log ) 237 ( 3.2 ) nm ; ir ( kbr ) max 3355 , 2922 , 1730 , 1073 , 1020 cm ; h and c nmr ( 400 mhz , cdcl3 ) , see
table 1 ; hresims m / z 171.1010 [ m + h ] ( calcd for c9h15o3 , 171.1016 ) . colorless gum ; [ ]d 7.5
( c 0.02 , meoh ) ;
uv ( meoh ) max nm 243 ( 3.1 ) nm ; ir ( kbr ) max 2944 , 1658 , 1023 cm ; h
and c nmr ( 400 mhz , cdcl3 ) , see table 2 ; hresims m / z 197.0811
[ m + h ] ( calcd for c10h13o4 , 197.0808 ) . colorless gum ; [ ]d + 13.3 ( c 0.03 , meoh )
; uv ( meoh )
max ( log ) 241 ( 3.1 ) nm ; ir ( kbr ) max 3337 , 2952 , 2937 , 1733 , 1650 , 1017 cm ; h and c nmr ( 400 mhz , cdcl3 ) , see table 2 ; hresims m / z 215.0884 [ m + na ] ( calcd for c11h16o5na , 215.0890 ) .
colorless gum ; [ ]d + 18.5 ( c 0.02 , meoh ) ; uv ( meoh )
max ( log ) 244 ( 2.8 ) nm ; ir ( kbr ) max 3401 , 2952 , 1733 , 1089 , 1068 cm ; h and c nmr ( 400 mhz , cdcl3 ) , see
table 2 ; hresims m / z 193.0834 [ m + na ] ( calcd for c9h14o3na , 193.0835 ) .
cornusoside a ( 1 ) ( 2 mg )
was hydrolyzed by using 1 m hcl ( 0.4 ml ) at 100
c for 2 h under argon and neutralized with amberlite ir400 .
after drying under reduced pressure ,
the residue was dissolved in
pyridine ( 0.4 ml ) containing l - cysteine ethyl ester hydrochloride
( 2 mg ) and heated at 60 c for 1 h. a 0.4 ml solution of 3,5-dichlorophenyl
isothiocyanate ( 2 mg ) in pyridine was added to the mixture , which
was heated at 60 c for 1 h. the reaction mixture was directly
analyzed by analytical hplc on a shim - pak rp - c18 column ,
5 m , 4.6 250 mm , by eluting with a gradient of 3080%
ch3cn in h2o with 0.02% hcooh for 40 min and
subsequent washing of the column with 100% ch3cn at a flow
rate 0.8 ml / min . in the acid hydrolysate of 1 , d - glucose was confirmed by comparison of the retention times of their
derivatives with those of l - glucose and d - glucose
derivatives prepared in the same way , which showed retention times
of 34.8 and 34.0 min , respectively . | a new iridoid glucoside , cornusoside
a ( 1 ) , and four
new natural product iridoid aglycones , cornolactones a d ( 25 ) , together with 10 known compounds
were isolated from the leaves of cornus florida .
the structures of compounds 15 were
established by interpretation of their spectroscopic data .
cornolactone
b ( 3 ) is the first natural cis - fused
tricyclic dilactone iridoid containing both a five- and a six - membered
lactone ring .
a biosynthesis pathway is proposed for cornolactones
c ( 4 ) and d ( 5 ) , the c-6 epimers of compounds 13 . | Experimental Section |
familial mediterranean fever ( fmf ) is an autosomal recessive , autoinflammatory disease predominantly affecting populations of mediterranean origin .
the clinical picture is characterized by recurrent episodes of inflammation and serositis causing fever , peritonitis , pleuritis , and arthritis .
the gene associated with fmf is the mediterranean fever gene ( mefv ) located on chromosome 16 , which encodes for a protein called pyrin .
mutations of pyrin protein lead to uncontrolled attacks of inflammation , while subclinical inflammation continues during attack - free intervals .
the most devastating complication of fmf is renal amyloidosis , leading to nephrotic syndrome and chronic renal failure .
lifelong use of colchicine provides complete remission or marked reduction of the inflammatory attacks and prevents amyloidosis .
if not treated , type aa amyloidosis can cause multi - organ dysfunctions because of the systemic spread .
other genes , such as the saa1 ( serum amyloid a1 ) and the mica ( major histocompatibility chain related gene a ) , also might influence the clinical picture .
some patients with fmf do not experience inflammatory attacks , and present with nephrotic syndrome due to amyloidosis , which is called phenotype 2 disease .
this leads us to wonder if there is more to the pathophysiology of fmf other than being a classical autosomal recessive disease , and to look for the role of innate immunity in this autoinflammatory disease .
natural killer ( nk ) cells are cytotoxic lymphocytes that participate in innate immunity . in addition to their cytotoxic response
the cytotoxicity of nk cells is controlled by surface molecules that are either activator or inhibitor receptors .
killer cell immunoglobulin - like receptors ( kir ) are the regulatory receptors expressed on nk cells and cd8 lymphocytes .
kirs determine target cells by hla class i molecules to help provide selectivity to cellular cytotoxicity [ 1012 ] .
this polymorphic variation may affect the immune response of the nk cell by altering hla selectivity and ligand affinity .
sixteen genes are present in the kir gene cluster ; 6 of these genes ( 3ds1 , 2ds15 ) encode receptors triggering activation and 7 of them ( 3dl13 , 2dl13 , 2dl5 ) encode receptors triggering inhibition of the immune response .
another member of this cluster , 2dl4 , may either activate or inhibit the immune response .
the last 2 kir genes , 2dp1 and 3dp1 , are pseudogenes that do not encode the surface receptors .
activation of innate immunity might be related to pathogenesis of fmf since there is an incomplete penetrance of the disease .
the aim of the current study is to define immunogenetic determinants in the pathogenesis of fmf and determine if kirs are related to susceptibility to disease or complications such as renal amyloidosis .
the study groups were 105 nonconsanguineous turkish patients with fmf ( 56 female , 49 male ; mean age : 29.8 years ) diagnosed by the departments of nephrology at gaziosmanpasa university faculty of medicine in tokat and rheumatology / immunology at cukurova university in adana ; and 100 age- and sex - matched , nonrelated , healthy volunteers ( 53 female , 47 male ; mean age : 29.4 ) from the same ethnic background .
fmf patients were diagnosed according to the tel hashomer clinical criteria and mefv mutations were identified by exon sequencing .
the control group never experienced clinical features of fmf and had no family history for autoinflammatory diseases .
therefore , they were not tested for mefv mutations . with the informed consent of all participants ,
the study was permitted by the ethics review board at gaziosmanpasa university faculty of medicine .
then , 5 milliliters of peripheral blood was obtained from all subjects for kir genotyping .
all fmf patients were attack - free and without active infection at the time of sample collection .
dna from the venous - edta blood sample of all subjects was extracted by a dna isolation kit ( qiaamp dna blood mini kit , cat no : 51104 , qiagen vertriebs gmbh , vienna , austria ) .
genotyping of kir genes were performed by the multiplex kir - sso kit which is a product of tepnel lifecodes corporation immucor , inc .
the product includes locus - specific oligonucleotide probes coupled with color coded microspheres ( luminex corp . ) and 2 pcrs to amplify the kir exons .
allel frequencies of kir haplotypes group a and group b were taken from data pool ( http://www.allelefrequencies.net ) .
individuals having the characteristic gene content of group a haplotypes ( kir2dl1 , 2dl3 , 2dl4 , 2dp1 , 3dp1 , 3dl1 , 3dl2 , 3dl3 and 2ds4 ) were considered as aa genotype .
if any gene among kir2dl2 , 2dl5 , 2ds1 , 2ds2 , 2ds3 , 2ds5 , and 3ds1 were present , the individual was considered to be b haplotype ( genotype bx ) .
each kir gene that is present in any subject is counted directly and the percentage of each kir gene was calculated for the patient and the control groups .
fisher s exact test was used to analyze the results for categorical variables and p values less than 0.05 were accepted as statistically significant .
the study groups were 105 nonconsanguineous turkish patients with fmf ( 56 female , 49 male ; mean age : 29.8 years ) diagnosed by the departments of nephrology at gaziosmanpasa university faculty of medicine in tokat and rheumatology / immunology at cukurova university in adana ; and 100 age- and sex - matched , nonrelated , healthy volunteers ( 53 female , 47 male ; mean age : 29.4 ) from the same ethnic background .
fmf patients were diagnosed according to the tel hashomer clinical criteria and mefv mutations were identified by exon sequencing .
the control group never experienced clinical features of fmf and had no family history for autoinflammatory diseases .
therefore , they were not tested for mefv mutations . with the informed consent of all participants ,
the study was permitted by the ethics review board at gaziosmanpasa university faculty of medicine .
then , 5 milliliters of peripheral blood was obtained from all subjects for kir genotyping .
all fmf patients were attack - free and without active infection at the time of sample collection .
dna from the venous - edta blood sample of all subjects was extracted by a dna isolation kit ( qiaamp dna blood mini kit , cat no : 51104 , qiagen vertriebs gmbh , vienna , austria ) .
genotyping of kir genes were performed by the multiplex kir - sso kit which is a product of tepnel lifecodes corporation immucor , inc .
the product includes locus - specific oligonucleotide probes coupled with color coded microspheres ( luminex corp . ) and 2 pcrs to amplify the kir exons .
allel frequencies of kir haplotypes group a and group b were taken from data pool ( http://www.allelefrequencies.net ) .
individuals having the characteristic gene content of group a haplotypes ( kir2dl1 , 2dl3 , 2dl4 , 2dp1 , 3dp1 , 3dl1 , 3dl2 , 3dl3 and 2ds4 ) were considered as aa genotype .
if any gene among kir2dl2 , 2dl5 , 2ds1 , 2ds2 , 2ds3 , 2ds5 , and 3ds1 were present , the individual was considered to be b haplotype ( genotype bx ) .
each kir gene that is present in any subject is counted directly and the percentage of each kir gene was calculated for the patient and the control groups .
fisher s exact test was used to analyze the results for categorical variables and p values less than 0.05 were accepted as statistically significant .
the framework genes ( kir2dl4 , 3dl2 , 3dl3 , and 3dp1 ) were present in all patients and controls . additionally , a pseudogene , kir2dp1 , was present in all of the patients and in 95% of the controls .
the frequencies of activator kir genes except kir2ds4 were lower than 58% , while the inhibitory genes kir2dl1 and 3dl1 were more frequent in all samples ( > 71% ) ( figure 1 ) .
thirty - eight different genotypes were found in 105 patients with fmf . among the patients ,
23.8% had aa genotypes ( aa1 , aa156 , aa180 ) , which consist of only 1 activating gene , 2ds4 .
seven patients ( 6% ) had all of the 16 kir genes , which is called genotype bx6 , while 20 different genotypes were seen only once .
other genotypes that were seen contained 8 to 15 kir genes ( figure 2 ) .
frequencies of 16 kir genes were compared between fmf patients and healthy controls using fisher s exact test ( table 1 ) .
kir2ds2 gene was significantly more common in the patients compared to the controls ( p=0.036 ) .
the rate of aa and bx genotypes were not different in both groups ( p>0.05 ) ( figure 2 ) .
distribution of individual kir genes / genotypes was evaluated in fmf patients with and without amyloidosis and arthritis .
additionally , relationship between individual kir genes / genotypes and the age of disease onset , serum c - reactive protein ( crp ) levels , erythrocyte sedimentation rate ( esr ) , and mefv mutations ( m694v and r202q ) were evaluated .
there were no statistically significant differences between kir gene frequencies and the rate of aa and bx genotypes of patients with ( n=9)/without amyloidosis ( n=96 ) groups and with ( n=53)/without arthritis ( n=52 ) groups ( p>0.05 ) ( table 2 ) . among our patients ,
when we compared kir gene frequencies of fmf patients with high serum crp levels to those with low serum crp levels , we found that kir3dl1 genes were more common in patients with high serum crp .
a statistically significant difference was found between these 2 groups ( p=0.016 ) ( figure 3 ) . as we compared kir gene frequencies of patients who were carrying m694v and r202q mutations with patients who were not carrying these mutations , we could not find any relationship between kir gene polymorphism and those mutations .
additionally , the kir genes and genotype were not associated with esr and age of disease onset .
the framework genes ( kir2dl4 , 3dl2 , 3dl3 , and 3dp1 ) were present in all patients and controls . additionally , a pseudogene , kir2dp1 , was present in all of the patients and in 95% of the controls .
the frequencies of activator kir genes except kir2ds4 were lower than 58% , while the inhibitory genes kir2dl1 and 3dl1 were more frequent in all samples ( > 71% ) ( figure 1 ) .
thirty - eight different genotypes were found in 105 patients with fmf . among the patients ,
23.8% had aa genotypes ( aa1 , aa156 , aa180 ) , which consist of only 1 activating gene , 2ds4 .
seven patients ( 6% ) had all of the 16 kir genes , which is called genotype bx6 , while 20 different genotypes were seen only once .
other genotypes that were seen contained 8 to 15 kir genes ( figure 2 ) .
frequencies of 16 kir genes were compared between fmf patients and healthy controls using fisher s exact test ( table 1 ) .
kir2ds2 gene was significantly more common in the patients compared to the controls ( p=0.036 ) .
the rate of aa and bx genotypes were not different in both groups ( p>0.05 ) ( figure 2 ) .
distribution of individual kir genes / genotypes was evaluated in fmf patients with and without amyloidosis and arthritis .
additionally , relationship between individual kir genes / genotypes and the age of disease onset , serum c - reactive protein ( crp ) levels , erythrocyte sedimentation rate ( esr ) , and mefv mutations ( m694v and r202q ) were evaluated .
there were no statistically significant differences between kir gene frequencies and the rate of aa and bx genotypes of patients with ( n=9)/without amyloidosis ( n=96 ) groups and with ( n=53)/without arthritis ( n=52 ) groups ( p>0.05 ) ( table 2 ) . among our patients ,
when we compared kir gene frequencies of fmf patients with high serum crp levels to those with low serum crp levels , we found that kir3dl1 genes were more common in patients with high serum crp .
a statistically significant difference was found between these 2 groups ( p=0.016 ) ( figure 3 ) . as we compared kir gene frequencies of patients who were carrying m694v and r202q mutations with patients who were not carrying these mutations , we could not find any relationship between kir gene polymorphism and those mutations .
additionally , the kir genes and genotype were not associated with esr and age of disease onset .
nk cell - mediated cytotoxicity is regulated by surface receptors that can either be inhibitors or activators . two types of receptors provide the nk cells with target selectivity by interacting with hla class i molecules .
c - type lectin - like receptors ( cd94/nk62 heterodimers ) bind to the hla - e and some peptides derived from other hla class 1 molecules , while the kirs are immunoglobulin - like receptors which can recognize polymorphic hla - a , -b , or -c determinants .
alternations in kir expression on the nk cells can be related to the development of some malignancies , as well as autoimmune and inflammatory diseases [ 1922 ] .
normally , inhibition and activation cycles of kirs are kept in balance , but an increase in the activation of nk cells may result in proliferation and change the immune response .
activator kir genes bind to hla determinants and enhance the activation of nk cells and cd8 + t cells , which leads to strengthened cellular cytotoxicity and increased cytokine production .
genetic variations in the kir gene family may also affect the immune response of the nk cell and contribute to the phenotype of inflammatory diseases like fmf .
kir genotype is quite variable among different ethnic groups . in the current study we investigated the kir gene frequencies and genotype in 105 unrelated turkish fmf patients
this is the first study to demonstrate the kir genes and genotype of turkish patients with fmf and the first study to compare the kir genes with the clinical features of fmf .
we found kir2ds2 to be more frequent in our fmf patients compared to in heathy controls ( p=0.036 ) .
since it is an activator gene , we think that the presence of kir2ds2 might be associated with susceptibility to fmf .
a previous study by mahfouz et al . showed a higher prevalence of a pseudogene kir 3dp1 * 003 in 56 lebanese fmf patients .
not only did we have a larger group of fmf patients , but also the ethnicity was different .
therefore , we suggest the importance of kir2ds2 in fmf for further research with on ethnic background . our study also aimed to search for possible relations between kir genes and genotype with complications of fmf particularly renal amyloidosis .
type aa amyloidosis is the most important complication of fmf causing nephrotic syndrome and end - stage renal disease ( esrd ) .
amyloidosis might develop in a small subset of fmf patients even if they are treated with colchicine .
this leads researchers to investigate other factors contributing to the susceptibility to disease and disease complications , like renal amyloidosis .
we did not find any study about kir genes and amyloidosis in our literature search .
all 9 patients with amyloidosis were bx genotype ; exhibiting 1 or more of the activator kir genes , but there was no significant relation for kir genotype and renal amyloidosis .
fmf patients with amyloidosis also exhibited kir2ds2 at a rate higher than the patients without amyloidosis ( 66.7% over 54% ) , but the difference was not significant .
the effect of kir2ds2 in predisposition to renal amyloidosis could be studied with a larger group of fmf patients with amyloidosis .
the acute - phase proteins crp and saa levels are elevated as a result of inflammation during febrile attacks in fmf patients . although colchicine prophylaxis prevents febrile attacks in fmf , crp and/or saa levels may persist to be higher than normal in fmf patients during the attack - free period
. we did not measure saa levels of the patients , but we measured serum crp levels during attack - free periods . in patients with high serum crp levels ( > 0.8 mg / dl ) ,
kir3dl1 , which is an inhibitor gene , was significantly more common ( p=0.016 ) .
although crp is not involved in effector - target cell - mediated recognition , it has been demonstrated that crp is present on at least certain nk effectors , thus crp or a molecule that co - caps with crp is required for optimal nk cell function .
our finding of inhibitor kir3dl1 association with higher crp in fmf patients may indicate some direct or indirect preventive role of kirs in inflammation .
it has been proposed that evolutionary pressures , such as pathogen - mediated immune evasion strategies , may drive kir3dl1 diversification .
we can speculate that inhibitor kir3dl1 might provide a selective advantage in fmf patients with high crp to prevent hazardous effects of inflammation , such as increased cardiovascular risk or even renal amyloidosis . further studies of crp measurements , as well as kir gene studies , in larger fmf patient groups and healthy populations are needed to obtain a more precise conclusions .
finally , we want to point out some other studies indicating activator kir genes and inflammatory conditions .
a recent study zal et al . showed that cd4 + t cells exhibited increased kir2ds2 and reduced kir2dl3 expression in patients with end - stage renal disease compared to nondialysis - dependent chronic kidney disease patients .
they suggested that immune activation through kir expression might increase with progressive renal impairment and contribute to cardiovascular risk .
the latter study , which indicates the enhancing role of activator gene kir2ds2 for the immune response denotes that patients with ra positive for kir2ds2 and kir3ds1 , showed risk associations .
another activator gene , kir2ds4 , was found to be associated with acute rejection in renal transplant recipients by kunierczyk , and the relation was stronger for patients whose esrd was caused by glomerulonephritis .
interestingly , kir2ds5 , which is also an activator , was found to be protective against acute rejection in the same study . a meta - analysis provided evidence that kir2dl1 , kir2ds4 , kir2ds5 and kir3ds1 gene variations may contribute to susceptibility to ankylosing spondylitis .
the association with kir2dl1 is interesting yet it is an inhibitory receptor for nk cell activities .
we could conceivably think that combinations of variations in activating , inhibitory , and pseudo kir genes is crucial for some diseases .
the innate immunity at the receptor level may be an accelerator for the disease process .
these variations may cause a genetic imbalance and change the course of some diseases . in this study we found kir2ds2 to be more frequent in 105 fmf patients , for the first time demonstrating the kir genotype of turkish patients with fmf .
we suggest that the presence of kir2ds2 , which is an activator gene for nk cell functions , might be related to the autoinflammation in fmf .
none of the kir genes was associated with common mefv or age of disease onset .
there was no significant difference between kir gene frequencies and genotypes of the patients with renal amyloidosis .
the potential effect of kir genes on predisposition to renal amyloidosis and other clinical features requires studies with larger sample sizes . | backgroundfamilial mediterranean fever ( fmf ) is an autosomal recessive autoinflammatory disease predominantly affecting mediterranean populations .
the gene associated with fmf is the mefv gene , which encodes for a protein called pyrin .
mutations of pyrin lead to uncontrolled attacks of inflammation , and subclinical inflammation continues during attack - free intervals .
killer cell immunoglobulin - like receptor ( kir ) genes encode hla class i receptors expressed by nk cells .
the aim this study was to look for immunogenetic determinants in the pathogenesis of fmf and find out if kir are related to susceptibility to disease or complications like renal amyloidosis.material/methodsone hundred and five patients with fmf and 100 healthy individuals were involved in the study .
isolated dna from peripheral blood was amplified by sequence specific pcr probes and analyzed by luminex for kir genotypes .
fisher exact test was used to evaluate the variation of kir gene distribution.resultsall patients and healthy controls expressed the framework genes .
an activator kir gene , kir2ds2 , was significantly more frequent in fmf patients ( p=0.036 ) .
renal amyloidosis and presence of arthritis were not associated with kir genes and genotype .
kir3dl1 gene was more common in patients with high serum crp ( p=0.016).conclusionsaccording to our findings , we suggest that presence of kir2ds2 , which is an activator gene for nk cell functions , might be related to the autoinflammation in fmf .
the potential effect of kir genes on amyloidosis and other clinical features requires studies with larger sample sizes . | Background
Material and Methods
Study group selection and samples
KIR genotype
Prediction of haplotype and genotype
Statistical Analysis
Results
KIR gene and genotype distribution in FMF patients and healthy controls
Relationship of KIR genes with FMF
Distribution of KIR genes/genotypes of FMF patients with different clinical features
Discussion
Conclusions |
most of the evidence which suggests that myocarditis and inflammatory cardiomyopathy are autoimmune diseases come from animal models [ 1 , 2 ] .
the macrophages and t lymphocytes were found to be directly engaged in cell - mediated immune mechanism in experimental autoimmune myocarditis ( eam ) [ 35 ] .
gold standard for myocarditis and both cells mentioned above might be assessed in it [ 6 , 7 ] . to increase the diagnostic sensitivity ebm immunohistochemistry
may be used for the identification of the inflammatory infiltration and for the detection of hla - dr upregulation .
progression from myocarditis to dilated cardiomyopathy ( dcm ) seems to occur predominantly in patients with histologically confirmed persistent ( chronic ) inflammation . besides cell - mediated myocytes damage
, various cytokines including interleukin-1 ( il-1 ) , interleukin il-2 ( il-2 ) , interleukin il-6 ( il-6 ) , interferon- ( ifn- ) , and tumor necrosis factor- ( tnf- ) have multiple biological activities and modulate immune response [ 10 , 11 ] .
neopterin ( npt ) arising from guanosine triphosphate ( gtp ) is mainly secreted by activated macrophages . ifn- and
tnf- were found to be the most frequent inducers of its synthesis . elevated ntp level was observed in infectious diseases , allograft rejection , disorders with potential autoimmune pathogenesis , and atherosclerosis [ 1315 ] . despite npt being produced locally
-2 microglobulin ( -2 m ) is a light chain of hla class i and its elevated serum concentration indicates cell surface expression of hla .
increased level of -2 m was reported in viral infections and in chronic inflammatory and lymphoproliferative disorders [ 16 , 17 ] .
accordingly , the aim of this study was to assess the usefulness of npt and -2 m serum concentration as the potential biomarkers that may reflect upregulation of immune system in dcm patients .
we recruited 172 patients hospitalized due to dilated cardiomyopathy and diagnosed according to the who criteria who underwent endomyocardial biopsy .
chronic heart failure was recognized if dyspnea or fatigue at rest or on exertion in association with an ejection fraction ( ef ) 45% was present .
all patients were clinically stable and received optimal conventional heart failure therapy including ace inhibitors , -blockers , digitalis , and diuretics for at least 6 months .
patients were excluded from the study if they had at least one the following : any changes in coronary vessels as assessed by coronary angiography , valvular disease except relative mitral regurgitation , connective tissue disease , endocrine disorders , renal insufficiency ( estimated glomerular filtration rate using modification of diet in renal disease - mdrd formula < 60 ml / min/1.73 m ) , infectious disease , malignancy , moderate or severe hypertension , and alcohol abuse .
echocardiographic images were acquired in standard views as recommended by the american society of echocardiography committee . left ventricular end - diastolic volume ( edv ) and end - systolic volume ( esv ) were obtained from the apical 4- and 2-chamber views by modified simpson 's method . left ventricular ejection fraction ( ef )
was calculated in a standard manner as follows : edv esv 100/edv to assess ventricular systolic function .
the new york heart association ( nyha ) classification was used to assess functional capacity . to rule out coronary artery disease all patients underwent coronary angiography .
blood samples for laboratory assessments were obtained from the patients at time of the biopsy .
serum was separated by centrifugation at 1500 g for 10 minutes and was frozen at 70c and protected from light .
neopterin and -2 microglobulin concentrations were determined in serum by elisa method using commercially available kit manufactured by ibl , hamburg ( germany ) .
serum for -2 microglobulin assay was diluted in a ratio of 1 : 50 .
the intra- and interassay coefficient of variation were < 11.6% and < 15.5% . additionally , we also determined serum creatinine , fibrinogen , and hs - crp concentrations using routine techniques . at endomyocardial biopsy procedure
a minimum of four specimens was obtained from the right side of the ventricular septum .
histopathological examination was performed by light microscopy according to dallas classification and immunohistologically as described previously . for immunohistochemistry
, frozen sections were incubated with murine monoclonal antihuman antibodies : anti - hla class ii ( dr antigens ) , alpha chain ( clone tal.1b5 ) , anti - hla class i ( abc antigens , clone w6/32 ) , anti - cd3 for t lymphocytes ( clone t3 - 4b5 ) , and antimacrophages ( clone ebm11 ) .
the bound primary antibody was detected using the envision method ( dako envision kit / dako a / s ) .
each specimen was evaluated qualitatively and quantitatively for cd 3 lymphocytes and macrophages count and semiquantitatively for hla expression on the previously defined and presented below immunoreactivity scoring system ir : 0 lack of focal staining on the endothelial and interstitial cells , 1 + focal staining on the endothelial and other interstitial cells , 2 + multifocal staining restricted to interstitial cells , 3 + diffuse endothelial and concomitant focal cardiac myocyte staining , 4 + diffuse endothelial and myocyte staining .
0 lack of focal staining on the endothelial and interstitial cells , 1 + focal staining on the endothelial and other interstitial cells , 2 + multifocal staining restricted to interstitial cells , 3 + diffuse endothelial and concomitant focal cardiac myocyte staining , 4 + diffuse endothelial and myocyte staining .
ir 3 + was assessed as positive for major histocompatibility complex . for the purpose of this study patients
group a is patients with positive ir 3 + for hla abc or hla dr , considered as having strong immune mediated inflammation .
group b is patients with weak immunoreactivity , staining of hla complex ir of 02 + .
group c is control group for neopterin and -2 microglobulin assay comprised of healthy volunteers from hospital staff ( 8 male and 22 female , aged 39.03 9.86 years ) .
normally distributed data are described as mean and sd , but nonnormally distributed data are presented as median and interquartile range .
frequencies of categorical variables were compared using chi - square test with yates correction ( ) .
continuous variables between groups were compared using student 's t - test but nonnormally distributed data were compared using mann - whitney u test .
a nonparametric kruskal - wallis h test was used to analyze differences of neopterin and -2 microglobulin concentrations among examined groups of patients and post hoc mann - whitney u test was applied .
spearman 's rank correlation coefficients ( r ) were computed for assessing mutual association among biochemical , biopsy and clinical results .
the strong expression of hla abc or hla dr ( ir 3 + ) was detected in 46 patients group a , low expression ( ir 1 or 2 + for at least one hla class ) was indicated , respectively , in 123 patients group b , and lack of expression ( hla abc and hla dr ir = 0 ) was detected in 3 patients who were excluded from analysis .
clinical characteristic of all dcm patients and groups a and b is presented in table 1 . in patients with stronger hla expression ,
lymphocytes cd3 and macrophages infiltrations were significantly more often presented in biopsy specimens . in group
a 26.1% ( 12/46 ) patients were diagnosed as active myocarditis according to dallas criteria in comparison to 6.5% ( 8/123 ) patients from group b ( p < 0.001 ) . as indicated , patients with stronger hla expression presented worse functional capacity assessed by nyha class ( p < 0.01 ) , but other clinical and echocardiographic parameters did not differ .
the concentrations of serum cell immune activation and inflammation markers are shown in tables 2 and 3 .
neopterin concentration was increased in patients with strong activation of hla molecule compared to the control group , p < 0.05 . the difference in neopterin concentrations between dcm groups
a and b was not significant ( table 2 ) . on the other hand , among groups according to the dallas criteria , neopterin level was higher in the group of patients with mci ( combined borderline and active ) than in patients without mci and control where the difference was statistically significant ( table 3 ) .
-2 microglobulin concentration was significantly higher in both dcm groups than in control , respectively : group a p < 0.001 and group b p < 0.00 . additionally -2 microglobulin level was nonsignificantly elevated in patients with active myocarditis according to the dallas criteria than in dcm patients without myocarditis .
higher serum neopterin levels were observed in patients with functional classes nyha iii and iv than in combined i and ii nyha functional classes ( figure 1 ) .
spearman 's rank correlations among npt and -2 m levels and acute phase proteins and echocardiographic parameters in all dcm subjects are presented in table 4 .
only neopterin concentration had significant correlation with count of macrophages in endomyocardial biopsy specimens ( figure 2 ) .
significant correlation of left ventricular ejection fraction and nyha class with neopterin ( p < 0.05 ) and nonsignificant trend towards significance of -2 microglobulin with ejection fraction ( p = 0.09 ) were observed .
there was positive correlation between serum concentration of cellular immune markers and acute phase proteins ( figures 3 , 4 , and 5 ) .
patients with heart failure due to dcm , especially if the conventional therapy is ineffective for them , need further diagnostic procedure including endomyocardial biopsy .
immune - mediated myocarditis may be identified in about one - third of dcm patients particularly in young adults and in cases of immune activation without presence of viruses immunosuppression therapy may be used . up to now
no available serum markers have been acknowledged as helpful in diagnostic decision making and choice of the best treatment option .
we decided to assess whether serum npt and -2 m may be useful for diagnostic procedure in dcm patients because those parameters are valuable tools for detection , staging , and monitoring of other diseases [ 13 , 22 , 23 ] .
moreover , npt and -2 m are stable biomarkers of cellular immune activation , easy to measure , and not expensive .
some recent analyses have shown that serum levels of npt are elevated also in patients with coronary artery disease and carotid and peripheral artery disease .
the concentration of this molecule might predict adverse cardiovascular events in patients with coronary and carotid arteries disease .
in addition , npt level is related to the development of heart failure [ 24 , 25 ] .
the local and systemic inflammations play an important role in the development of atherosclerosis and are responsible for the instability in coronary artery disease .
therefore we intended to investigate the group of nonischemic cardiomyopathy to exclude influence of atherosclerosis on inflammatory markers .
it should be emphasized that the biopsy procedure was performed at patients with chronic and clinically stable heart failure treated optimally for at least 6 months without complete recovery .
it is relatively late according to current clinical practice guidelines and partly explains the low percentage of active myocarditis .
the increased concentrations of npt are found in dilated cardiomyopathy group with strong immune activation
hla expression in endomyocardium in comparison to controls , but the difference with the group of weak immune activation was not significant .
elevated npt concentrations in patients with heart failure due to idiopathic or inflammatory dcm were described previously , especially in patients with active myocarditis assessed according to the dallas criteria [ 2729 ] . in our patients
active myocarditis was diagnosed only in twenty patients and npt concentration was increased in mci group including active and borderline compared to controls .
did not find any differences either in mean value or in frequency of abnormal results of npt among idiopathic cardiomyopathy , ischemic cardiomyopathy patients , and controls , but patients with myocarditis according to the dallas criteria were excluded from the study .
data concerning -2 m concentrations in dilated cardiomyopathy are different . in keeping with our results
found elevated -2 m plasma level correlating with parameters of stimulated renin - angiotensin system and with t cellular hyperresponsiveness in dcm patients .
observed the higher mean level and the proportion of abnormal results of -2 m in ischaemic but not in idiopathic cardiomyopathy . in our study
we failed to find associations between serum npt or -2 m levels and hla expression or lymphocytes cd3 count in endomyocardial specimens ; only correlation of npt with macrophages infiltrations was found .
similarly , correlations between serum npt or -2 m levels and hla class i and ii and icam-1 expression in patients with idiopathic non- inflammatory cardiomyopathy was not found .
lack of correlations between concentration of -2 m , light invariant chain of the class major histocompatibility molecule , and hla i expression in immunohistological staining endomyocardium may be surprising .
some data obtained so far indicate that there is no simple correlation among hla i complex on the surface of cells , its soluble form , and its part -2 microglobulin in serum [ 32 , 33 ] .
most of hla class i molecules on the surface of cells are connected with presented antigens during interaction with t lymphocytes .
serum npt and -2 m concentrations depend on their production in states of lymphocytes t stimulation and renal elimination .
previous studies have shown that serum level of npt in group with creatinine clearance > 60 ml / min was comparable to healthy control and significantly higher in group with creatinine clearance 1060 ml / min . -2 m is an indicator of gfr , but some nonrenal factors were found independent of kidney function predictors of its concentration in healthy elderly ( e.g. , crp level and white blood cells count ) . in patients with heart failure glomerular filtration
may be decreased , so we excluded patients with egfr < 60 ml / min/1.73 m. we observed that increased npt concentrations were associated with higher nyha class .
similar observations of elevated npt and/or -2 m concentrations in dilated cardiomyopathy were noticed earlier [ 2931 ] . in our dcm group like in previous data higher serum npt and -2 m concentrations correlated with lower left ventricular ejection fraction [ 30 , 31 ] .
the participation of inflammation and immune reactions in pathogenesis of heart failure was confirmed in clinical and experimental investigations .
the concentrations of tnf- and inf- were reported to be raised in patients with heart failure [ 36 , 37 ] . both are known stimulators of npt synthesis , hla expression , and turnover with -2 m secretion into blood [ 38 , 39 ] .
the mechanisms of immune activation and the place of cytokines , npt , and -2 m generation are not precisely established .
increased concentrations of cytokines may be associated with the immune reactions in cardiac tissue during viral infection and inflammation stage or the excessive load of the heart . beside the heart , their source may be other organs for example , intestines .
disturbances resulting from circulatory insufficiency such as hypoxia or congestion may cause inflammation by free radicals reactions and bacterial penetration through intestinal wall , therefore in some reports congestive heart failure is considered a state of chronic low - grade inflammation .
the positive correlation between neopterin and acute phase proteins concentration indicated in our study might confirm these data . on the other hand
, the experimental data indicated that npt might be a pathogenic factor in the development of cardiac dysfunction in chronic disease states with high neopterin levels secondary to activation of the immune system .
the upregulation of hla on cardiac tissue and their damage is a criterion which makes it possible to recognize active chronic myocarditis . despite various data , concerning the effectiveness of immunosuppressive treatment
, we can offer this therapy to patients with chronic myocarditis with increased hla expression as an efficient method to improve the systolic function of left ventricle .
it is worth noting that an active inflammation and strong immune response were revealed in some cases of relatively long - lasting dilated cardiomyopathy .
perhaps biopsy should be considered in dilated cardiomyopathy patients with elevated immune and inflammatory markers especially neopterin concentration .
increased serum concentrations of neopterin reflect strong inflammatory - immunological response in endomyocardium of patients with dilated cardiomyopathy . -2
microglobulin concentration is elevated in all patients with dcm independently on immune activation in myocardium .
correlations between above markers of cellular immune activation with acute phase proteins level and left ventricle ejection fraction suggest the participation of low grade inflammatory state in pathophysiology of heart failure potentially related to its severity . |
background .
the aim of the study was to assess the relationships among serum neopterin ( npt ) , 2-microglobulin ( 2-m ) levels , clinical status , and endomyocardial biopsy results of dilated cardiomyopathy patients ( dcm ) .
methods .
serum npt and -2 m were determined in 172 nonischaemic dcm patients who underwent right ventricular endomyocardial biopsy and 30 healthy subjects ( elisa test ) .
the cryostat biopsy specimens were assessed using histology , immunohistology , and immunochemistry methods ( hla abc , hla dr expression , cd3 + lymphocytes , and macrophages counts ) .
results .
the strong increase of hla abc or hla dr expression was detected in 27.2% patients group a being low in 72.8% patients group b. neopterin level was increased in patients in group a compared to healthy controls 8.11 ( 4.5012.57 ) versus 4.99 ( 2.668.28 ) nmol / l ( p < 0.05 ) .
-2 microglobulin level was higher in dcm groups a ( 2.60 ( 1.713.58 ) ) and b ( 2.52 ( 1.513.72 ) ) than in the control group 1.75 ( 1.281.96 ) mg / l , p < 0.001 .
neopterin correlated positively with the number of macrophages in biopsy specimens ( p < 0.05 ) acute phase proteins : c - reactive proteins ( p < 0.05 ) ; fibrinogen ( p < 0.01 ) ; and nyha functional class ( p < 0.05 ) and negatively with left ventricular ejection fraction ( p < 0.05 ) .
conclusions .
neopterin but not -2 microglobulin concentration reflected immune response in biopsy specimens .
neopterin correlated with acute phase proteins and stage of heart failure and may indicate a general immune and inflammatory activation in heart failure . | 1. Introduction
2. Study Group and Methods
3. Results
4. Discussion
5. Conclusion |
gemcitabine is a chemotherapeutic agent frequently used by for the treatment of several malignancies both in the adjuvant and metastatic setting .
although myelosuppression is the most adverse event of this therapy , gemcitabine might induce severe pulmonary toxicities .
we describe a case of pulmonary veno - occlusive disease ( pvod ) related to gemcitabine .
the patient was an 83-year - old man with a metastatic pancreatic cancer who was treated by gemcitabine as first - line therapy .
a dose of 1000 mg / m of gemcitabine was administered over a 30-minute intravenous infusion on days 1 , 8 , and 15 of a 28-day cycle . after a period of 6 months , a complete response was observed .
nevertheless , the patient developed a severe dyspnea , with arterial hypoxemia and very low lung diffusion for carbon monoxide .
a ct scan showed diffuse ground glass opacities with septal lines , bilateral pleural effusion , and lymph node enlargement .
on echocardiography , there was a suspicion of pulmonary hypertension with elevated systolic pulmonary artery pressure and normal left ventricular pressures .
a symptomatic treatment was started . at last follow - up , patient was in functional class i with near - normal of ct scan , arterial blood gases , and echocardiography .
it is relatively well tolerated , and pulmonary toxicities are usually mild and self - limiting .
although up to 25% of patients treated with gemcitabine may develop dyspnea , severe pulmonary adverse events remain rare and include mainly diffuse alveolar damage , acute respiratory distress syndrome , interstitial pneumonitis , or noncardiogenic pulmonary edema requiring steroid therapy.13 recently , roychowdhury et al analyzed the incidences of pulmonary toxicity events in 4,448 patients treated with gemcitabine in clinical trial databases.4 the authors reported 0.27% of other serious pulmonary toxicities . based on an estimated 217,400 patients treated with commercial gemcitabine worldwide , the crude incidences of dyspnea and other serious pulmonary toxicity events were 0.02% and 0.06% , respectively.4 however , these publications lack a precise description of gemcitabine - induced lung toxicities . the clinical presentation of these pulmonary diseases is mostly a sub - acute clinical syndrome and is frequently nonspecific
. there are several possible diagnoses , and the delay between gemcitabine introduction and the onset of pulmonary symptoms is variable as described in previous case reports ( table 1).115 we report a case of pulmonary veno - occlusive disease ( pvod ) related to gemcitabine .
written informed consent was obtained from the next of kin of the patient for publication of this case report and accompanying images .
an 83-year - old man , without a history of cardiopulmonary disease , was admitted to our institution for epigastric pain .
only an antecedence of high blood pressure was reported and the concomitant medications were plavix , cholesterol - lowering therapy , finasteride for a benign prostatic hypertrophy , and an antihypertensive medication . of note ,
after extensive medical tests , a pancreatic tumor was diagnosed , measuring 42 mm and associated with the involvement of lymph nodes . a biopsy revealed the presence of an undifferentiated adenocarcinoma of the pancreas .
doses of 1000 mg / m of gemcitabine were administered as a monotherapy over a 30-minute intravenous infusion on days 1 , 8 , and 15 of a 28-day cycle .
a total of 24 injections ( eight cycles ) of gemcitabine were administered without any extra - pulmonary side effects .
six months after the beginning of chemotherapy , an assessment of his disease was performed by a ct scan , which showed a complete response on liver metastases and partial response on primary pancreatic tumor .
seven months following gemcitabine introduction , the patient developed dyspnea on exertion ( new york heart association [ nyha ] class iii ) and hemoptysis , slowly increasing for several weeks .
the patient needed hospitalization , and an oxygen therapy was required . on physical examination ,
electrocardiogram showed a right bundle branch block with a left anterior hemiblock . a ct scan identified diffuse ground glass opacities with thickening of the interlobular septa and mild pleural effusion .
infectious causes were ruled out because of the absence of fever and negative microbiological testing .
bronchoscopy was performed , and analysis of the bronchoalveolar lavage showed neutrophilia ( 49% ) and small eosinophilia ( 10% ) .
there was a moderate chronic alveolar hemorrhage with siderophages ( golde score = 63 ) .
pulmonary function tests after this procedure showed a slight restrictive pattern ( vital capacity 65% , normal tiffeneau ratio ) , whereas diffusing capacity of the lung for carbon monoxide ( dlco ) was reduced to 32% of the predicted value .
right ventricular cavity was dilated , and systolic pulmonary artery pressure was estimated at 80100 mmhg .
precapillary pulmonary hypertension with mean pulmonary artery pressure at 45 mmhg and pulmonary artery occlusion pressure at 7 mmhg .
cardiac index was maintained at 2.7 l / min / ml and venous oxygen saturation ( svo2 ) was measured as 57% .
blood was taken during right heart catheterization after occlusion of a pulmonary artery for cytological examination , but no circulating tumor cells were found .
the diagnosis of pvod ( group 1 of the nice classification ) was then hypothesized and gemcitabine was stopped .
high doses of diuretics and curative doses of heparin were started . in consequence , the clinical status of the patient was stabilized .
the patient was still in complete remission 24 months after the diagnosis of his pancreatic carcinoma .
eighteen months after stopping chemotherapy , an improvement in dyspnea ( nyha ii ) was noted , and moderate physical activity began once again .
gemcitabine ( 2,2-difluoro-2-deoxycytidine [ dfdc ] ) is a deoxycytidine analog with multiple modes of action inside the cell . as a prodrug , dfdc ,
the principal pharmacological activity of the drug is its incorporation into dna during replication in the s phase of the cell cycle .
the induction of apoptosis through caspase signaling is also another important mechanism of action.16 the precise molecular mechanisms determining tumor cell responses to gemcitabine and the impact of mechanistic interactions with other chemotherapeutic agents remain unelucidated.17 gemcitabine is generally well tolerated , with common side effects including nausea and vomiting , rash , fever , reversible elevation of liver transaminases , flu - like symptoms , and peripheral edema .
myelosuppression is the most common dose - limiting toxicity.18 various pulmonary toxicities of gemcitabine have been reported .
pulmonary toxicities due to gemcitabine have been reported , including bronchospasm , capillary leak syndrome , non - cardiogenic pulmonary edema , hypersensitivity reaction , acute respiratory distress syndrome , diffuse alveolar damage , pleural effusion , and chronic interstitial pneumonitis .
belknap has reviewed the data from spontaneous reports to research on adverse drug events and reports of pharmacovigilance ( radar ) program and appraised the clinical feature of gemcitabine - associated severe acute lung injury . among 178 reports of gemcitabine - induced pulmonary injury ( 55 from clinical trials and 92 from spontaneous reports ) , dyspnea , fever , and pulmonary infiltrate were the most frequent symptoms .
eleven phase ii or phase iii clinical trials enrolling 317 patients identified pulmonary injury rates > 10%.19 the present case report provides further evidence that chemotherapy may produce pvod . to our knowledge , this is the second case of pvod probably related to gemcitabine administration.20 in the first case , the patient was treated for lung cancer .
dyspnea appeared during the second cycle , and clinical and radiological signs were the same as in our patient .
the right catheterization confirmed the diagnosis because the mean pulmonary artery pressure was elevated to 35 mmhg .
pvod is uncommon and belongs to group 1 in new classification of pulmonary arterial hypertension ( pah).21 it is characterized by elevated pulmonary artery pressure leading to right heart failure .
several risk factors for pvod have been described , including infection , genetic factors , autoimmune disorders , congenital heart disease , and exposure to toxins .
pvod has also been found in association with a variety of different tumors treated with chemotherapy protocols that mostly contained mitomycin , bleomycin , gemcitabine , or carmustine.19 formal diagnosis of pvod is based on histopathology22 and requires a lung biopsy or pathologic examination of pulmonary explants or postmortem lung samples .
histopathologic features are venous changes with intimal obstruction , congested capillaries , dilated lymphatics , and alveoli with hemosiderin - laden macrophages . however , lung biopsy is hazardous in patients with severe pulmonary hypertension , and this histological evidence is rarely available .
the diagnosis of pvod was considered highly probable if the patients fulfilled the characteristics previously described : hypoxemia , post - capillary pulmonary hypertension confirmed by right heart catheterization ( with an increase of pulmonary pressure and a pulmonary artery occlusion pressure normal ) , presence of radiological abnormalities on high - resolution ct ( hrct ) of the chest ( including lymph node enlargement , centrilobular ground - glass opacities , and septal lines ) , low dlco , and occult alveolar hemorrhage .
the diagnosis of drug - induced lung disease is made by the exclusion of other potential causes , including congestive heart failure , infections , auto - immune disease , or lymphangitic carcinomatosis .
it has generally been accepted that a diagnosis of chemotherapy - induced pulmonary toxicity can be made when it develops shortly after the initiation of treatment , when there is lack of an alternative explanation of respiratory and/or cardiac failure , and , in some cases , when there is the resolution of symptoms after corticosteroid treatment and withdrawal of the presumed agent.23 in the present case report , the diagnosis was proposed on the basis of the presence of a severe pulmonary hypertension associated with typical clinical and radiological features .
differential diagnoses were ruled out by physical , biological , and radiological examinations . indeed , an hrct scan did not support any arguments in favor of pulmonary embolism , and immune infiltration of the lung
the absence of tumor cells in the bronchoscopy and the absence of disease progression during the follow - up ruled out a cancer - related lung infiltration . finally , tumor emboli were unlikely because anatomo - pathological examination of arterial blood was negative and usually described with a precapillary pulmonary hypertension .
the evolution was an argument for drug toxicity because the clinical condition of patient improved after discontinuing gemcitabine .
moreover , no other therapy was introduced that could have been responsible for the development of respiratory problems .
no specific treatment of pah ( such as epoprostenol ) was introduced , which might have produced an adverse event .
a score of 7 was obtained using the naranjo adverse drug reaction probability scale , suggesting gemcitabine as the most probable cause of pvod in our patient.24 clinical course of pvod is often unfavorable , leading to death of the patient .
various treatment options have been recommended for pvod , such as vasodilators , immunosuppressive medications , anticoagulant or antithrombotic agents , and , most of the time , oxygen therapy .
while specific pah therapies such as intravenous prostacyclin have established efficacy in the treatment of pah , benefits of these treatments in patients with pvod are unclear , as these patients may be refractory to pah - specific therapy and may even deteriorate with it .
lung transplantation is the only curative treatment for pah.25 unfortunately , this treatment can only rarely be proposed and contraindicated in cases of cancer .
although this chemotherapy is very well tolerated , pvod might be considered during the investigations performed to characterize any serious pulmonary toxicity of gemcitabine . this serious complication is rare , but can potentially be fatal or have a significant detrimental effect , resulting in severely reduced functional capacity and dependency on supplemental oxygen .
this report illustrates the importance of early detection and treatment in maintaining the quality of life for patients . | introductiongemcitabine is a chemotherapeutic agent frequently used by for the treatment of several malignancies both in the adjuvant and metastatic setting .
although myelosuppression is the most adverse event of this therapy , gemcitabine might induce severe pulmonary toxicities .
we describe a case of pulmonary veno - occlusive disease ( pvod ) related to gemcitabine.case presentationthe patient was an 83-year - old man with a metastatic pancreatic cancer who was treated by gemcitabine as first - line therapy .
he was in good health and received no other chemotherapy .
a dose of 1000 mg / m2 of gemcitabine was administered over a 30-minute intravenous infusion on days 1 , 8 , and 15 of a 28-day cycle . after a period of 6 months , a complete response was observed .
nevertheless , the patient developed a severe dyspnea , with arterial hypoxemia and very low lung diffusion for carbon monoxide .
a ct scan showed diffuse ground glass opacities with septal lines , bilateral pleural effusion , and lymph node enlargement .
on echocardiography , there was a suspicion of pulmonary hypertension with elevated systolic pulmonary artery pressure and normal left ventricular pressures .
right heart catheterization confirmed pulmonary hypertension and normal pulmonary artery occlusion pressure .
diagnosis of pvod was made , and a gemcitabine - induced toxicity was suspected .
a symptomatic treatment was started . at last follow - up
, patient was in functional class i with near - normal of ct scan , arterial blood gases , and echocardiography .
a gemcitabine - induced pvod is the more likely diagnosis . | INTRODUCTION
CASE PRESENTATION
Introduction
Case Presentation
Discussion
Conclusion |
halogen and hydrogen bonding plays an essential role in chemistry and biochemistry [ 114 ] . recently , the halogen bonding , a xb ( x halogen atom , b
lewis base , a electronegative atom ) , attracted considerable attention due to its strong , selective and directional character [ 414 ] .
these features make them very important in supramolecular crystal engineering and in determination of biological structures [ 413 ] .
therefore , many efforts are made to describe the halogen bonding by theoretical methods of quantum chemistry ; this in turn can help in rational design of new halogen bonded systems with desired molecular properties [ 414 ] . the pioneering experimental studies reporting on the existence of halogen bonding dates back to the mid nineteenth century [ 1519 ] .
the first theoretical analysis of the resonance structures has been proposed by mulliken to explain the molecular spectra of halogen bonded complexes .
one decade later hassel and co - workers expanded this area by developing new systems containing halogen bonds [ 1921 ] . in his nobel lecture hassel highlighted that the electrophilic part of halogen atom can be very crucial in molecular self - assembly phenomena .
in addition , it has been stressed the importance of charge transfer in halogen bonded systems .
the novel concept that explains the origin of halogen bonding was proposed by politzer and coworkers [ 69 , 13 ] .
the authors noticed for the first time , based on the molecular electrostatic potential , that there exists an electron deficiency at the outer part of the halogen atom , so called -hole , what leads in turn to the electrostatic attraction with lewis bases .
hence , the halogen bonding is driven mainly by the electrostatic term [ 69 ] . very recently
hennemann and coworkers extended the -hole concept to analysis of polarized hydrogen bonds . in the present study
we will characterize the halogen bonding in selected molecules in terms of quantitative role of the electronic ( the charge transfer and covalency ) and electrostatic factors .
the recently developed ets - nocv scheme will be used , that originates from a combination of the extended transition state ( ets ) energy decomposition approach with the natural orbitals for chemical valence ( nocv ) analysis [ 2329 ] .
it was shown that ets - nocv is able to extract and directly quantify the crucial components ( , , , etc . )
that constitute various types of chemical bonds including donor - acceptor [ 23 , 24 , 2629 ] , covalent , intra - molecular agostic [ 30 , 31 ] and inter - molecular hydrogen bonding [ 3235 ] .
we will first apply the ets - nocv scheme in a description of relatively weak halogen bonds ( 1-nh3 and 1-cph3 , see panels a and b of fig . 1 ) .
ammonia and divalent carbon ( 0 ) ligands [ c(ph3)2 ] will be used as electron donating species , whereas cf3i molecule as electron acceptor .
the cl2 ligands ( where l is an electron donor ) have recently attracted considerable attention due to untypical oxidation state of carbon atom [ 3638 ] .
ets - nocv approach will also be used for the first time to describe the -hole at iodine atom of cf3i , based on deformation density contributions obtained from the nocv analysis of the bond between the cf3 and i fragments . in the next step , we will focus our attention on the catalytic system [ c6h4(c3h2n2i)2][otf]2 ( 2 in fig . 1 ) .
it has been proven experimentally that this halogen bond donor is a very strong electrophilic species that is able to break covalent c br bond in benzhydryl bromide ( ph2hc br ) . accordingly , as the second objective of our study we will perform pioneering theoretical analysis of the interaction between isopropyl bromide ( the methyl groups were used instead of phenyl rings ) and the model [ c6h4(c3h2n2i)2 ] system ( the counter anion otf has been omitted in the calculations ) , see 2-br in fig . 1 .
further , we will demonstrate that very high electrophilicity of 2 can lead to the formation of dihydrogen complex ( 2-h2 in fig . 1 ) . finally , the halogen ( cl i ) and hydrogen bonds ( cl hn ) co - existing within the same molecule ( 3-cl in fig .
this novel urea - based system has been recently developed experimentally by chudzinski and coworkers .fig .
a thick black line represents the way of fragmentation used in a description of bonding halogen bonded systems studied in the present work .
all the dft calculations presented here are based on the amsterdam density functional ( adf2009 ) program in which ets - nocv scheme was implemented [ 4145 ] .
the becke - perdew exchange - correlation functional [ 46 , 47 ] was applied with an inclusion of the dispersion correction ( bp86-d ) . a standard triple - zeta sto basis containing two sets of polarization functions
auxiliary s , p , d , f , and g sto functions , centered on all nuclei , were used to fit the electron density and to obtain accurate coulomb potentials in each scf cycle .
the contours of deformation densities were plotted based on adf - gui interface . in our analysis of halogen bonding
each of the systems are divided up into two individual fragments as shown schematically by thick black lines in fig .
1 . then we used the ets - nocv method to study the interaction between these subsystems .
thus , our analysis is based on the bonding between the two close shell molecular fragments .
bonding analysis presented in this study is based on the ets - nocv approach which is a combination of the extended transition state ( ets ) method with the natural orbitals for chemical valence ( nocv ) scheme [ 2329 ] .
historically , the natural orbitals for chemical valence ( nocv ) have been derived from the nalewajski - mrozek valence theory [ 5056 ] as eigenvectors of the deformation density matrix .
it was shown [ 24 , 26 ] that the natural orbitals for chemical valence pairs ( -k,,k ) decompose the differential density into nocv - contributions ( k):1\documentclass[12pt]{minimal }
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\begin{document}$$ \delta \rho ( r ) = \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } } } { { v_k } [ - \psi _ { { - k}}^2(r ) + \psi_k^2 } ( r ) ] = \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } } } { \delta { \rho_k}(r ) } , $ $ \end{document}where k and m stand for the nocv eigenvalues and the number of basis functions , respectively .
visual inspection of deformation density plots ( k ) helps to attribute symmetry and the direction of the charge flow .
negative values of this function are marked by red color ( outflow of electrons due to bond formation ) , whereas positive values of k are in blue color ( charge accumulation ) .
in addition , within ets - nocv scheme , the deformation - density based picture is enriched by the energetic estimations , \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^k $ $ \end{document } , for each k . in the ets energy decomposition scheme ,
the interaction energy eint between the fragments ( exhibiting geometries as in the combined molecule ) is divided into three components:2\documentclass[12pt]{minimal }
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\begin{document}$$ \delta { e_{\text{int } } } = \delta { e_{\text{elstat } } } + \delta { e_{\text{pauli } } } + \delta { e_{\text{orb}}}. $ $ \end{document } the first term , eelstat , corresponds to the classical electrostatic interaction between the fragments as they are brought to their positions in the final molecule .
the second term , epauli , accounts for the repulsive pauli interaction between occupied orbitals on the fragments in the combined molecule .
the third stabilizing term , eorb , represents the interactions between the occupied molecular orbitals of one fragment with the unoccupied molecular orbitals of the other fragments as well as mixing of occupied and virtual orbitals within the same fragment ( inner - fragment polarization ) .
this energy term may be linked to the electronic bonding effect coming from the formation of a chemical bond . in the combined ets - nocv scheme the orbital interaction term ( eorb )
is expressed in terms of nocv s eigenvalues ( vk ) as:3\documentclass[12pt]{minimal }
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\begin{document}$$ \delta { e_{{orb } } } = \sum\limits_k { \delta e_{{orb}}^k = } \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } } } { { v_k } [ - f _ { { - k , - k}}^{{ts } } } + f_{{k , k}}^{{ts } } ] , $ $ \end{document}where \documentclass[12pt]{minimal }
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\begin{document}$$ f_{{i , i}}^{{ts } } $ $ \end{document } are diagonal kohn - sham matrix elements defined over nocv with respect to the transition state ( ts ) density ( at the midpoint between density of the molecule and the sum of fragment densities ) .
the above components \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^k $ $ \end{document } provide energetic estimation of k , thus , they characterize the importance of a particular electron flow channel for the bonding between considered molecular fragments .
as has been already mentioned , we will use the dispersion corrected bp86-d functional [ 4648 ] , hence , the dispersion correction ( edisp ) will be added to eint values .
we will start our discussion from the nocv s based description of halogen bonding in 1-nh3 .
2a that the leading deformation density channel , 1 , with the corresponding electronic stabilization \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right . } $ $ \end{document } , constitutes the halogen bonding between ammonia and cf3i molecule .
qualitatively , 1 is based on the donation from the lone electron pair of nitrogen to the empty *(i - c ) .
this transfer leads to elongation of c i bond , by 0.015 , as compared to non - bonded cf3i species .
it is important to note that of similar importance is the covalent contribution that originates from the electron transfer from both the nitrogen and iodine atoms to the i
it is noteworthy that , due to formation of halogen bond , the accumulation of electron density is also observed at the carbon atom of cf3i , which is in line with an increase in s character of carbon ; this issue has been deeply analyzed recently by grabowski using nbo method .
the charge transfer , 1 , characterized by \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right .
} $ $ \end{document } nearly covers the total orbital interaction term \documentclass[12pt]{minimal }
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\begin{document}$$ \left ( { \delta { e_{{orb } } } = { { { - 9.2kcal } } \left/ { { mol } } \right . } } \right ) $ $ \end{document } , see table 1 .
the remaining part of \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^{{rest } } = { { { - 1.0kcal } } \left/ { { mol } } \right . } $ $ \end{document } corresponds to cf3i nh3 back - donation ( ca .
0.5 kcal mol ) and the intra - fragment polarization ( ca . 0.5 kcal mol).fig .
2the contour of deformation density contribution
1 describing formation of the halogen bonding in 1-nh
3 ( part a ) and in 1-cph
3 ( part b ) .
in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown .
the numerically smallest contour values are 0.0005 a.u.table 1ets - energy decomposition ( in kcal mol ) characterizing the halogen bonded systems ( bp86-d / tz2p)systems
e
pauli
e
elstat
e
orb
e
disp
e
int
1-nh3
18.3 - 15.2 - 9.2 - 1.3 - 7.41-cph3
34.0 - 27.6 - 16.8 - 2.8 - 13.22-br22.8 - 20.3 - 19.6 - 2.8 - 19.92-h2
3.0 - 1.6 - 2.2 - 1.0 - 1.83-cl48.0 - 63.8 - 44.1 - 2.5 - 62.4
eint=epauli+eelstat+edisp+eorb
the labeling corresponds to fig . 1
the contour of deformation density contribution
1 describing formation of the halogen bonding in 1-nh
3 ( part a ) and in 1-cph
3 ( part b ) .
in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown .
ets - energy decomposition ( in kcal mol ) characterizing the halogen bonded systems ( bp86-d / tz2p )
eint=epauli+eelstat+edisp+eorb
the labeling corresponds to fig . 1
an analysis of the data presented in table 1 for 1-nh3 leads to the conclusion that the electrostatic contribution ( eelstat = -15.2 kcal mol ) is visibly more important than the orbital - interaction factor ( covalent + charge transfer , as discussed above , eorb = -9.2 kcal mol ) .
thus , our ets - nocv result supports the role of electrostatic factor disclosed in the previous studies based on the other theoretical approaches ( electrostatic potential , symmetry adapted perturbation theory - based energy partitioning ) [ 69 , 57 , 58 ] .
however , it should be emphasized that both , the electrostatic and orbital - interaction components are crucial for total energetic stabilization .
the total interaction energy that includes all bonding contributions , is stabilizing : eint = -7.4 kcal mol .
domination of the electrostatic component can be understood in terms of -hole concept that has been introduced by politzer and coworkers and extensively studied during recent years [ 59 ] . according to this concept halogen bonding is based on the electrostatic attraction between the lewis base and lewis acid parts ( -hole ) of the halogen atom [ 59 ] .
such electron deficiency in the outer part of iodine atom in cf3i ( -hole ) is manifested by positive molecular electrostatic potential , as can be seen from fig .
the contour of deformation density contribution
1 describing the formation of c i bond in cf3i molecule ( part b ) .
the contour of deformation density contribution
1 describing the formation of c i bond in cf3i molecule ( part b ) .
namely , whether we are able to visualize the formation of -hole using deformation density contributions originating from nocv . for this purpose we have analyzed the bonding between the iodium atom and the cf3 radical ( each carrying one unpaired electron with the opposite spin polarizations ) .
3b , the leading deformation density channel , 1 , with the corresponding energy \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 98.6kcal } } \left/ { { mol } } \right . }
in addition , it is gratifying to see an outflow of electron density from the outer area of iodine atom , which clearly corresponds to the formation of -hole .
it should further be noted that apart from the above components , the charge accumulation at iodine is observed due to formation of c i bond , which confirms significant charge anisotropy around this atom [ 414 ] .
the presence of such a anisotropy is important for the reactivity , as the halogen atom can simultaneously act as electron donor and acceptor [ 414 ] .
let us now briefly discuss the bonding of divalent carbon ( 0 ) ligand , c(ph3)2 , with cf3i moiety , in 1-cph3 , see fig .
qualitatively , the deformation density channel , 1 , exhibits similar features as in the case of 1-nh3 .
c contribution as well as the charge transfer from the lone electron pair of carbon to the empty *(c
however , these transfers correspond to significantly higher stabilization , \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 14.2kcal } } \left/ { { mol } } \right . }
$ $ \end{document } , as compared to 1-nh3 ( \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right . }
this is probably due to stronger overlap of orbitals involved in the halogen bonding interaction .
divalent carbon ( 0 ) ligands appeared to be strong electron donors , which has been recently extensively studied by tonner and frenking .
a significant overlap leading to notable electronic stabilization in 1-cph3 also causes the electrostatic contribution to be more pronounced when compared to 1-nh3 ( by 12.4 kcal mol ) , see table 1 .
accordingly , the total interaction energy , eint , becomes more stabilizing , by 5.8 kcal mol for 1-cph3 .
after we discussed the character of halogen bonding in simple molecules , let us consider now the bonding of isopropyl bromide to the model system of catalyst [ c6h4(c3h2n2i)2][otf]2 , see 2-br in fig .
it has been proven experimentally that [ c6h4(c3h2n2i)2][otf]2 is able to break a strong carbon - bromide bond .
authors of this work suggested that the activation of c br bond is induced by the formation of halogen bond between bromine and the iodine atoms , see fig .
we have found indeed , based on dft / bp86-d / tz2p calculations , the minimum on the potential energy surface for the complex 2-br , where the bromine center forms two halogen connections with iodine atoms .
energy decomposition method ( ets ) indicates that isopropyl bromide is strongly bonded to [ c6h4(c3h2n2i)2 ] moiety , i.e. , the total interaction energy is eint = -19.9 kcal mol .
br bond becomes significantly elongated , by 0.08 ( as compared to non - bonded isopropyl bromide ) , due to the formation of two br
interestingly , in this case the orbital - interaction contribution ( eorb = -19.6 kcal mol ) appeared to be of the same importance compared to the electrostatic stabilization ( eelstat = -20.3 kcal mol ) .
it is an important result in light of the common view that the charge transfer ( electronic factor ) is a rather inferior factor in the halogen bonding [ 9 , 21 ] .
that two deformation density channels , 1 and 2 , build up the two i br connections .
ets - nocv allows to conclude that the one contribution is stronger ( \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.5kcal } } \left/ { { mol } } \right . }
$ $ \end{document } ) than the other one ( \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^2 = { { { - 6.5kcal } } \left/ { { mol } } \right . }
figure 4a shows that , similar to the previously considered examples , both halogen bonds contain the covalent i
br components as well as the charge transfer contributions originating from the lone electron pair donation to the empty *(c
further mechanistic and kinetic studies leading to the heterolytic c br bond cleavage are under way.fig .
4the contours of deformation density contributions describing the halogen bonding in 2-br ( part a ) and in 2-h
2 ( part b ) .
in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown the contours of deformation density contributions describing the halogen bonding in 2-br ( part a ) and in 2-h
2 ( part b ) .
in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown keeping in mind the above important experimental finding that a strongly electrophilic system can activate covalent bond , we decided to check whether the same system can form a chemical bond with chemically inert hydrogen molecule , h2 . to our partial surprise
we found a stable complex of dihydrogen with [ c6h4(c3h2n2i)2 ] moiety , see 2-h2 in fig . 1 .
ets energy decomposition method indicated that the hydrogen unit is weakly bonded to iodine atom , eint = -1.9 kcal mol , see table 1 .
interestingly , neither the electrostatic term ( eelstat = -1.6 kcal mol ) , nor the dispersion interaction ( edisp = -1.0 kcal mol ) , are the leading contributions to eint .
it appears that the electronic factor ( eorb = -2.2 kcal mol ) is the most important for the bonding in 2-h2 , see table 1 .
deformation density channel originating from nocv s , 1 , presented in the right side of fig .
4b , shows that the electronic stabilization originates predominantly from the electron donation from the occupied (h - h ) orbital to the i
we believe that this result is promising in terms of searching for new reactivity patterns of molecular hydrogen .
it should be added that it has already been found that transition metal - based dihydride systems can form stable complexes with halogen - containing molecules .
finally , we will end our study by a brief discussion of the binding of chloride anion to a recently developed system that contains two iodine atoms and two n h groups as electron acceptors , see 3-cl in fig . 1 .
in line with expectation , the simultaneous presence of two hydrogen ( cl hn ) and two halogen ( cl i ) interactions , leads to very high total stabilization energy , eint = -62.4 kcal mol , see table 1 .
it is the result of significant electrostatic ( eelstat = -63.8 kcal mol ) and electronic
it is important to highlight that nocv deformation density contributions combined with ets scheme allowed to estimate separately the electronic strength of hydrogen ( cl hn ) , 2 , and halogen ( cl i ) interactions , 1 , see fig .
i bonding , in terms of the orbital interaction component , is more than three times stronger ( \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 22.3kcal } } \left/ { { mol } } \right . } $ $ \end{document } ) than the hydrogen bond connection ( \documentclass[12pt]{minimal }
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\begin{document}$$ \delta e_{{orb}}^1 = { { { - 6.3kcal } } \left/ { { mol } } \right . } $ $ \end{document } ) . such separated information on
the strength of halogen and hydrogen connections within the same molecule can help to modulate the selectivity and accordingly lead to rational design of new acceptor molecules.fig .
5the contours of deformation density contributions describing the halogen (
1 ) and hydrogen bonding (
1 ) in 3-cl .
in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown .
the contours of deformation density contributions describing the halogen (
1 ) and hydrogen bonding (
1 ) in 3-cl .
in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown .
in the present study we have characterized the halogen bonding in selected molecules , 1-nh3 , 1-cph3 , 2-br , 2-h2 and 3-cl , based on the recently proposed ets - nocv procedure .
we have chosen the examples containing from one ( 1-nh3 , 1-cph3 ) up to four bonding connections ( 3-cl ) . in the ets energy decomposition scheme ,
the interaction energy between the fragments is divided into three well defined components : electrostatic , pauli repulsion , and the orbital interaction .
however , it should be emphasized that any energy partitioning method includes some arbitrariness due to the fact that the contributions to the total interaction energy are not physical observables .
nevertheless , the results of the present analysis , in agreement with previous studies [ 69 , 57 , 58 ] demonstrate the indisputable role of the electrostatic stabilization in halogen bonding . we have found based on the nocv - deformation density contours ( i ) that in each analyzed system the halogen bonding c xb ( x - halogen atom , b - lewis base ) , contains a large degree of covalent contribution ( charge transfer to x
b inter - atomic region ) supported further by the electron donation from base atom b to empty *(c - x ) orbital .
it should be emphasized that ets - nocv approach allowed to clearly visualize the formation of -hole at iodine atom of cf3i molecule .
thus , the nocv - analysis confirms the vital importance of the presence of -hole for halogen bonding [ 69 ] ; an electron - density deficiency at outer part of halogen atom ( x ) along c x bond leads to both effects , electrostatic stabilization of the c xb interaction , but as well to a charge flow from a base to electron - deficient region .
an importance of the observed electron - density displacements is further demonstrated by the ets orbital - interaction energy , eorb , dominated in each case by one contribution eorb corresponding to main charge transfer channel 1 . finally , in all of the analyzed systems the dispersion interaction appeared to be less important for halogen bonding . due to the fact that in each case we noted a large degree of covalency of halogen bond , we believe that the term non - covalent interaction , frequently used in literature , is not fully appropriate , at least for the systems studied in the present work .
these results suggest further that in addition to the dominant electrostatic component , depending on the system , other terms can be relatively important .
this observation supports the new set of criteria proposed recently by legon for definition of halogen bonding .
we have also demonstrated that strongly electrophilic species , [ c6h4(c3h2n2i)2][otf]2 , ( 2 ) , can activate chemically inert molecules via formation of halogen bonding , e.g. , isopropyl bromide ( eint = -19.9 kcal mol ) .
interestingly , it has also been noted that ( 2 ) can bind the hydrogen molecule , 2-h2 , eint = -1.8 kcal mol , which seems to be a promising result in terms of future findings of new reactivity patterns of hydrogen molecule .
finally , ets - nocv approach allowed to qualitatively and quantitatively characterize separately the halogen ( cl i ) and hydrogen bonds ( cl hn ) within the same acceptor molecule ( 3-cl ) . | in the present study we have characterized the halogen bonding in selected molecules h3n
icf3 ( 1-nh3 ) , ( ph3)2c icf3 ( 1-cph3 ) , c3h7br(in2h2c3)2c6h4 ( 2-br ) , h2(in2h2c3)2c6h4 ( 2-h2 ) and cl(ic6f5)2c7h10n2o5 ( 3-cl ) , containing from one halogen bond ( 1-nh3 , 1-cph3 ) up to four connections in 3-cl ( the two cl hn and two cl
i ) , based on recently proposed ets - nocv analysis .
it was found based on the nocv - deformation density components that the halogen bonding c x b ( x - halogen atom , b - lewis base ) , contains a large degree of covalent contribution ( the charge transfer to x
b inter - atomic region ) supported further by the electron donation from base atom b to the empty *(c x ) orbital .
such charge transfers can be of similar importance compared to the electrostatic stabilization .
further , the covalent part of halogen bonding is due to the presence of -hole at outer part of halogen atom ( x ) .
ets - nocv approach allowed to visualize formation of the -hole at iodine atom of cf3i molecule .
it has also been demonstrated that strongly electrophilic halogen bond donor , [ c6h4(c3h2n2i)2][otf]2 , can activate chemically inert isopropyl bromide ( 2-br ) moiety via formation of br
i bonding and bind the hydrogen molecule ( 2-h2 ) .
finally , ets - nocv analysis performed for 3-cl leads to the conclusion that , in terms of the orbital - interaction component , the strength of halogen ( cl i ) bond is roughly three times more important than the hydrogen bonding ( cl hn).figureets - nocv reprezentation of -hole at iodine together with the molecular electrostatic potential picture | Introduction
Computational details
Computational methods
Results and discussion
Conclusions |
presbyphagia refers to changes in the
sensorimotor nervous system related to swallowing caused by aging .
with progression of
aging , changes in the motor nervous system , such as a decreased muscle mass and size , that
are attributable to sarcopenia can occur1 , 2 .
sarcopenia presents with a decrease in
overall muscle strength and affects the muscles related to swallowing .
in particular ,
weakening of the orofacial muscles , including the tongue , buccinator , and orbicularis oris ,
and the submental muscles located in the anterior neck region can cause many problems
associated with residual bolus in the oral cavity and spillage from the lips , bolus
formation , mastication , and reduced hyoid bone movement3 , 4 . therefore , swallowing
management is essential in elderly individuals who are susceptible to presbyphagia .
accordingly , the present study aimed to investigate the effects of dysphagia therapy in an
elderly individual with a chief complaint of swallowing difficulty .
the subject was a 72-year - old man who was admitted with the chief complaint of spillage
through the lips during swallowing , difficulty with residue on oral cavity , and aspiration .
the purpose of the study were explained to the participant before study inclusion , and
informed consent was obtained from the participant according to the principles of the
declaration of helsinki .
he had no history of stroke or other neurological disorder with no
cognitive problems .
we assessed strength of tongue and lip muscle using iowa oral performance instrument ( iopi )
( iopi medical llc , carnation , wa , usa ) .
swallowing function was assessed by using the
videofluoroscopic dysphagia scale ( vds ) and penetration - aspiration scale ( pas ) based on a
videofluoroscopic swallowing study ( vfss ) .
strength measurement revealed tongue and lip
muscle strengths of 35 and 18 kpa , respectively .
the vds score was 14 of 40 in the oral
phase , indicating problems in tongue - palate contact , mastication , lip closure , and oral
transit time . in the pharyngeal phase , the score was 19 of 60 , with problems of laryngeal
elevation , vallecular residue , and penetration . based on the vfss results , the subject was
diagnosed as having swallowing difficulties caused by aging or presbyphagia .
dysphagia therapy performed conventional dysphagia therapy ( thermal - tactile stimulation ,
orofacial exercise ) during electrical stimulation ( es ) .
es was performed using the vitalstim
( chattanooga group , hixson , tn , usa ) .
two pair of electrodes were placed in the suprahyoid
region targeting the suprahyoid muscles , and the inter - electrode distance was set at 2.5 mm .
the es provided 2 channels of bipolar electrical stimulation at a fixed 80-hz pulse rate and
a fixed biphasic pulse duration of 700 s .
the stimulation intensity was increased until the
patients felt a grabbing sensation in neck .
subject received the treatment for 30 minutes / day , 5 day / week , for 4 weeks .
tongue and lip muscle strengths improved after the intervention , from 35 to 39 kpa and from
18 to 23 kpa , respectively .
vds scores decreased in both the oral ( tongue - palate contact ,
mastication , and lip closure ) and pharyngeal phases ( laryngeal elevation and vallecular
residue ) after the intervention , from 14 to 8 and from 19 to 8.5 , respectively .
the pas
score also decreased after the intervention , from 4 to 2 points .
this study performed exercises for the tongue , lip , and facial muscles for improvement of
oral functions , and es was applied on swallowing - related muscles in the anterior neck
region . according to a previous study , resistance exercise for the tongue is an effective
method for reducing aspiration5 .
moreover ,
facial muscles contribute to masticatory activities , while the lip muscles contribute to
maintaining oral closure during swallowing to generate appropriate intraoral pressure6 .
improving the strength of these oral
muscles can increase intraoral pressure during swallowing and have a positive effect on the
pharyngeal phase as well .
a previous study reported that electrical stimulation in patients with dysphagia can
activate the swallowing muscles .
it is an effective method for inducing antero - superior
movement of the hyoid , which has a direct impact on the airway protection mechanism , and can
help decrease aspiration7 .
we performed electrical stimulation and conventional
dysphagia therapy in an old man with swallowing difficulty and demonstrated improvements in
both oral muscle strength and swallowing function . | [ purpose ] the purpose of the present study was to investigate the effects of dysphagia
therapy in an old man with difficulty swallowing in the oral and pharyngeal phases .
[ subjects and methods ] the subject was a 72-year - old man with no history of neurological
disorders .
he was admitted to local hospital because of the complaint of swallowing
difficulty .
the interventions performed were electrical stimulation and conventional
dysphagia therapy .
we assessed the tongue and lip muscle strengths . swallowing function
was evaluated by using the videofluoroscopic dysphagia and penetration - aspiration scales .
[ results ] after the intervention , the tongue and lip muscle strengths increased from 35 to
39 kpa and from 18 to 23 kpa , respectively .
moreover , the oral and pharyngeal phases of
the videofluoroscopic dysphagia scale were improved . furthermore , aspiration decreased
from 4 to 2 points in the penetration - aspiration scale .
[ conclusion ] our results suggest
that electrical stimulation and conventional dysphagia therapy were effective in improving
the swallowing function in an elderly individual with dysphagia . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
gastroenterologists and surgeons who specialize in the treatment of crohn disease ( cd ) and chronic ulcerative colitis ( cuc ) have become increasingly aware of the implications of different forms of medical therapy on surgical outcomes .
this is especially important in the era of top - down and multi - agent therapy for inflammatory bowel disease ( ibd ) .
although relatively little literature exists regarding the relationship between pre - operative corticosteroid use and post - operative complications , several authors have found that increasingly higher doses of such immunosuppressive medications may result in an increased rate of post - operative septic complications .
similarly , there is increasing concern that combination immunomodulatory therapy with multiple agents may also be associated with an increase in post - operative infectious complications .
given reports of the association of monoclonal anti - tumor necrosis factor - alpha ( anti - tnf- ) antibody therapy with pulmonary infections , this concern has specifically been focused on infliximab ( ifx ) , which until recently has been the only fda - approved biologic agent for ibd [ 7 - 11 ] . a recent meta - analysis of the association between immunomodulatory therapy and post - operative complications after surgery for ibd , which included studies of azathioprine , cyclosporine a , and three studies of ifx , found that the available evidence did not support this association . since then , several studies have been published suggesting that ifx is indeed associated with an increased risk of post - operative complications .
thus , the potential association between ifx and surgical complications is controversial ; studies both support and refute this potential relationship [ 5,13 - 19 ] .
in a study of 270 patients by colombel et al . , 52 patients experienced intra - abdominal septic complications ( iascs ) after bowel resection for cd
. analysis did not reveal any increased risk in the ifx - treated subgroup , the moderate - to - high - dose corticosteroid subgroup , or the immunomodulator subgroup .
likewise , a matched case - control study from belgium of 31 cd patients also concluded that pre - operative ifx use did not result in a significantly increased rate of post - operative complications , or increased hospital length of stay , as compared to a control group of ifx - nave patients .
that study did note , however , a trend towards increased early post - operative infections in the ifx group .
more recently , a study of 413 ibd patients ( 156 with cd ) , of which 101 had received ifx within 12 weeks of surgery , also did not find any relationship between pre - operative ifx therapy and post - operative complications .
that study , similar to others , was relatively limited by the absolute number of complications .
in contrast , a more recent study from appau et al . found an association between pre - operative ifx therapy for cd and post - operative complications .
they concluded that ifx therapy within 3 months of surgery was associated with an increased rate of iascs , and of hospital re - admissions .
they also found that patients who received a diverting stoma had a lower risk of iascs , thus prompting the observation that a diverting stoma may be prudent when surgeons are faced with cd patients who have recently received ifx . in light of this study ,
the risks associated with stoma reversal surgery must be weighted against the potential increase in complications associated with ifx for cd . in a study of 151 patients with cuc , 17 ( 10% ) of whom failed ifx therapy and went to surgery and 134 of whom were ifx - nave , no association between ifx and complications was observed .
however , the authors observed that ifx patients who had concurrent cyclosporine a treatment were at increased risk for overall and infectious complications . in a larger study by selvasekar et al .
in which 47 cuc patients received pre - operative [ ileal pouch - anal anastomosis ( ipaa ) ] ifx therapy and 254 did not , those who received ifx were more likely to have anastomotic leak , and after multivariate adjustment for both disease severity and other medication use , ifx remained independently associated with an increased risk of ileal pouch - related and infectious complications .
finally , a recent study of 85 patients with cuc who received ifx pre - operatively also found that patients who receive pre - operative ifx were at increased risk of post - operative septic complications as well as late complications .
importantly , the authors also noted that patients who received ifx were more likely to have undergone a 3-stage ipaa , likely due to surgeon reluctance to perform an anastomosis in the setting of pre - operative ifx administration .
disease severity determined retrospectively is often inaccurate and surrogate covariates may be inadequate substitutions for validated methods of assessing disease activity .
however , it is interesting to note that in the studies of cuc , those that adjusted for disease activity showed a relationship between ifx and complications , while the converse was true for those studies that did not .
another major limitation of several of these studies is the relatively long pre - operative ifx window , often 12 weeks or more ( table 1 ) .
recent studies of the pharmacokinetics of ifx in ibd suggest that the elimination half - life is between 7 and 18.5 days . by 12 weeks ( 84 days , or 4.5 half - lives )
a window of 12 weeks used by several of the studies published to date may theoretically produce negative results regarding the occurrence of post - operative outcomes if a significant proportion of patients had an interval this long between ifx administration and surgery .
ideally , the duration between last infusion and surgery should be included as a continuous variable , but outpatient infusion often makes these data unavailable retrospectively .
referral practice patterns may too account in part for the heterogenous findings of these studies , as these may differ substantially in various regions of the world , as demonstrated by at least a 10% variation in the proportion of surgical ibd patients who received ifx .
thus , as is usually true of most retrospective studies , results from a single institution may not be broadly generalizable ; the institutions themselves may be considered as a potential confounding factor when comparing results of different studies to each other . in one of the largest studies of the association of ifx and serious infections and mortality , the treat registry study of 6290 patients found that after adjustment for corticosteroid use and disease severity , ifx was not independently associated with increased risk , although both corticosteroids and disease severity were associated with those adverse outcomes .
although this study did not include surgical endpoints , the implications are nonetheless important , especially given the lack of large - scale surgical data .
in a study of 270 patients by colombel et al . , 52 patients experienced intra - abdominal septic complications ( iascs ) after bowel resection for cd
. analysis did not reveal any increased risk in the ifx - treated subgroup , the moderate - to - high - dose corticosteroid subgroup , or the immunomodulator subgroup .
likewise , a matched case - control study from belgium of 31 cd patients also concluded that pre - operative ifx use did not result in a significantly increased rate of post - operative complications , or increased hospital length of stay , as compared to a control group of ifx - nave patients .
that study did note , however , a trend towards increased early post - operative infections in the ifx group .
more recently , a study of 413 ibd patients ( 156 with cd ) , of which 101 had received ifx within 12 weeks of surgery , also did not find any relationship between pre - operative ifx therapy and post - operative complications .
that study , similar to others , was relatively limited by the absolute number of complications .
in contrast , a more recent study from appau et al . found an association between pre - operative ifx therapy for cd and post - operative complications .
they concluded that ifx therapy within 3 months of surgery was associated with an increased rate of iascs , and of hospital re - admissions .
they also found that patients who received a diverting stoma had a lower risk of iascs , thus prompting the observation that a diverting stoma may be prudent when surgeons are faced with cd patients who have recently received ifx . in light of this study ,
the risks associated with stoma reversal surgery must be weighted against the potential increase in complications associated with ifx for cd .
in a study of 151 patients with cuc , 17 ( 10% ) of whom failed ifx therapy and went to surgery and 134 of whom were ifx - nave , no association between ifx and complications was observed .
however , the authors observed that ifx patients who had concurrent cyclosporine a treatment were at increased risk for overall and infectious complications . in a larger study by selvasekar et al .
in which 47 cuc patients received pre - operative [ ileal pouch - anal anastomosis ( ipaa ) ] ifx therapy and 254 did not , those who received ifx were more likely to have anastomotic leak , and after multivariate adjustment for both disease severity and other medication use , ifx remained independently associated with an increased risk of ileal pouch - related and infectious complications . finally , a recent study of 85 patients with cuc who received ifx pre - operatively also found that patients who receive pre - operative ifx were at increased risk of post - operative septic complications as well as late complications .
importantly , the authors also noted that patients who received ifx were more likely to have undergone a 3-stage ipaa , likely due to surgeon reluctance to perform an anastomosis in the setting of pre - operative ifx administration .
many of these retrospective studies suffer from common limitations . first is lack of adjustment for disease severity ( table 1 ) .
disease severity determined retrospectively is often inaccurate and surrogate covariates may be inadequate substitutions for validated methods of assessing disease activity .
however , it is interesting to note that in the studies of cuc , those that adjusted for disease activity showed a relationship between ifx and complications , while the converse was true for those studies that did not .
another major limitation of several of these studies is the relatively long pre - operative ifx window , often 12 weeks or more ( table 1 ) .
recent studies of the pharmacokinetics of ifx in ibd suggest that the elimination half - life is between 7 and 18.5 days . by 12 weeks ( 84 days , or 4.5 half - lives )
a window of 12 weeks used by several of the studies published to date may theoretically produce negative results regarding the occurrence of post - operative outcomes if a significant proportion of patients had an interval this long between ifx administration and surgery .
ideally , the duration between last infusion and surgery should be included as a continuous variable , but outpatient infusion often makes these data unavailable retrospectively .
referral practice patterns may too account in part for the heterogenous findings of these studies , as these may differ substantially in various regions of the world , as demonstrated by at least a 10% variation in the proportion of surgical ibd patients who received ifx .
thus , as is usually true of most retrospective studies , results from a single institution may not be broadly generalizable ; the institutions themselves may be considered as a potential confounding factor when comparing results of different studies to each other . in one of the largest studies of the association of ifx and serious infections and mortality , the treat registry study of 6290 patients found that after adjustment for corticosteroid use and disease severity , ifx was not independently associated with increased risk , although both corticosteroids and disease severity were associated with those adverse outcomes .
although this study did not include surgical endpoints , the implications are nonetheless important , especially given the lack of large - scale surgical data .
whether pre - operative ifx use is associated with post - operative complications after surgery for ibd remains controversial ; current evidence favors this association after surgery for cuc but does not favor an increased risk after surgery for cd .
the potentially practice - changing implications of this debate are occurring now ; increasingly some surgeons are waiting until anti - tnf- agents are washed out before operating or , if that is not possible , performing resections for cd with temporary diverting stomas or advising 3- rather than 2-staged ipaa .
larger studies and/or formal meta - analysis of the relationship between ifx and post - operative complications after surgery for ibd are needed before definitive treatment recommendations can be made . the decision of whether or not to perform staged surgery for ibd patients on ifx needs to be made on an individual basis , based on the clinical context and other known risk factors , such as hypoalbuminemia and high - dose corticosteroid treatment . | conflicting data exist regarding the association between pre - operative monoclonal anti - tumor necrosis factor - alpha antibody therapy with infliximab for crohn disease and chronic ulcerative colitis , and the occurrence of post - operative complications .
this report reviews the current literature that supports and refutes this association . | Introduction and context
Recent advances
Crohn disease
Chronic ulcerative colitis
Common limitations
Implications for clinical practice
Competing interests |
since the early 1970s there have been dramatic improvements in the availability and quality of treatment for persons with hemophilia ( mannucci and tuddenham 2001 ) . as a result of this progress , barring the consequences of the human immunodeficiency virus ( hiv ) epidemics in the 1980s , the life span of hemophiliacs has progressively become similar to that of males in the general population , at least in more developed countries ( mejia - carvajal et al 2006 ) .
accordingly , a considerable number of hemophiliacs now reach old age : in italy , for instance , nearly 8% of persons with severe hemophilia a or b are 65 years old or older ( tagliaferri et al 2006 ) .
however , with age , persons with hemophilia develop medical and surgical diseases ( eg , cardiovascular diseases , prostatic hypertrophy , cancers , renal disease ) not previously seen in this group .
this aspect , together with the management of these patients , is discussed in the first part of this review .
the second part addresses the main features and clinical management of acquired hemophilia due to anti - factor viii antibodies , an acquired disease that may occur in elderly people .
however , the vast majority of them have evaluated only subgroups of patients , not the whole population .
the mortality of patients infected by the hepatitis c virus ( hcv ) or hiv has been specially analyzed .
for example , darby et al ( 1997 ) analyzed mortality in hemophiliacs in the united kingdom infused with blood products contaminated with hcv .
other studies evaluated the mortality rate among hiv - infected patients in spain ( del amo et al 2006 ) , the united kingdom ( darby et al 2004a ) and canada ( arnold et al 2006 ) . finally , another study investigated the effect of factor viii and ix inhibitors on mortality in a hemophilia population ( darby 2004b ) .
although only a small number of studies have so far evaluated mortality in the whole population of hemophiliacs ( street et al 2006 ) , these are interesting because they cover the whole history of the clinical - therapeutic approach to hemophilia , from the dramatic increase in life expectancy between the 1940s and 1980s with the advent of replacement therapy with plasma - derived clotting factor to the catastrophic contamination of the plasma pool by hiv and hcv in the 1980s ( street et al 2006 ) . in parallel with the therapeutic progresses , the causes of death have changed in hemophilia , hemorrhage being replaced by the acquired immunodeficiency syndrome ( aids ) as the leading cause of death ( mejia - carvajal et al 2006 ) .
indeed , the mortality curve in hemophiliacs over the years has a biphasic form with two peaks : one before the 1960s ( before the widespread availability of factor replacement therapy ) and the other in the 1980s1990s ( after the occurrence of aids and of the clinical manifestations of earlier hcv infections ) .
this trend has been confirmed by a recent survey that analyzed the mortality rate and causes of death in 967 dutch hemophiliacs ( plug et al 2006 ) . during the period of the study ( 19922001 ) the mortality rate was 2.3 times higher in patients with hemophilia than in the general male population .
moreover , comparing these data with those previously reported by the same group ( rosendaal et al 1989 ; triemstra et al 1995 ) , the life expectancy of patients with severe hemophilia decreased from 63 years in the period 19731986 to 61 and 59 years in the period 19861992 and 19922001 , respectively .
however , the exclusion of virus - related deaths resulted in a life expectancy at birth of 74 years , similar to that of the unaffected dutch male population .
similar trends were observed in studies involving scottish ( ludlam 2000 ) and canadian ( walker 1998 ) hemophiliacs . from 1900 to 1942 the life expectancy of severe hemophiliacs in sweden was 16.5 years ; this increased to 23.2 years between 1943 and 1957 ( ramgren 1962 ) and to 50 years between 1957 and 1980 ( larsson and wiechel 1983 ) . for comparison , the life expectancy of unaffected swedish men during the latter period was 75 years
another study analyzed 163 patients with severe hemophilia a living in finland in the period between 193079 ( ikkala 1982 ) . during 50 years of observation
, the mean age at death increased from 7.8 years in 193039 to 25.5 years in 197079 .
the effect of hiv infection on age and causes of death among persons with hemophilia a was analyzed in a study conducted in the united states ( chorba et al 2001 ) . the median age at death decreased from 55 years in 19791982 to 40.5 years in 19871990 and increased to 46 years in 19951998 , although the median age at death of patients with hemophilia a and hiv - related disease in the latter period was 33 years , compared to 72 years for those without hiv - related disease . during the same period ,
hemophilia a - associated deaths decreased by 41% , with a 78% decrease among those who had hiv - related disease .
this decline in hiv mortality was consistent with that observed in the general population of hiv - infected persons and reflected improvements in anti - retroviral therapy .
on the other hand , another study that analyzed the causes of death among hemophiliacs not infected by hiv found that the leading cause of death was hemorrhage , followed by liver disease ( hepatitis c and b ) , stroke and cancer .
currently , children with hemophilia look forward to a normal life expectancy and excellent health - related quality of life ( gringeri et al 2006 ) .
the most important factors contributing to the improved quality of life , reduced morbidity and increased life expectancy are the availability for replacement therapy of high - quality factor concentrates ( ie , virus inactivated plasma - derived clotting factor concentrates and recombinant products ) , insights into the management of hemophilic arthropathy and liver disease ( ie , surveillance of patients with chronic hepatitis , especially with respect to cancer and liver failure , and newer treatment options such as antiviral treatment against hiv and hcv and liver transplantation ) and the improved medical management provided by specialized hemophilia treatment centers .
high - quality factor concentrates are important not only for reducing the likelihood of death from hemorrhage but also for improving the quality of life , especially through prophylactic regimens and home treatment .
secondary prophylaxis , in particular , is becoming frequently prescribed among hemophiliacs , although only few data are available in this setting ( tagliaferri et al 2006a ) .
the impact of comprehensive care has been demonstrated by soucie et al ( 2006 ) who found that hemophiliacs who had received care in a specialized hemophilia treatment center had a significantly decreased risk of death .
the impact of comprehensive care is further exemplified by the analysis of 164 patients treated at the international haemophilia training centre at bangkok with blood and blood components from 1971 to 2000 .
the estimated probability of survival beyond 13 years of age of patients with severe hemophilia increased from 0.85 during the first decade of observation to 0.94 and 1 in the second and third decades ( chuansumrit et al 2004 ) .
the mortality rate fell from 30% to 14% and 5% over the three decades of observation . despite the lack of treatment with high quality clotting factor
concentrates in this center , the patients outcome improved with the development of a comprehensive approach to management . on behalf of the italian association of hemophilia centers ( aice )
, we are conducting a multicenter study to evaluate the health status and health - related quality of life ( hr - qol ) of severely affected italian hemophiliacs of 65 years or older , recruited from italian hemophilia centers ( tagliaferri et al 2006b ) .
another primary goal of the study is to provide information on whether or not there are differences in the global health status and hr - qol between healthy elderly individuals and persons with hemophilia , who started their lives in the first part of the last century when the availability and quality of treatment were poor .
this study will also allow us to establish whether or not the burden of diseases other than hemophilia ( including cognitive impairment measured using instruments of geriatric medicine ) is similar or different in cases and controls , and to understand which factors help people with severe hemophilia to cope with their disease and reach old age .
hemophilia is associated with bleeding episodes , which often occur into the joints or muscles . over time , recurrent bleeding into joints and muscles can cause permanent problems such as arthritis , chronic pain and joint damage resulting in poor control of balance and falls which ultimately worsen quality of life .
thus joint disease remains the leading cause of morbidity in those elderly hemophiliacs who had little or no access to prophylactic regimens and home treatment when they were younger .
an ongoing australian study is evaluating the degree of joint - related dysfunction in persons with hemophilia older than 30 years and the feasibility of home exercise programs targeting balance training ( street 2006 ) .
we have recently reported the case of a 72-year - old hemophiliac with severe joint bleeding in whom treatment was converted from on - demand to a secondary prophylaxis regimen ( tagliaferri et al 2006a ) .
the latter significantly reduced the number of bleeding episodes in target joints , slowing the progression of the arthropathy and improving the patient s perceived well - being .
however , further studies , dealing with the approach to replacement therapy and the development of preventive physical therapy programs for elderly patients , are warranted .
attempts have been made to determine whether or not hemophilia protects from the development of atherosclerosis and cardiovascular events , but the results are contradictory .
studies of carotid and femoral artery intimalmedial thickness as a surrogate of atherosclerosis burden showed that hemophilia does not protect from atherosclerosis ( sramek et al 2001 ) . on the other hand ,
hemophiliacs have a decreased mortality rate due to ischemic heart disease , supporting the views that they are protected from arterial thrombosis ( sramek et al 2003 ) .
recently , girolami et al ( 2006 ) reviewed reported cardiovascular events in patients with hemophilia a. among the 42 cases published so far , nine occurred in individuals aged 65 or over . analyzing the literature data
, the authors concluded that the bleeding diathesis did not help to prevent the occurrence of arterial occlusions .
thus factors which are known to predispose to atherothrombosis ( eg , hypercholesterolemia , smoking , diabetes , hypertension , aging ) appear to overcome the coagulation defect .
concomitant replacement therapy , especially with bypassing agents ( ie , activated or non - activated prothrombin complex concentrates and recombinant activated factor vii ) , was identified as having a predominant role in thrombotic manifestations in the majority of the cases ( aledort 2004 ) .
a number of successful cardiovascular interventions in elderly hemophiliacs have been carried out ( bovenzi et al 2003 ; de bels et al 2004 ) .
however , these cases raise the question of how to conduct anticoagulant therapy in such naturally anticoagulated patients .
the management of cardiovascular events in elderly hemophiliacs necessarily differs from that of non - hemophilic patients .
thus , while the treatment of acute coronary syndromes in hemophiliacs requires a combination of antithrombotic and replacement therapies , thrombolytic therapy for myocardial infarctions is unjustified in these patients due to the unacceptable risk of hemorrhage and should be substituted by alternative techniques such as percutaneous coronary angioplasty ( mafrici et al 2003 ) .
similarly , long - term anticoagulation should be avoided whenever possible by using , for example , a bioprosthetic valve in valve surgery .
elderly people with hemophilia are also at increased risk of chronic renal disease ( kulkarni et al 2003 ) .
this is probably due to chronic renal bleeding associated with the inherited coagulopathy which leads to structural renal abnormalities .
moreover , the concomitant presence of viral infections ( eg , hiv and hcv ) or exposure to nephrotoxic antiviral therapy may increase the incidence of renal dysfunction over time .
chronic renal disease is responsible for hypertension ( scully et al 2005 ) , which is observed more frequently in elderly hemophiliacs than in the general population and is a risk factor not only for cardiac disease but also for cerebral hemorrhage .
although most inhibitors occur at a young age in patients with severe hemophilia , patients with mild hemophilia may develop inhibitors at an advanced age when they receive intensive factor viii replacement therapy on the occasion of a surgical or invasive procedure for age - related problems such as prostatic hypertrophy , cardiovascular disease or cancer ( franchini et al 2006a ) .
indeed , there are some concerns on the use of bypassing agents in those patients considered at higher risk of developing thrombotic complications .
however , leebek et al ( 2006 ) have recently reported the safe and effective use of recombinant activated factor vii in three elderly patients with mild hemophilia a and high - titer inhibitors .
however , the vast majority of them have evaluated only subgroups of patients , not the whole population .
the mortality of patients infected by the hepatitis c virus ( hcv ) or hiv has been specially analyzed .
for example , darby et al ( 1997 ) analyzed mortality in hemophiliacs in the united kingdom infused with blood products contaminated with hcv .
other studies evaluated the mortality rate among hiv - infected patients in spain ( del amo et al 2006 ) , the united kingdom ( darby et al 2004a ) and canada ( arnold et al 2006 ) . finally , another study investigated the effect of factor viii and ix inhibitors on mortality in a hemophilia population ( darby 2004b ) .
although only a small number of studies have so far evaluated mortality in the whole population of hemophiliacs ( street et al 2006 ) , these are interesting because they cover the whole history of the clinical - therapeutic approach to hemophilia , from the dramatic increase in life expectancy between the 1940s and 1980s with the advent of replacement therapy with plasma - derived clotting factor to the catastrophic contamination of the plasma pool by hiv and hcv in the 1980s ( street et al 2006 ) . in parallel with the therapeutic progresses , the causes of death have changed in hemophilia , hemorrhage being replaced by the acquired immunodeficiency syndrome ( aids ) as the leading cause of death ( mejia - carvajal et al 2006 ) .
indeed , the mortality curve in hemophiliacs over the years has a biphasic form with two peaks : one before the 1960s ( before the widespread availability of factor replacement therapy ) and the other in the 1980s1990s ( after the occurrence of aids and of the clinical manifestations of earlier hcv infections ) .
this trend has been confirmed by a recent survey that analyzed the mortality rate and causes of death in 967 dutch hemophiliacs ( plug et al 2006 ) . during the period of the study ( 19922001 ) the mortality rate was 2.3 times higher in patients with hemophilia than in the general male population .
moreover , comparing these data with those previously reported by the same group ( rosendaal et al 1989 ; triemstra et al 1995 ) , the life expectancy of patients with severe hemophilia decreased from 63 years in the period 19731986 to 61 and 59 years in the period 19861992 and 19922001 , respectively .
however , the exclusion of virus - related deaths resulted in a life expectancy at birth of 74 years , similar to that of the unaffected dutch male population .
similar trends were observed in studies involving scottish ( ludlam 2000 ) and canadian ( walker 1998 ) hemophiliacs . from 1900 to 1942 the life expectancy of severe hemophiliacs in sweden was 16.5 years ; this increased to 23.2 years between 1943 and 1957 ( ramgren 1962 ) and to 50 years between 1957 and 1980 ( larsson and wiechel 1983 ) . for comparison , the life expectancy of unaffected swedish men during the latter period was 75 years
another study analyzed 163 patients with severe hemophilia a living in finland in the period between 193079 ( ikkala 1982 ) . during 50 years of observation
, the mean age at death increased from 7.8 years in 193039 to 25.5 years in 197079 .
the effect of hiv infection on age and causes of death among persons with hemophilia a was analyzed in a study conducted in the united states ( chorba et al 2001 ) . the median age at death decreased from 55 years in 19791982 to 40.5 years in 19871990 and increased to 46 years in 19951998 , although the median age at death of patients with hemophilia a and hiv - related disease in the latter period was 33 years , compared to 72 years for those without hiv - related disease . during the same period ,
hemophilia a - associated deaths decreased by 41% , with a 78% decrease among those who had hiv - related disease .
this decline in hiv mortality was consistent with that observed in the general population of hiv - infected persons and reflected improvements in anti - retroviral therapy .
on the other hand , another study that analyzed the causes of death among hemophiliacs not infected by hiv found that the leading cause of death was hemorrhage , followed by liver disease ( hepatitis c and b ) , stroke and cancer .
currently , children with hemophilia look forward to a normal life expectancy and excellent health - related quality of life ( gringeri et al 2006 ) .
the most important factors contributing to the improved quality of life , reduced morbidity and increased life expectancy are the availability for replacement therapy of high - quality factor concentrates ( ie , virus inactivated plasma - derived clotting factor concentrates and recombinant products ) , insights into the management of hemophilic arthropathy and liver disease ( ie , surveillance of patients with chronic hepatitis , especially with respect to cancer and liver failure , and newer treatment options such as antiviral treatment against hiv and hcv and liver transplantation ) and the improved medical management provided by specialized hemophilia treatment centers .
high - quality factor concentrates are important not only for reducing the likelihood of death from hemorrhage but also for improving the quality of life , especially through prophylactic regimens and home treatment .
secondary prophylaxis , in particular , is becoming frequently prescribed among hemophiliacs , although only few data are available in this setting ( tagliaferri et al 2006a ) .
the impact of comprehensive care has been demonstrated by soucie et al ( 2006 ) who found that hemophiliacs who had received care in a specialized hemophilia treatment center had a significantly decreased risk of death .
the impact of comprehensive care is further exemplified by the analysis of 164 patients treated at the international haemophilia training centre at bangkok with blood and blood components from 1971 to 2000 .
the estimated probability of survival beyond 13 years of age of patients with severe hemophilia increased from 0.85 during the first decade of observation to 0.94 and 1 in the second and third decades ( chuansumrit et al 2004 ) .
the mortality rate fell from 30% to 14% and 5% over the three decades of observation . despite the lack of treatment with high quality clotting factor
concentrates in this center , the patients outcome improved with the development of a comprehensive approach to management . on behalf of the italian association of hemophilia centers ( aice )
, we are conducting a multicenter study to evaluate the health status and health - related quality of life ( hr - qol ) of severely affected italian hemophiliacs of 65 years or older , recruited from italian hemophilia centers ( tagliaferri et al 2006b ) .
another primary goal of the study is to provide information on whether or not there are differences in the global health status and hr - qol between healthy elderly individuals and persons with hemophilia , who started their lives in the first part of the last century when the availability and quality of treatment were poor .
this study will also allow us to establish whether or not the burden of diseases other than hemophilia ( including cognitive impairment measured using instruments of geriatric medicine ) is similar or different in cases and controls , and to understand which factors help people with severe hemophilia to cope with their disease and reach old age .
hemophilia is associated with bleeding episodes , which often occur into the joints or muscles . over time , recurrent bleeding into joints and muscles can cause permanent problems such as arthritis , chronic pain and joint damage resulting in poor control of balance and falls which ultimately worsen quality of life .
thus joint disease remains the leading cause of morbidity in those elderly hemophiliacs who had little or no access to prophylactic regimens and home treatment when they were younger .
an ongoing australian study is evaluating the degree of joint - related dysfunction in persons with hemophilia older than 30 years and the feasibility of home exercise programs targeting balance training ( street 2006 ) .
we have recently reported the case of a 72-year - old hemophiliac with severe joint bleeding in whom treatment was converted from on - demand to a secondary prophylaxis regimen ( tagliaferri et al 2006a ) .
the latter significantly reduced the number of bleeding episodes in target joints , slowing the progression of the arthropathy and improving the patient s perceived well - being .
however , further studies , dealing with the approach to replacement therapy and the development of preventive physical therapy programs for elderly patients , are warranted .
attempts have been made to determine whether or not hemophilia protects from the development of atherosclerosis and cardiovascular events , but the results are contradictory .
studies of carotid and femoral artery intimalmedial thickness as a surrogate of atherosclerosis burden showed that hemophilia does not protect from atherosclerosis ( sramek et al 2001 ) . on the other hand ,
hemophiliacs have a decreased mortality rate due to ischemic heart disease , supporting the views that they are protected from arterial thrombosis ( sramek et al 2003 ) .
recently , girolami et al ( 2006 ) reviewed reported cardiovascular events in patients with hemophilia a. among the 42 cases published so far , nine occurred in individuals aged 65 or over .
analyzing the literature data , the authors concluded that the bleeding diathesis did not help to prevent the occurrence of arterial occlusions .
thus factors which are known to predispose to atherothrombosis ( eg , hypercholesterolemia , smoking , diabetes , hypertension , aging ) appear to overcome the coagulation defect . concomitant replacement therapy , especially with bypassing agents ( ie , activated or non - activated prothrombin complex concentrates and recombinant activated factor vii ) , was identified as having a predominant role in thrombotic manifestations in the majority of the cases ( aledort 2004 ) .
a number of successful cardiovascular interventions in elderly hemophiliacs have been carried out ( bovenzi et al 2003 ; de bels et al 2004 ) .
however , these cases raise the question of how to conduct anticoagulant therapy in such naturally anticoagulated patients .
the management of cardiovascular events in elderly hemophiliacs necessarily differs from that of non - hemophilic patients .
thus , while the treatment of acute coronary syndromes in hemophiliacs requires a combination of antithrombotic and replacement therapies , thrombolytic therapy for myocardial infarctions is unjustified in these patients due to the unacceptable risk of hemorrhage and should be substituted by alternative techniques such as percutaneous coronary angioplasty ( mafrici et al 2003 ) .
similarly , long - term anticoagulation should be avoided whenever possible by using , for example , a bioprosthetic valve in valve surgery .
elderly people with hemophilia are also at increased risk of chronic renal disease ( kulkarni et al 2003 ) .
this is probably due to chronic renal bleeding associated with the inherited coagulopathy which leads to structural renal abnormalities .
moreover , the concomitant presence of viral infections ( eg , hiv and hcv ) or exposure to nephrotoxic antiviral therapy may increase the incidence of renal dysfunction over time .
chronic renal disease is responsible for hypertension ( scully et al 2005 ) , which is observed more frequently in elderly hemophiliacs than in the general population and is a risk factor not only for cardiac disease but also for cerebral hemorrhage .
although most inhibitors occur at a young age in patients with severe hemophilia , patients with mild hemophilia may develop inhibitors at an advanced age when they receive intensive factor viii replacement therapy on the occasion of a surgical or invasive procedure for age - related problems such as prostatic hypertrophy , cardiovascular disease or cancer ( franchini et al 2006a ) .
indeed , there are some concerns on the use of bypassing agents in those patients considered at higher risk of developing thrombotic complications .
however , leebek et al ( 2006 ) have recently reported the safe and effective use of recombinant activated factor vii in three elderly patients with mild hemophilia a and high - titer inhibitors .
acquired hemophilia is an uncommon but potentially life - threatening clinical syndrome characterized by the sudden onset of bleeding in patients with a negative family and personal history .
acquired hemophilia is caused by auto - antibodies that , in the vast majority of cases , are directed against functional epitopes of factor viii causing its neutralization and/or accelerated clearance from the plasma ( delgado et al 2003 ) .
the incidence of acquired hemophilia has been estimated to be 0.21.0 cases per 1 million persons per year , with a mortality rate estimated to be in the range of 8 to 22% ( franchini et al 2005a ) .
the age distribution of autoantibodies is typically biphasic , with a small peak between 20 and 30 years ( mainly postpartum inhibitors ) and a larger peak in patients aged 6880 years .
factor viii inhibitors are distributed equally between sexes , although females predominate in the younger age group because of the association with pregnancy while males constitute the majority of patients with inhibitors over the age of 60 ( boggio and green 2001 ) . in approximately 50% of cases
factor viii autoantibodies occur in patients without concomitant diseases ( spontaneous antibodies ) , and in nearly 10% of cases autoantibodies appear during the postpartum period , usually in primiparous women within 3 months of delivery ( green and lechner 1981 ; solymoss 1998 ) .
however , several other conditions and diseases ( eg , autoimmune disorders , malignancies and drugs ) have been associated with the development of factor viii inhibitors ( bossi et al 1998 ) .
the clinical picture of acquired hemophilia differs from that of classical hereditary hemophilia a. more than 80% of patients with factor viii autoantibodies bleed into the skin , muscles or soft tissues ( including retroperitoneal hematoma ) and mucous membranes ( eg , epistaxis , gastrointestinal and urological bleeds ) , whereas hemarthroses , a typical manifestation of congenital factor viii deficiency , are unusual . not rarely the hemorrhages in acquired hemophilia are serious or life - threatening , as in the case of cerebral hemorrhage or rapidly progressive retroperitoneal hematomas ( franchini et al 2005a ) . the diagnosis of acquired hemophilia is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time , not corrected by incubating the patient s plasma with equal volumes of normal plasma ( mixing study ) , associated with low factor viii levels and evidence of a factor viii inhibitor ( which can be titrated in bethesda units ( bu)/ml ) , in a patient with no previous personal or family history of bleeding ( cohen and kessler 1996 ) .
figure 1 reports the laboratory algorithm for the diagnosis of acquired hemophilia . the treatment of acquired hemophilia is classically divided into two phases : acute and chronic .
acute phase treatment is focused on the bleeding symptoms ( ludlam et al 1994 ) .
efficient hemostasis can be achieved with a variety of methods such as correction of fviii deficiency ( plasma - derived or recombinant fviii concentrates , desmopressin ) or bypassing the inhibitor ( activated prothrombin complex concentrate [ apcc ] , recombinant activated fvii [ rfviia ] ) ( roberts 1999 ) .
the choice of antihemorrhagic therapy depends mainly on the site and the entity of the bleeding . in patients with a high titer of inhibitor and severe hemorrhage , extracorporeal removal of the autoantibody by therapeutic plasmapheresis or immunoadsorption of immunoglobulins to staphylococcal protein
the aim of the chronic phase of treatment is to eradicate the inhibitor , which is achieved by using immunosuppressive agents including corticosteroids and such drugs as cyclophosphamide , azathioprine , 6-mercaptopurine and vincristine ( green et al 1993 ) .
high - dose immunoglobulins and cyclosporin have also been shown to be effective in acquired hemophilia and can be considered as second - line therapy for those patients who do not respond to standard immunosuppressive regimens ( schwartz et al 1995 ; petrovic et al 2000 ) .
more recently , serotherapy with anti - cd20 monoclonal antibody ( rituximab ) has been successfully used in immune disorders resulting from autoantibody formation , including acquired hemophilia ( wiestner et al 2002 ) .
finally , immune tolerance induction protocols have been proposed for the eradication of autoantibodies against coagulation factors ( nemes and pitlik 2000 ) . as reported above , with the exception of post - partum cases ,
although acquired hemophilia is considered a very rare occurrence , its true incidence may be underestimated .
indeed , patients may be seen by several specialists and undergo dangerous invasive investigations and interventions before the correct diagnosis is made .
the high mortality rate reported previously is in part related to a delay in the diagnosis . besides misdiagnoses
, acquired hemophilia may be underdiagnosed as many cases of clinically silent low titer inhibitors may be unrecognized unless patients undergo surgery or trauma . in approximately 50% of the elderly patients with acquired hemophilia ,
no underlying primary disorders are identified ( idiopathic forms ) , while most of the remaining cases are associated with autoimmune diseases or cancer , which increase with age ( hauser et al 1999 ; yee et al 2000 ) .
the prognosis of acquired hemophilia in elderly patients is strongly linked to the associated primary disorder , which should be always searched for and , if possible , cured .
idiopathic cases with a low inhibitor titer and mild bleeding may be managed with desmopressin alone and may not require eradicating therapy but only a watch and wait strategy .
similarly , drug - induced inhibitors may require nothing else than close observation , since these inhibitors tend to disappear spontaneously within a few months after drug discontinuation .
on the other hand , it is unlikely that cases associated with autoimmune disorders will remit spontaneously and thus they require more aggressive eradication therapy with steroids , whether or not combined with cytotoxic agents such as cyclophosphamide ( franchini et al 2005b ) . however , particular care must be taken when using these agents in the elderly and the choice of drugs and dosages should be tailored to the individual patient s age .
an analysis of the data from the european acquired hemophilia registry ( baudo et al 2004 ) showed that infections related to immunosuppressive therapy are the first cause of death in patients with acquired hemophilia .
thus , less toxic agents should be preferred for inhibitor eradication in elderly patients . in this context
we have recently reviewed the literature data on the use of rituximab in acquired hemophilia and have collected 50 cases , including many elderly patients ( franchini et al 2006b ) .
although most of the reports were isolated cases , there was a high%age of complete or partial responses ( 86% ) when rituximab was used alone or in association with immunosuppressive therapy .
thus , if confirmed by further controlled studies , this agent could be very useful in the management of such patients .
the presence of an underlying malignancy was associated with a poorer prognosis in a recent meta - analysis by delgado et al ( 2003 ) .
however , sallah et al ( 2001 ) , in a previous review of literature data on 41 cases of patients with cancer - associated factor viii inhibitors , including patients with solid and hematologic malignancies , found a 70% rate of complete responses to treatment of the inhibitor .
low titer inhibitors associated with early stage tumors were more likely to disappear after treatment than high titer inhibitors and the eradication of autoantibody was associated with a higher overall survival .
the same authors also observed that 22% of responders achieved complete remission after treatment of the cancer . on the basis of this high response rate
, it appears that the treatment of the primary malignancy in patients with cancer - associated factors viii inhibitors is of great importance , because this facilitates eradication of the antibody .
the presence of an underlying cancer is not a contraindication to the use of immunosuppressive therapy aimed at eradicating the autoantibody , so that these patients should be treated in the same manner as other patients with acquired hemophilia .
although only a few thrombotic adverse events have been recorded in patients with acquired hemophilia following the use of bypassing agents ( guillet et al 2002 ; roberts et al 2004 ) , particular care should be exercised when these drugs are used in elderly patients who should be monitored not only for clinical evidence of hemorrhage but also for the possible onset of thrombotic complications .
concomitant cardiovascular diseases should be ruled out before using desmopressin in elderly patients with congenital or acquired hemophilia ( mannucci 1997 ) .
acquired hemophilia is an uncommon but potentially life - threatening clinical syndrome characterized by the sudden onset of bleeding in patients with a negative family and personal history .
acquired hemophilia is caused by auto - antibodies that , in the vast majority of cases , are directed against functional epitopes of factor viii causing its neutralization and/or accelerated clearance from the plasma ( delgado et al 2003 ) .
the incidence of acquired hemophilia has been estimated to be 0.21.0 cases per 1 million persons per year , with a mortality rate estimated to be in the range of 8 to 22% ( franchini et al 2005a ) .
the age distribution of autoantibodies is typically biphasic , with a small peak between 20 and 30 years ( mainly postpartum inhibitors ) and a larger peak in patients aged 6880 years .
factor viii inhibitors are distributed equally between sexes , although females predominate in the younger age group because of the association with pregnancy while males constitute the majority of patients with inhibitors over the age of 60 ( boggio and green 2001 ) . in approximately 50% of cases
factor viii autoantibodies occur in patients without concomitant diseases ( spontaneous antibodies ) , and in nearly 10% of cases autoantibodies appear during the postpartum period , usually in primiparous women within 3 months of delivery ( green and lechner 1981 ; solymoss 1998 ) .
however , several other conditions and diseases ( eg , autoimmune disorders , malignancies and drugs ) have been associated with the development of factor viii inhibitors ( bossi et al 1998 ) .
the clinical picture of acquired hemophilia differs from that of classical hereditary hemophilia a. more than 80% of patients with factor viii autoantibodies bleed into the skin , muscles or soft tissues ( including retroperitoneal hematoma ) and mucous membranes ( eg , epistaxis , gastrointestinal and urological bleeds ) , whereas hemarthroses , a typical manifestation of congenital factor viii deficiency , are unusual . not rarely the hemorrhages in acquired hemophilia are serious or life - threatening , as in the case of cerebral hemorrhage or rapidly progressive retroperitoneal hematomas ( franchini et al 2005a ) . the diagnosis of acquired hemophilia is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time , not corrected by incubating the patient s plasma with equal volumes of normal plasma ( mixing study ) , associated with low factor viii levels and evidence of a factor viii inhibitor ( which can be titrated in bethesda units ( bu)/ml ) , in a patient with no previous personal or family history of bleeding ( cohen and kessler 1996 ) .
figure 1 reports the laboratory algorithm for the diagnosis of acquired hemophilia . the treatment of acquired hemophilia is classically divided into two phases : acute and chronic .
acute phase treatment is focused on the bleeding symptoms ( ludlam et al 1994 ) .
efficient hemostasis can be achieved with a variety of methods such as correction of fviii deficiency ( plasma - derived or recombinant fviii concentrates , desmopressin ) or bypassing the inhibitor ( activated prothrombin complex concentrate [ apcc ] , recombinant activated fvii [ rfviia ] ) ( roberts 1999 ) .
the choice of antihemorrhagic therapy depends mainly on the site and the entity of the bleeding . in patients with a high titer of inhibitor and severe hemorrhage , extracorporeal removal of the autoantibody by therapeutic plasmapheresis or immunoadsorption of immunoglobulins to staphylococcal protein
the aim of the chronic phase of treatment is to eradicate the inhibitor , which is achieved by using immunosuppressive agents including corticosteroids and such drugs as cyclophosphamide , azathioprine , 6-mercaptopurine and vincristine ( green et al 1993 ) .
high - dose immunoglobulins and cyclosporin have also been shown to be effective in acquired hemophilia and can be considered as second - line therapy for those patients who do not respond to standard immunosuppressive regimens ( schwartz et al 1995 ; petrovic et al 2000 ) .
more recently , serotherapy with anti - cd20 monoclonal antibody ( rituximab ) has been successfully used in immune disorders resulting from autoantibody formation , including acquired hemophilia ( wiestner et al 2002 ) .
finally , immune tolerance induction protocols have been proposed for the eradication of autoantibodies against coagulation factors ( nemes and pitlik 2000 ) .
as reported above , with the exception of post - partum cases , a large number of patients with acquired hemophilia are elderly .
although acquired hemophilia is considered a very rare occurrence , its true incidence may be underestimated .
indeed , patients may be seen by several specialists and undergo dangerous invasive investigations and interventions before the correct diagnosis is made .
the high mortality rate reported previously is in part related to a delay in the diagnosis . besides misdiagnoses
, acquired hemophilia may be underdiagnosed as many cases of clinically silent low titer inhibitors may be unrecognized unless patients undergo surgery or trauma . in approximately 50% of the elderly patients with acquired hemophilia ,
no underlying primary disorders are identified ( idiopathic forms ) , while most of the remaining cases are associated with autoimmune diseases or cancer , which increase with age ( hauser et al 1999 ; yee et al 2000 ) .
the prognosis of acquired hemophilia in elderly patients is strongly linked to the associated primary disorder , which should be always searched for and , if possible , cured .
idiopathic cases with a low inhibitor titer and mild bleeding may be managed with desmopressin alone and may not require eradicating therapy but only a watch and wait strategy .
similarly , drug - induced inhibitors may require nothing else than close observation , since these inhibitors tend to disappear spontaneously within a few months after drug discontinuation .
on the other hand , it is unlikely that cases associated with autoimmune disorders will remit spontaneously and thus they require more aggressive eradication therapy with steroids , whether or not combined with cytotoxic agents such as cyclophosphamide ( franchini et al 2005b ) . however , particular care must be taken when using these agents in the elderly and the choice of drugs and dosages should be tailored to the individual patient s age .
an analysis of the data from the european acquired hemophilia registry ( baudo et al 2004 ) showed that infections related to immunosuppressive therapy are the first cause of death in patients with acquired hemophilia .
thus , less toxic agents should be preferred for inhibitor eradication in elderly patients . in this context
we have recently reviewed the literature data on the use of rituximab in acquired hemophilia and have collected 50 cases , including many elderly patients ( franchini et al 2006b ) .
although most of the reports were isolated cases , there was a high%age of complete or partial responses ( 86% ) when rituximab was used alone or in association with immunosuppressive therapy .
thus , if confirmed by further controlled studies , this agent could be very useful in the management of such patients .
the presence of an underlying malignancy was associated with a poorer prognosis in a recent meta - analysis by delgado et al ( 2003 ) .
however , sallah et al ( 2001 ) , in a previous review of literature data on 41 cases of patients with cancer - associated factor viii inhibitors , including patients with solid and hematologic malignancies , found a 70% rate of complete responses to treatment of the inhibitor .
low titer inhibitors associated with early stage tumors were more likely to disappear after treatment than high titer inhibitors and the eradication of autoantibody was associated with a higher overall survival .
the same authors also observed that 22% of responders achieved complete remission after treatment of the cancer . on the basis of this high response rate
, it appears that the treatment of the primary malignancy in patients with cancer - associated factors viii inhibitors is of great importance , because this facilitates eradication of the antibody .
the presence of an underlying cancer is not a contraindication to the use of immunosuppressive therapy aimed at eradicating the autoantibody , so that these patients should be treated in the same manner as other patients with acquired hemophilia .
although only a few thrombotic adverse events have been recorded in patients with acquired hemophilia following the use of bypassing agents ( guillet et al 2002 ; roberts et al 2004 ) , particular care should be exercised when these drugs are used in elderly patients who should be monitored not only for clinical evidence of hemorrhage but also for the possible onset of thrombotic complications .
concomitant cardiovascular diseases should be ruled out before using desmopressin in elderly patients with congenital or acquired hemophilia ( mannucci 1997 ) .
with their greatly prolonged life expectancy , persons with congenital hemophilia are now developing medical and surgical conditions previously not seen in this group .
the new challenge for physicians in hemophilia centers is to provide optimal care for this aging population of patients .
their optimal management requires a hemophilia team with close cooperation among physicians from different specialties such as hematology , oncology , cardiology , and nephrology .
particular care must be taken when choosing the inhibitor eradication regimen , which should be tailored to the patient s age . | after the increasing rate of deaths observed during the 1980s due to human immunodeficiency virus ( hiv ) infection , the health - related quality of life and life expectancy of persons with hemophilia have improved , mainly due to the progresses of replacement therapy and antiviral drugs and to the improvement of the global comprehensive care provided by specialized centers . as a consequence
, an increasing number of hemophiliacs have reached an older age and nowadays physicians in hemophilia centers find that they must handle age - related clinical problems never previously observed in this population .
the management of elderly persons with congenital hemophilia is discussed in the first part of this review .
the second part describes the general aspects of acquired hemophilia due to anti - factor viii autoantibodies , focusing on the clinical management of elderly patients , one of the groups most frequently affected by this acquired bleeding disorder . | Introduction
Management of congenital hemophilia in the elderly
Life-expectancy, causes of death and quality of life
Clinical management of congenital hemophilia in elderly patients
Management of acquired hemophilia in the elderly
General aspects of acquired hemophilia
Clinical management of acquired hemophilia in elderly patients
Conclusions |
undescended testis is one of the most common disorders of childhood , with a rate of 3.68% among full - term infants managed by surgical correction .
surgical intervention is warranted during early infancy to avoid secondary degeneration of the testis , to improve fertility later , to help with the detection of malignancy , and to reduce the chance of testicular torsion .
the inguinal approach is the traditional method for correcting undescended testis . in this approach ,
two incisions are made : one inguinal or groin incision to open the inguinal canal to visualize the cord structure and a second scrotal incision to fix the testes within the scrotum .
it was believed that inguinal incision is helpful for sufficient mobilization of the spermatic cord , separation of the processus vaginalis or hernia sac , high ligation of the hernia sac , and to achieve an adequate length for the testes to be relocated to the dependent portion of the scrotum .
however , bianchi and squire introduced the high scrotal incision orchiopexy technique for a palpable cryptorchid testis to decrease the potential morbidity of traditional inguinal incision orchiopexy .
until now , few prospective studies have been reported regarding the success rate of this technique compared with traditional inguinal orchiopexy for undescended palpable testis . in the present study , therefore , we evaluated a single scrotal incision technique for palpable undescended testis within the inguinal canal or distal to the external inguinal ring compared with traditional inguinal orchiopexy .
from january 2007 to december 2010 , a total of 292 children ( 398 testes ) with palpable undescended testes were randomly assigned to two groups : single scrotal incision orchiopexy ( group i , 147 children with 201 testes ) or traditional inguinal incision orchiopexy ( group ii , 145 children with 197 testes ) . patients
were assigned to the scrotal or inguinal group in a 1:1 ratio through a simple randomization procedure .
1 ) . a total of 107 children ( 146 testes ) underwent single scrotal incision orchiopexy ( group i ) and 105 children ( 141 testes ) underwent traditional inguinal incision orchiopexy ( group ii ) .
they were followed up and evaluated until 12 months after the operation and included in the final data analysis .
the patients ' mean ages ( months ) at the time of operation in groups i and ii were 40.110.3 and 41.811.4 , respectively ( table 1 ) .
all patients were seen at least 1 week postoperatively to evaluate the possible occurrence of wound infection or skin problems and to assess for any other operation - related complications . at 3 months and 12 months after the operation ,
the patients were followed up for evaluation of long - term complications , overall success rate , and parents ' satisfaction rate .
success was defined as no complications , postoperative intrascrotal location of the testis , and no conversion to the other method .
the parents ' satisfaction with the cosmetic results of the operation was assessed by use of a simple questionnaire that consisted of the answers ' satisfied , ' ' not fully satisfied , ' and ' unsatisfied . '
all parents signed an informed consent form before participation in this study allowing the use of the patients ' medical records for a scientific purpose .
children who had undergone a previous inguinal or pelvic surgery or who had a secondary ascending testis , ectopic testis , or undescended testis related to ambiguous genitalia or intersex condition were excluded from the study .
patients with primary and secondary hypogonadism and a detected hormonal abnormality or history of hormonal treatment were not included .
all children were examined twice , preoperatively in the supine position by the primary surgeon and again after induction of general anesthesia to exclude retractile testis .
the first surgical step of the single scrotal incision orchiopexy after induction of general anesthesia was a transverse skin incision that was commonly made along the high scrotal skin fold .
the dartos pouch was adequately created through this incision for later relocation of the affected testis .
the assistant manipulated the testis and held it between the thumb and index finger in a stable position , and in sequence the surgeon used blunt and sharp dissection of the subcutaneous tissues to approach the testis .
the scrotal wound was retracted in an upward direction to facilitate easier dissection , and the surgeon divided the various covering and adhesive tissues of the cord .
the dissection was carried out to the most cephalad to secure sufficient cord length and to possibly enter the lower half of the inguinal canal from below .
the gubernacular attachments were released to enable identification of the testes within the cremasteric fibers , a patent processus vaginalis , and the cord structures .
the cremasteric fibers and hernia sac were carefully separated from the cord structures , and the cranial sac was mobilized under traction into the canal and was ligated with a suture , as in traditional inguinal incision orchiopexy .
when additional cord length was required , additional dissection was done through this incision by opening the external ring and canal , as necessary . despite the additional dissection , if more length was required , the surgeon converted to the traditional inguinal orchiopexy method .
the testis was then relocated into the dartos pouch , and two fixing sutures were made between the testicular tunica albuginea and inner scrotal wall medially and laterally to prevent ascent .
after inguinal skin incision , the cord structure and testis were taken out of the inguinal incision site and then sufficiently dissected for mobilization .
high ligation was done with the processus vaginalis , and the surgeon made an incision along the scrotal crease and relocated the testis in the scrotum .
the student 's t - test and chi - square test were used for data analysis .
from january 2007 to december 2010 , a total of 292 children ( 398 testes ) with palpable undescended testes were randomly assigned to two groups : single scrotal incision orchiopexy ( group i , 147 children with 201 testes ) or traditional inguinal incision orchiopexy ( group ii , 145 children with 197 testes ) . patients
were assigned to the scrotal or inguinal group in a 1:1 ratio through a simple randomization procedure .
1 ) . a total of 107 children ( 146 testes ) underwent single scrotal incision orchiopexy ( group i ) and 105 children ( 141 testes ) underwent traditional inguinal incision orchiopexy ( group ii ) .
they were followed up and evaluated until 12 months after the operation and included in the final data analysis .
the patients ' mean ages ( months ) at the time of operation in groups i and ii were 40.110.3 and 41.811.4 , respectively ( table 1 ) .
all patients were seen at least 1 week postoperatively to evaluate the possible occurrence of wound infection or skin problems and to assess for any other operation - related complications . at 3 months and 12 months after the operation ,
the patients were followed up for evaluation of long - term complications , overall success rate , and parents ' satisfaction rate .
success was defined as no complications , postoperative intrascrotal location of the testis , and no conversion to the other method .
the parents ' satisfaction with the cosmetic results of the operation was assessed by use of a simple questionnaire that consisted of the answers ' satisfied , ' ' not fully satisfied , ' and ' unsatisfied . '
all parents signed an informed consent form before participation in this study allowing the use of the patients ' medical records for a scientific purpose .
children who had undergone a previous inguinal or pelvic surgery or who had a secondary ascending testis , ectopic testis , or undescended testis related to ambiguous genitalia or intersex condition were excluded from the study .
patients with primary and secondary hypogonadism and a detected hormonal abnormality or history of hormonal treatment were not included .
all children were examined twice , preoperatively in the supine position by the primary surgeon and again after induction of general anesthesia to exclude retractile testis .
all operations were performed by 1 surgeon ( kim so ) . the first surgical step of the single scrotal incision orchiopexy after induction of general anesthesia was a transverse skin incision that was commonly made along the high scrotal skin fold .
the dartos pouch was adequately created through this incision for later relocation of the affected testis .
the assistant manipulated the testis and held it between the thumb and index finger in a stable position , and in sequence the surgeon used blunt and sharp dissection of the subcutaneous tissues to approach the testis .
the scrotal wound was retracted in an upward direction to facilitate easier dissection , and the surgeon divided the various covering and adhesive tissues of the cord .
the dissection was carried out to the most cephalad to secure sufficient cord length and to possibly enter the lower half of the inguinal canal from below .
the gubernacular attachments were released to enable identification of the testes within the cremasteric fibers , a patent processus vaginalis , and the cord structures .
the cremasteric fibers and hernia sac were carefully separated from the cord structures , and the cranial sac was mobilized under traction into the canal and was ligated with a suture , as in traditional inguinal incision orchiopexy .
when additional cord length was required , additional dissection was done through this incision by opening the external ring and canal , as necessary . despite the additional dissection ,
if more length was required , the surgeon converted to the traditional inguinal orchiopexy method .
the testis was then relocated into the dartos pouch , and two fixing sutures were made between the testicular tunica albuginea and inner scrotal wall medially and laterally to prevent ascent .
after inguinal skin incision , the cord structure and testis were taken out of the inguinal incision site and then sufficiently dissected for mobilization .
high ligation was done with the processus vaginalis , and the surgeon made an incision along the scrotal crease and relocated the testis in the scrotum .
the student 's t - test and chi - square test were used for data analysis .
at the 12-month follow up , a total of 107 children ( 146 testes ) who underwent single scrotal incision orchiopexy ( group i ) and 105 children ( 141 testes ) who underwent traditional inguinal incision orchiopexy ( group ii ) were included in the final data analysis .
the mean follow - up time was 12.93.4 months in group i and 12.73.3 months in group ii , and the evaluation is still ongoing ( table 1 ) . when the overall success rate was compared between the groups , it was not significantly different : 92.5% ( 135/146 testes ) in group i and 96.5% ( 136/141 testes ) in group ii ( p=0.86 ) at 12 months ( table 2 ) .
there was a significant difference between groups i and ii in terms of the period of hospitalization ( days ) ( 2.10.8 vs 2.50.7 ; p=0.03 ) and the operation time ( minutes ) ( 40.525.9 vs 62.335.6 ; p<0.001 ) ( table 2 ) . in nine cases in group
i , the dissected cord length was insufficient because of severe adhesion despite a distal location in seven cases and a higher location in two cases , and we converted to the traditional inguinal approach in these cases . in the converted cases , the period of hospitalization ( days ) was 3.30.7 and the operation time ( minutes ) was 5530.3 . thus , it seemed to take more time compared with the rest of group i , but we did not perform statistical analysis .
ii , traditional incision failed because of the need for laparoscopic exploration of hidden testes in two cases and the need for orchiectomy due to already atrophied testes in two cases .
postoperative scrotal hematoma was found in one case in group i , and wound dehiscence was found in one case in each group .
other complications including wound infection , testicular ascension , and testicular atrophy were not detected even after long - term follow - up .
both groups were pleased with the cosmetic result of the operation in terms of the parents ' subjective satisfaction rate : 96.6% in group i and 96.5% in group ( table 2 ) .
single scrotal incision orchiopexy for a palpable undescended testis is well tolerated and has a cosmetically satisfactory result .
compared with traditional inguinal incision orchiopexy , single incision orchiopexy showed several benefits such as a shorter operation time and shorter hospital stay .
the results of this study suggest that single incision orchiopexy is a useful method in terms of simplicity without significant surgical difficulties .
also , the success rate of single incision orchiopexy was as high as 92.5% ; only 11 testes required conversion to traditional inguinal incision orchiopexy or had postoperative complications .
traditional inguinal incision orchiopexy was previously regarded as a mandatory procedure for obtaining adequate mobilization of the spermatic cord , but requires two standard skin incisions for direct visualization of the cord structures , and separation and high ligation of commonly associated inguinal hernia is not easy without opening the inguinal canal
furthermore , in the pediatric population , there is good mobility of the skin incision and a relatively short distance from the external to the internal inguinal ring .
these points led others to believe that one scrotal incision rather than two may be sufficient for orchiopexy in patients with a palpable , low - lying undescended testis .
bianchi and squire proposed that moving the incision by retraction and the short distance from the internal to the external ring made it possible to dissect the hernia sac without opening the canal .
moreover , they suggested that achieving adequate palpable testis cord length was dependent more on releasing the hernial sac from the cord than on dissection around the spermatic cord vessels .
the suggested that the benefits of using one incision in the scrotal skin fold included decreased pain , improved cosmesis , and a shorter operative time with less incision needed to close the wound window .
caruso et al evaluated the bianchi single scrotal incision technique for orchiopexy in patients with a palpable undescended testis distal to the external inguinal ring and reported that this technique is simple and safe in such cases .
only 1 of 42 testicles approached in this manner required a conversion to the traditional inguinal incision orchiopexy to gain adequate cord length .
an important point in their report was that , after opening the inguinal canal when they converted to the inguinal approach , they found that the ligated hernia sac was retracted well into the low retroperitoneum above the level of the internal ring .
their findings support previous results showing that a palpable undescended testis may be surgically relocated into the dependent scrotum without sacrificing the traditional principles of orchiopexy .
the present study is distinct from previous descriptions of scrotal orchiopexy . in previous reports ,
single scrotal incision orchiopexy was rarely tried in patients with an undescended testis in the inguinal canal and the exact success rate and satisfaction rate were rarely calculated . bianchi and squire performed single scrotal incision orchiopexy in 120 patients with undescended testis and reported a 95.8% success rate .
they reported that the testicular locations of failed cases were in high areas such as the inguinal canal .
dayan et al prospectively evaluated the success rate with or without inguinal hernia in patients with an undescended testis within the inguinal canal or beyond the external inguinal ring .
they divided the patients into two groups : one with the testis located within the inguinal canal and the other with the testis located beyond the external inguinal ring .
scrotal orchiopexy was performed successfully ( 97.6% ) in 42 of 43 testes in the distal to the external inguinal ring group , and only 1 patient required conversion to a traditional inguinal incision .
the average operating time was 18 minutes , and no hydrocele or hernia was noted . in the 29 testes that were located within the inguinal canal ,
3 cases needed conversion to traditional inguinal orchiopexy , with an average operative time of 25 minutes and a success rate of 89.7% . in the present study , the overall success rate was 92.5% ( 135/146 ) in the single scrotal incision orchiopexy group and 96.5% ( 136/141 ) in the traditional inguinal incision orchiopexy group , thus showing no statistically significant difference between the two procedures .
however , the operation time and hospital stay period were significantly shorter in the single scrotal incision group than in the traditional inguinal incision orchiopexy group , as in the previous study . in our study , in two of the nine cases that were converted to traditional orchiopexy
, the testes were located in the higher inguinal canal , and 7 were severely adhered with adjacent tissues . in those cases ,
the main reason for conversion was insufficient length of cord . in the failed orchiopexy cases in the traditional orchiopexy group ,
according to the results of our study , we suggest that possible conversion to traditional orchiopexy should be considered before the operation when the testis is located in the inguinal canal or higher .
our results show that single scrotal orchiopexy can be safely performed through a high scrotal incision .
an additional inguinal incision is only required in a small number of subjects in whom the palpable testis has a high location and the vascular length is insufficient or the processus vaginalis is not sufficient . a possible controversy regarding this scrotal approach technique is whether the dissection is high enough to easily allow for adequate lengthening of the cord and placement of the testis into the scrotum without tension . also , there is concern that a single scrotal approach may not allow sufficient ligation of the processus vaginalis to avoid hernia or hydrocele formation after the operation .
several others have used the high scrotal incision technique to correct abnormalities of the patent processus vaginalis , such as hernia and hydrocele . moreover , many other studies have suggested that failed orchiopexy from a scrotal incision is not due to incomplete division of the hernia sac from the spermatic vessels . despite the controversy over the relationship between the success rate of single scrotal incision orchiopexy and ligation of the patent processus vaginalis , we successfully ligated the processus vaginalis in all cases .
thus , it could be said that the scrotal incision orchiopexy was cosmetically feasible . in the single scrotal incision group compared with the traditional inguinal incision group , objective indexes such as operation time and hospital
the results of the present study confirm our belief that single scrotal incision orchiopexy is a simple , safe , and cosmetically satisfactory technique .
this study had some limitations , such as the relatively small number of patients included in the study .
also , important variables in this analysis , such as testis volume , were not considered in the follow - up evaluation .
the results of our study showed that single scrotal incision orchiopexy is a simple technique associated with a short operation time and hospital stay and is a cosmetically feasible method .
most palpable testes can be safely approached through a scrotal incision , but an additional inguinal incision should be considered in some cases of high inguinal testes where insufficient dissection of vascular length or the processus vaginalis is encountered . | purposewe prospectively evaluated the surgical outcomes of single scrotal incision orchiopexy in children with a palpable undescended testis compared with the traditional two incision orchiopexy.materials and methodsa total of 398 orchiopexies ( 292 children ) were included and randomly assigned to the single scrotal incision orchiopexy group ( group i , 147 children , 201 testes ) or the traditional inguinal incision orchiopexy group ( group ii , 145 children , 197 testes ) .
the final number of patients enrolled ( excluding those lost to follow - up ) was 107 children ( 146 testes ) in group i and 105 children ( 141 testes ) in group ii .
success was defined as no complications , postoperative intrascrotal location of the testis , and no conversion to the traditional inguinal approach .
surgical outcomes and complications were compared between the two groups .
testicular location , complications , and subjective satisfaction rate were assessed at the follow - up evaluation at least 12 months postoperatively.resultsthe overall success rate in group i was 92.5% in 135 of 146 testes ; the remaining 9 testes required conversion to traditional two incision orchiopexy . in group ii , orchiopexy was successful in 136 of 141 testes ( 96.5% ) .
the operation time and hospital stay were significantly shorter in group i ( 40.525.9 minutes , 2.10.8 days ) than in group ii ( 62.335.6 minutes , 2.50.7 days ) , respectively ( p<0.001 , p=0.03 ) .
postoperative complications were found in two cases ( hematoma , wound dehiscence ) in group i and in one case ( wound dehiscence ) in group ii ; all cases with complications recovered with conservative care .
the subjective rate of satisfaction with the cosmetic result was 96.6% in group i and 96.5% in group ii ( p=0.97).conclusionswe conclude that single scrotal incision orchiopexy is a simple technique that is associated with a shorter operation time and hospital stay than the traditional method and that is more feasible cosmetically . | INTRODUCTION
MATERIALS AND METHODS
1. Study design
2. Exclusion criteria
3. Surgical procedure
4. Statistics
RESULTS
DISCUSSION
CONCLUSIONS |
diabetic complications have become a serious issue in japan , and the dramatic rise in the number of patients with diabetes has exacerbated this problem
. however , awareness of one serious health condition , namely , diabetic foot disease , is inadequate among patients and healthcare providers [ 1 , 2 ] . in 2008 , the japanese government introduced a management fee for diabetic complications to facilitate the prevention of diabetic foot ulcers .
consequently , the number of foot care outpatient clinics that specialize in preventing diabetic foot ulcers has risen along with the increase in the number of patients with diabetes in japan . however , only a few japanese reports are available that describe investigations into the long - term occurrence of diabetic foot ulcer and its risk factors .
although diabetic neuropathy and angiopathy have been reported as classical risk factors for diabetic foot ulcer [ 1 , 4 ] , few studies ' findings demonstrate the association between the occurrence of diabetic foot ulcer and microangiopathy complications , including albuminuria ( alb ) and diabetic retinopathy ( dr ) .
indeed , patients with advanced dr or alb are very likely to develop diabetic neuropathy or angiopathy .
moreover , dr reduces visual acuity in adults , and the findings from previous studies have shown that a reduction in visual acuity is a risk factor for diabetic foot ulcer [ 4 , 5 ] .
in addition , alb and elevated serum creatinine ( scr ) levels have been reported as risk factors for diabetic foot ulcer [ 6 , 7 ] . to determine the diabetic foot ulcer incidence and examine the association between diabetic foot ulcer and microangiopathy complications , namely , dr and alb , we conducted a retrospective study of 1,305 patients with type 2 diabetes who had received in - hospital foot care education .
all subjects who had undergone a 2-week education program for diabetic patients between january 1999 and may 2005 were considered for inclusion in the study . during hospitalization ,
the following exclusion criteria were applied : ( 1 ) the presence of an active diabetic foot ulcer at the time of inclusion , ( 2 ) an estimated glomerular filtration rate ( egfr ) < 30 ml / min/1.73 m or a strong suspicion of renal disease other than diabetic nephropathy , for example , glomerulonephritis , and ( 3 ) a follow - up period < 1 year . of the 1,958 study candidates , 648
were transferred to other facilities for medical or personal reasons , or they withdrew from the study for unknown reasons within 1 year , 3 subjects had egfrs < 30 ml / min/1.73 m , and 2 subjects had active diabetic foot ulcers .
data relating to the patients ' ages , sexes , body mass indexes , diabetes durations , and smoking statuses were collected from the medical records at the time of hospitalization .
past histories of cardiovascular disease , including the occurrences of myocardial infarctions , coronary interventions , and ischemic strokes , and the presence of diabetic neuropathy and angiopathy were determined from the patients ' medical records .
blood samples were collected in the early morning of day 2 of hospitalization following an overnight fast . the glycosylated hemoglobin ( hba1c )
( % ; ngsp ) levels on admission were determined using high - performance liquid chromatography .
the presence of diabetic neuropathy was evaluated using the semmes - weinstein monofilament test and the vibratory sensation test .
the semmes - weinstein monofilament test involved assessing a foot 's sensation using a 10 g monofilament at five sites on the plantar aspect of each foot , namely , the hallux , and the first , second , third , and fifth metatarsal heads .
the results from the vibratory sensation test were considered abnormal if the perception of vibration ceased within 10 s after a 128-hz tuning fork was applied to the medial malleolus .
if the sensations from the 10-g monofilament or the vibration sensation tests were reduced , the patient was diagnosed as having sensory neuropathy . when the patients had histories of intermittent claudication and rest pain , peripheral arterial disease , or absent or reduced pedal pulses , ankle brachial index ( abi ) measurements were performed .
every patient rested in a supine position for at least 5 min in a quiet room before the abi was measured .
if either or both abi values were 0.9 , the patient was diagnosed with angiopathy .
in addition , 24-hour urine samples were collected from the patients from day 2 until day 3 of hospitalization .
the clinical staging of diabetic nephropathy was based mainly on the urinary albumin excretion rate ( aer ) and , at the advanced stage , it was based on reductions in the egfr .
alb was defined as a urinary aer > 20 g / min that was determined during the 24-hour urine sample collection .
the egfr , which was used as an index of nephropathy , was obtained using the following formula : ( 1)egfr=194scr1.094age0.287woman0.739 . all of the subjects underwent funduscopic examinations that were carried out by a qualified ophthalmologist during or just before hospital admission .
the subjects were categorized into four groups based on their diabetic microangiopathy complications at baseline , as follows : category 1 , normoalbuminuria without dr ( n = 712 ) ; category 2 , alb without dr ( n = 195 ) ; category 3 , normoalbuminuria with dr ( n = 185 ) ; and category 4 , alb with dr ( n = 213 ) .
the subjects were treated for diabetes mellitus and , when necessary , for coexisting diseases at our outpatient clinics at intervals of between 1 and 4 months , depending on the status of each disease .
the follow - up histories for incident diabetic foot ulcers were obtained from the medical records .
the study 's protocol abided by the japanese government 's ethical guidelines for epidemiological research , and it was reviewed and approved by the ethics committee of saiseikai central hospital ( protocol number 311 ) .
all of the procedures were undertaken in accordance with the ethical standards established by the institutional and national committees on human experimentation and in accordance with the declaration of helsinki .
the patients ' baseline characteristics were determined after they had been stratified into the four categories .
the differences among the categories were evaluated using a one - way analysis of variance .
the 5-year and 10-year cumulative diabetic foot ulcer incidence rates were estimated using the kaplan - meier method .
the log - rank test was used to examine the differences among the four patient categories in relation to the diabetic foot ulcer incidence .
we constructed cox proportional hazard models to estimate the associations between diabetic complications and the diabetic foot ulcer incidence , using the follow - up time as a dependent variable .
age- , sex- , and multivariable - adjusted hazard ratios ( hrs ) were calculated using cox proportional hazards regression models .
the multivariable - adjusted models were adjusted for the presence of diabetic neuropathy alone ( model 2 ) and for the presence of diabetic neuropathy and angiopathy ( model 3 ) .
the multivariable - adjusted hrs were used to calculate the relative risks of diabetic foot ulcer occurrence in each diabetic microangiopathy complications category . a p value < 0.05 was considered to indicate statistical significance .
stata version 10 ( statacorp lp , college station , tx , usa ) was used for the statistical calculations .
table 1 shows the baseline characteristics of the study participants who were grouped according to their diabetic microangiopathy complications .
the mean age of the study cohort was approximately 60 years , approximately 70% of the patients were men , and the mean diabetes duration was approximately 10 years .
of the 1,305 study participants , 54.6% ( n = 712 ) were assigned to category 1 , 14.9% ( n = 195 ) were assigned to category 2 , 14.2% ( n = 185 ) were assigned to category 3 , and 16.3% ( n = 213 ) were assigned to category 4 .
for example , the subjects in category 4 were older , were predominantly male , and they had longer disease durations compared with the subjects in the other categories .
of the 1,305 subjects evaluated , 93 died , 28 were transferred to other facilities for medical or personal reasons , and 201 withdrew for unknown reasons during the 10-year follow - up period .
the follow - up rate at 10 years was 75.3% . during the median follow - up period of 11.8 years
, 50 cases were diagnosed with new - onset diabetic foot ulcers , yielding 14,291 person - years ( table 2 ) .
the annual diabetic foot ulcer incidence rates were 1.6 , 1.5 , 3.4 , and 12.5 per 1,000 person - years in categories 1 , 2 , 3 , and 4 , respectively .
the kaplan - meier curves showed that the individuals in category 4 had a significantly higher diabetic foot ulcer incidence ( log - rank test , p < 0.001 ) ( figure 1 ) .
the 10-year cumulative diabetic foot ulcer incidence increased according to the microangiopathy complications category , with 1.7% , 1.2% , 4.2% , and 12.7% of subjects developing diabetic foot ulcers in categories 1 , 2 , 3 , and 4 , respectively ( table 2 ) .
associations between the presence of dr , alb , diabetic neuropathy , and angiopathy and the diabetic foot ulcer incidence were identified using cox regression analysis ( table 3 ) . after adjusting for age and sex ( model 1 ) ,
the presence of diabetic microangiopathy complications was positively and significantly associated with the diabetic foot ulcer incidence . in model 3 , which was additionally adjusted for the presence of diabetic neuropathy and angiopathy , the hrs and the 95% confidence intervals ( cis ) for the diabetic foot ulcer incidence were 0.66 ( 95% ci , 0.182.36 ) , 1.72 ( 95% ci , 0.674.42 ) , and 3.17 ( 95% ci 1.526.61 ; p = 0.002 ) for categories 2 , 3 , and 4 , respectively , compared with category 1 .
this study aimed to determine the diabetic foot ulcer incidence in patients with type 2 diabetes and to investigate the association between diabetic foot ulcer and microangiopathy . following in - hospital education ,
the 5-year and 10-year cumulative diabetic foot ulcer incidence rates in patients with type 2 diabetes were 1.9% and 3.7% , respectively .
importantly , the 10-year cumulative diabetic foot ulcer incidence rate in category 4 , which comprised patients with both dr and alb , was 12.7% and was up to threefold higher than the 10-year cumulative diabetic foot ulcer incidence rate in category 1 .
after adjusting for the presence of diabetic neuropathy and angiopathy , the presence of diabetic microangiopathy complications was positively and significantly associated with the occurrence of diabetic foot ulcers .
the findings from this study provide information about the risk of patients developing diabetic foot ulcers , and they enable more realistic risk estimations in relation to the occurrence of diabetic foot ulcers in individuals with dr and alb . although the number of foot care outpatient departments has grown in response to the increase in the number of patients with diabetes in japan , few long - term follow - up studies have investigated the diabetic foot ulcer incidence , and the results from short - term studies only are available in the literature .
reports from asian countries are particularly uncommon , and an association between the diabetic foot ulcer incidence and the presence of dr and/or alb has not been proposed .
diabetic microangiopathy progresses with the duration of a patient 's diabetes , and the diabetic complications observed are interrelated .
the results from this study may enable type 2 diabetes patients with dr and/or alb to understand the actual risk in relation to diabetic foot ulcer development .
moreover , the 10-year cumulative diabetic foot ulcer incidence rates in the patients in categories 3 and 4 , which included those with dr , were higher than the 10-year cumulative diabetic foot ulcer incidence rates in the patients in categories 1 and 2 , which contained patients who did not have dr , indicating that dr is a particularly important factor associated with the occurrence of diabetic foot ulcers . indeed , boyko et al . and leese et al
. found that poor vision , which was defined as a corrected visual acuity of 20/40 or lower , is a risk factor for diabetic foot ulcer .
in addition , dr is a risk factor for all causes of death , cardiovascular events , and arteriosclerosis . in their study ,
ohtomo et al . found that approximately 20% of the patients with dr had coronary artery disease , which may reflect impaired vascular perfusion caused by arteriosclerosis . in another study ,
ogawa et al . reported that diabetic neuropathy was present in all of the patients with type 2 diabetes who were hospitalized for diabetic foot ulcers and that dr and nephropathy were present in 96.4% and 78.6% of the patients , respectively .
the incidence of dr in japanese patients with type 2 diabetes is 38.3/1,000 person - years , and the dr progression rate has been reported as 21.1/1,000 person - years . since subjective symptoms are often absent , even when dr is at an advanced stage , it is important to encourage patients with definitively diagnosed or suspected diabetes to visit an ophthalmology department . the diabetic foot ulcer incidence has been investigated previously . from their study ,
peters and lavery reported that during the 3-year follow - up period , foot ulceration occurred in 14.3% , 18.8% , and 55.8% of the patients with diabetic neuropathy , diabetic neuropathy combined with foot deformities or peripheral arterial disease , and previous histories of foot ulceration , respectively .
moss et al . followed up with 906 patients who were diagnosed with type 2 diabetes before they were 30 years old and 984 patients who were diagnosed with type 2 diabetes after they were 30 years old for 14 years , and they found that amputations caused by diabetic foot ulcers were required in 7.2% and 9.9% of these patients , respectively , and they concluded that severe retinopathy was a risk factor for amputation . in our study cohort ,
the diabetic foot ulcer frequency was lower than the frequencies reported from these aforementioned studies , which could be a consequence of the in - hospital education that our patients received . in a japanese study , oe et al
. followed up with 578 patients with diabetes who visited a specialized outpatient department for 5 years , and they found diabetic foot ulcers in 6 ( 1.2% ) patients .
however , 70% of the subjects did not have diabetic neuropathy or angiopathy ; therefore , the patients ' backgrounds differed from those in the current study , in which 40% of the patients had diabetic neuropathy .
despite the patients in the current study attending a group lecture about foot care as part of an in - hospital education program , the patients with type 2 diabetes , dr , and alb developed diabetic foot ulcers , and a sharp increase in the cumulative diabetic foot ulcer incidence was observed after 5 years ( figure 1 ) .
therefore , since the complications associated with diabetes tend to progress with its duration , patients with microangiopathy may require recurring outpatient education programs .
first , this was a hospital - based cohort study ; hence , a selection bias can not be excluded .
second , a considerable percentage of the subjects were lost to follow - up during the study period .
in addition , changes in the glucose levels and changes to the therapeutic regimens were not considered during the outpatient treatment period .
furthermore , we did not identify an association between dr and visual acuity . finally , the impacts of the foot care and education programs on the study 's results are unknown
. future investigations into the occurrence of diabetic foot ulcer in japan should account for these limitations .
the findings from this study showed that diabetic individuals with dr and alb were at a significantly increased risk of developing diabetic foot ulcers .
from the perspective of a diabetic patient 's evaluation of risk , the presence of asymptomatic dr and/or alb may not be considered serious ; hence , early intervention and long - term follow - up programs are crucial to prevent diabetic foot ulcers . |
aim . to determine the diabetic foot ulcer incidence and examine its association with microangiopathy complications , including diabetic retinopathy ( dr ) and albuminuria ( alb ) , in type 2 diabetes patients
. methods .
this was a retrospective cohort study of 1,305 patients with type 2 diabetes who were assigned to the following groups : category 1 , normoalbuminuria without dr ( n = 712 ) ; category 2 , alb without dr ( n = 195 ) ; category 3 , normoalbuminuria with dr ( n = 185 ) ; and category 4 , alb with dr ( n = 213 ) .
cox proportional hazard models were used to compare the risks of developing diabetic foot ulcers across the categories .
results . during 14,249 person - years of follow - up
, 50 subjects developed diabetic foot ulcers , with incidence rates of 1.6/1,000 , 1.5/1,000 , 3.4/1,000 , and 12.5/1,000 person - years in categories 1 , 2 , 3 , and 4 , respectively .
after adjusting for the presence of diabetic neuropathy and macroangiopathy , the hazard ratios and 95% confidence intervals ( cis ) for the risk of diabetic foot ulcer development were 0.66 ( 95% ci , 0.182.36 ) , 1.72 ( 95% ci , 0.674.42 ) , and 3.17 ( 95% ci , 1.526.61 ) in categories 2 , 3 , and 4 , respectively , compared with category 1 . conclusion .
the presence of dr and alb significantly increases the risk of diabetic foot ulcer development . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
hypersensitivity to mosquito bites ( hmb ) is characterized by intense skin reactions at bite sites , which consist of not only erythema or bulla but also ulcer or scar , and is associated with general symptoms , such as high fever , general malaise , cramps and renal failure . while this disorder is very rare and reports are scarce in the western literature , some cases of hmb have been reported from japan and taiwan . more than 90% of adults have been exposed to epstein - barr virus ( ebv ) and the virus persists in b cells and epithelial cells in the oropharynx .
recently , ebv has been considered to be associated with not only b - cell malignancy but also t - cell malignancy and natural killer ( nk ) cell granular lymphocyte proliferative disorder ( glpd ) . here
we report the first case of hmb associated with nk cell - derived large granular lymphocyte ( nk - lgl ) lymphocytosis in korea .
a 19-year - old male was referred to our hospital with well - demarcated pustules on the erythematous base on the face / right ear ( figure 1 ) and with fever for 4 days .
he had suffered hypersensitive reactions to mosquito bites , such as skin lesions ( ex , nodules , pustules , ulcerations ) , edematous change on the whole body and the extremities and fever , from childhood .
laboratory tests showed white blood cell count 7.010/l , hemoglobin 15.0 g / dl , platelet 27210/l , biochemical profile , including lactate dehydrogenase , was in the normal range .
ebv anti - ea - dr igg , anti - ebna was positive and serum titer of igg against ebv vca increased .
immunophenotypic analysis demonstrated that cd16+cd56 + cells increased ( 79% ) and cd3 + , cd4 + , and cd8 + cells decreased ( 15% , 8% , 7% ) .
we found rearrangement of tcr-chain gene by pcr analysis in this case ( figure 3 ) , as in some cases of the glpd . in our case ,
pcr amplification with specific primers for tcr-chain gene demonstrated a single band of approximately 160190 bp for v-j products .
ebv , a widespread human herpes virus , infects > 90% of the population by adulthood and persists in b cells and epithelial cells in the oropharynx where reactivation and viral replication may intermittently occur . the virus has also been linked to various b cell , non - b cell neoplasms , such as endemic burkitt s lymphoma and nasopharyngeal carcinoma .
ebv can infect t cells and peripheral lymphoproliferation of cd3 + cells may occur under caebv and , also , ebv can infect nk cells and may induce nk cell lymphoproliferation in patients with caebv . if the activity of ebv is responsible for the lymphoproliferation , the anti - ebv antibody titers , especially anti - vca igg or anti - ea igg might be related with lymphoproliferation . however , if our patient 's data , such as ebv anti - vca igg and ebv anti - ea igg , could not indicate caebv directly , this patient may be considered as caebv because nk cell lymphocytosis associated with ebv infection is frequently detected . to confirm the caebv , further examination of ebv , serial check - up for serum levels of anti - vca igg , iga and igm , anti - ea igg , iga and igm , and anti - ebna are required . according to ishihara et al ,
31% of cases of caebv were complicated by hmb and suggested that the pathogenesis of hmb might be related to clonal lymphoproliferation of ebv dna - positive nk cells .
this immunohematological abnormality may induce the characteristic symptoms of hmb . on the other hand ,
ishihara et al suggested that hmb might be one of the factors that induce ebv - associated lymphoproliferative disease . in this study , while three patients who did not manifest lymphoproliferation did not exhibit this history of hmb , four of six patients who did manifest monoclonal or oligoclonal lymphoproliferation had hmb .
tokura et al have reported that nk cell - dominant mononuclear cells are infiltrated in mosquito bite sites of a severe hmb patient .
it could be speculated that accumulation of nk cells augments reactions on the skin and that these nk cells may directly or indirectly mediate the systemic symptoms by the mosquito bites .
recurrent and prolonged activated state of nk cells may induce additional genetic damage , including chromosomal changes that lead to the genesis of leukemias or lymphomas . in conclusion , we report the first case of hmb associated with nk - lgl lymphocytosis in korea , and further investigation of hmb pathogenesis and the relationship between ebv - infected nk cells and subsequent oncogenesis of nk - lgl lymphoma , including chronic nk - lgl lymphocytosis , is needed . | hypersensitivity to mosquito bites ( hmb ) is characterized by intense skin reactions at bite sites .
the pathogenesis of hmb might be related to clonal lymphoproliferation of epstein - barr virus dna - positive natural killer ( nk ) cells .
we report the first case of hmb possibly associated with nk cell - derived large granular lymphocyte ( nk - lgl ) lymphocytosis in korea . | INTRODUCTION
CASE
DISCUSSION |
in keeping with the mission of a liberal arts education , denison s neuroscience concentration provides our students with a challenging interdisciplinary perspective on the complex relationships between brain and behavior . in this way
, students who pursue the concentration are exposed to a number of perspectives within neuroscience , from cellular and molecular analysis to broader , more molar systems approaches to behavior .
students pursuing our neuroscience concentration are most often biology or psychology majors , although there have been a few biochemistry majors completing the concentration . since 2000 , we have had close to 70 students earn the concentration , with an additional nine students completing self - designed majors in neuroscience , cognitive neuroscience , social neuroscience , or psychobiology .
our curriculum is structured in such a way that all neuroscience students must complete introductory psychology and introduction to the science of biology prior to enrolling in our introduction to neuroscience ( neur 200 ) course .
neur 200 emphasizes the basics of the field , including cellular physiology , ionic movements , refractory periods , receptor dynamics , post - synaptic potentials , neuropharmacology , and neuroanatomy .
the two introductory courses are also required for the 200- and 300-level elective courses in psychology or biology .
students must also complete four courses ( including neur 200 ) designed to provide breadth in the concentration . throughout the remainder of their tenure at denison
, neuroscience students must complete biological psychology , biological psychology research , two upper - level electives depending upon the student s major and area(s ) of interest , and our capstone course , advanced neuroscience ( neur 400 ) . table 1 summarizes the neuroscience curriculum , identifying pre - requisite courses , courses required for breadth , and more advanced required and elective courses that offer depth in the concentration .
over the last several years , capstone courses have become increasingly more visible on college and university campuses .
indeed , the capstone experience is now fairly commonplace in smaller , private colleges , as well as in large public institutions ( badway and grubb , 1999 ) .
capstone courses are believed to provide valuable experiences for students , including opportunities for synthesis and integration of information ( e.g. , henscheid et al . , 2000 ) , further development of critical thinking and writing and speaking skills ( cuseo , 1998 ) , and an ideal environment for assessing student learning ( moore , 2005 ) . in our program , the capstone experience for all neuroscience students is advanced neuroscience ( neur 400 ) .
this course is designed for juniors and seniors who have completed the majority ( if not all ) of the courses required for the concentration .
typically , the course enrolls 1525 students , and is taught in the spring semester . from the inception of our neuroscience program , we envisioned neur 400 to be a course that brings together critical concepts and ideas from the students previous coursework in a format emphasizing discussion of primary literature and incorporating multiple learning activities and projects throughout the semester . unlike the introductory course in neuroscience , neur 400 addresses contemporary molar issues in neuroscience .
importantly , our advanced neuroscience course is team taught by faculty from biology , psychology , chemistry , philosophy and computer science . in this way
, students are exposed to cutting edge issues within a number of sub - fields of neuroscience by faculty whose primary interests reflect those issues and problems .
one faculty member serves as the instructor of record ( this person receives teaching credit for the course ) and is responsible for the course organization and administration ( syllabus creation , coordination among participating faculty , development of assignments , and grading ) . at denison ,
the instructor of record rotates every two years between the psychology and biology departments . in recent years
, the course topics and readings have examined visual attention and computational neuroscience , autism , biochemistry of memory formation , explicit and implicit memory systems , amnesia , face recognition and cognitive neuroscience , artificial intelligence and face recognition , neuroscience of consciousness , neurophilosophy , nervous tissue differentiation and central nervous system development , glia and glioma , stress and neurodegeneration , affective neuroscience , educational neuroscience , and cultural neuroscience .
each faculty participant assigns research articles and will typically lead either one , two , or three class meetings that can include lectures and research presentations , student - led discussions and presentations , or active learning exercises .
students are also asked to post on blackboard , an on - line course management system , two or three discussion questions or talking points on the reading assignments prior to each class meeting .
several different assignments have been designed to help students enhance their ability to read and critique neuroscience research , develop an integrative understanding of neuroscience core areas , examine the intersections that connect neuroscience with other disciplinary areas in sciences , arts , and humanities , and explore the relevance of neuroscience to contemporary issues and personal applications .
we present three of the activities below . the dissemination of neuroscience research to scientists , educators , professionals , and
the general public has increased through the availability of internet sites . in particular , the range and complexity of neuroscience research introduces unique challenges for the presentation of informative and useful research summaries to an informed lay audience .
thus , the goal of this activity is to provide students with an opportunity to read a primary research article in a neuroscience journal and then to prepare and deliver an oral presentation about that research article to classmates in a session that is known as neuroscience in the news .
each student selects a research article from any field of neuroscience published in the past three years based upon his or her personal interests .
then they prepare a 5-minute presentation that will model the format of a news release and that will be similar in content and style to the neuroscience news reports on the webpage of the british neuroscience association ( bna ; http://www.bna.org.uk/news/ ) . in the presentation , the students objectives are to explain why they selected the particular research article ; describe the most significant theoretical issues , methods , and results ; summarize the most meaningful conclusions including the importance or relevance of the research and/or its applications ; and identify how the research contributed to an enhancement of personal knowledge and interests in neuroscience .
it is stressed to the students that their neuroscience in the news presentation should be understandable to an informed lay audience as much as to neuroscientists .
students can augment their oral presentation with a small number of powerpoint slides or video clips in order to show results , equipment , or other pertinent information .
some of the recent neuroscience in the news presentations include does sleep deprivation put you in a better mood ? , exercise training increases the size of the hippocampus and improves your memory , videogames and cognitive training in the elderly , brain - machine - brain interfaces : a new way to connect to the world , brain imaging : visualizing the developing and maturing brain , and therapeutic potential of omega-3 pufas for peripheral nerve injuries . following each presentation
many of our neuroscience students have educational and career goals involving the application of basic neuroscience research to medical , clinical , educational , or other professional settings .
therefore , in this assignment , pairs of students work together to develop a brain briefing , a written document that explains how basic neuroscience research has relevance to a general audience of policy makers in clinical , health , educational , or other fields and professions ( e.g. , sports , law , economics , and robotics ) .
the model for this assignment is the brain briefings newsletter published online by the society for neuroscience ( sfn ) ( lom , 2005 ) and more recently posted at the website , brainfacts.org ( http://www.brainfacts.org/ ) , a public information initiative of the kavli foundation , the gatsby charitable foundation , and the sfn .
students first review several brain briefings on the website in order to get a grasp of the content , format , and style of these papers .
then , each pair of students identify a particular area of application ( e.g. , medical treatments for alzheimer s disease ) that can be informed by neuroscience and conduct an online search to find four to six relevant research articles .
the students brain briefing report is not meant to be a research abstract ; rather , their goal is to follow the format of the sfn brain briefings and provide information on recent neuroscience research that has exciting and valuable applications to neurological , psychological , and medical contexts .
each briefing has a limit of 1000 words and includes references and one or two visual objects ( e.g. , research data , brain images , or other illustrations ) .
the evaluation of the brain briefing is based upon how well students identify and explain the connections between the research outcomes with the potential applications .
examples of brain briefings produced in the most recent class include an eye toward the future : sight restoration through neuroengineering and visual prosthesis ,
neuromarketing , transcranial direct current stimulation : shocking new therapeutic possibilities , and the neural truth : religion as an anesthetic .
case studies are a type of non - experimental research methodology that typically investigate one individual in depth or over time .
recently , case studies have also been used effectively in neuroscience pedagogy ( meil , 2007 ) and are frequently included in textbooks and in popular books written by neuroscientists or neurologists ( e.g. , oliver sacks ) .
this assignment is a major and culminating project for the course and the overall learning goal is to develop a deep understanding of the neurological condition that is presented in the case study through the evaluation and synthesis of contemporary neuroscience research .
students choose a case study after examining the strange brains and case studies internet resource compiled by william meil and jeremy skipper , http://lablab.hamilton.edu/lab-teaching/strange-brains-and-case-studies , or other resources noted by the instructor ( e.g. , ramachandran , 2004 ; bogousslavsky and boller , 2005 ; sacks , 2007 , 2010 ) .
the 12-page written report for this project has three parts : 1 ) case description , 2 ) literature review , and 3 ) research proposal ( cf .
meil , 2007 ) . in part one , students are asked to describe the most salient aspects of the case .
for example , who was the person and what happened to them ? how did their condition influence their life ? why did you choose this case in order to deepen your study of neuroscience ?
in part two , the literature review should include articles that help to answer questions such as what are the typical symptoms and what are the neurobiological or neuropsychological basis of the condition ? how is the condition treated and what is the treatment prognosis ? in this section
the students are asked to evaluate and synthesize the results of at least eight empirical articles and to discuss how the literature review is relevant to course topics and readings . in the final section of the paper ,
students identify at least one unanswered question about the condition described in the case ( causes , symptoms , or treatment ) and then propose an experiment to address this question .
the research proposal includes rationale , hypotheses , participants , materials and apparatus , procedures , and statistical analyses .
finally , students are also asked to describe the potential significance of the proposed research for the field of neuroscience .
the final three class meetings of the semester are devoted to oral presentations of the case studies .
each presentation is 12 minutes in length and can include powerpoint slides . in the presentation , students describe the very most salient aspects of the case in terms of the underlying neurological issue .
they also explain their decision to choose the case and how the case is important for the study of neuroscience .
students briefly present the key research goals , methods , results , and conclusions from only two of the research articles selected from the literature review .
the students also explain why or how these two articles have relevance to an understanding of the case study .
finally , each student describes how the case study project has enhanced their study of neuroscience and the neuroscience topics that have been examined in the course .
the topics of the case studies chosen by students have included creutzfeld - jakob disease , capgras syndrome , post - traumatic stress disorder , autism , tourette s syndrome , amnesia , epilepsy , dissociative identity disorder , charles bonnet syndrome , and auditory and visual hallucinations .
the dissemination of neuroscience research to scientists , educators , professionals , and the general public has increased through the availability of internet sites . in particular
, the range and complexity of neuroscience research introduces unique challenges for the presentation of informative and useful research summaries to an informed lay audience .
thus , the goal of this activity is to provide students with an opportunity to read a primary research article in a neuroscience journal and then to prepare and deliver an oral presentation about that research article to classmates in a session that is known as neuroscience in the news .
each student selects a research article from any field of neuroscience published in the past three years based upon his or her personal interests .
then they prepare a 5-minute presentation that will model the format of a news release and that will be similar in content and style to the neuroscience news reports on the webpage of the british neuroscience association ( bna ; http://www.bna.org.uk/news/ ) . in the presentation , the students objectives are to explain why they selected the particular research article ; describe the most significant theoretical issues , methods , and results ; summarize the most meaningful conclusions including the importance or relevance of the research and/or its applications ; and identify how the research contributed to an enhancement of personal knowledge and interests in neuroscience .
it is stressed to the students that their neuroscience in the news presentation should be understandable to an informed lay audience as much as to neuroscientists .
students can augment their oral presentation with a small number of powerpoint slides or video clips in order to show results , equipment , or other pertinent information .
some of the recent neuroscience in the news presentations include does sleep deprivation put you in a better mood ? , exercise training increases the size of the hippocampus and improves your memory , videogames and cognitive training in the elderly , brain - machine - brain interfaces : a new way to connect to the world , brain imaging : visualizing the developing and maturing brain , and therapeutic potential of omega-3 pufas for peripheral nerve injuries .
following each presentation , there is a short period of questions and discussion among the students .
many of our neuroscience students have educational and career goals involving the application of basic neuroscience research to medical , clinical , educational , or other professional settings . therefore , in this assignment , pairs of students work together to develop a brain briefing , a written document that explains how basic neuroscience research has relevance to a general audience of policy makers in clinical , health , educational , or other fields and professions ( e.g. , sports , law , economics , and robotics ) .
the model for this assignment is the brain briefings newsletter published online by the society for neuroscience ( sfn ) ( lom , 2005 ) and more recently posted at the website , brainfacts.org ( http://www.brainfacts.org/ ) , a public information initiative of the kavli foundation , the gatsby charitable foundation , and the sfn .
students first review several brain briefings on the website in order to get a grasp of the content , format , and style of these papers .
then , each pair of students identify a particular area of application ( e.g. , medical treatments for alzheimer s disease ) that can be informed by neuroscience and conduct an online search to find four to six relevant research articles .
the students brain briefing report is not meant to be a research abstract ; rather , their goal is to follow the format of the sfn brain briefings and provide information on recent neuroscience research that has exciting and valuable applications to neurological , psychological , and medical contexts .
each briefing has a limit of 1000 words and includes references and one or two visual objects ( e.g. , research data , brain images , or other illustrations ) .
the evaluation of the brain briefing is based upon how well students identify and explain the connections between the research outcomes with the potential applications .
examples of brain briefings produced in the most recent class include an eye toward the future : sight restoration through neuroengineering and visual prosthesis ,
alzheimer s dementia , sleep and your emotions , neuromarketing , transcranial direct current stimulation : shocking new therapeutic possibilities , and the neural truth : religion as an anesthetic .
case studies are a type of non - experimental research methodology that typically investigate one individual in depth or over time .
recently , case studies have also been used effectively in neuroscience pedagogy ( meil , 2007 ) and are frequently included in textbooks and in popular books written by neuroscientists or neurologists ( e.g. , oliver sacks ) .
this assignment is a major and culminating project for the course and the overall learning goal is to develop a deep understanding of the neurological condition that is presented in the case study through the evaluation and synthesis of contemporary neuroscience research .
students choose a case study after examining the strange brains and case studies internet resource compiled by william meil and jeremy skipper , http://lablab.hamilton.edu/lab-teaching/strange-brains-and-case-studies , or other resources noted by the instructor ( e.g. , ramachandran , 2004 ; bogousslavsky and boller , 2005 ; sacks , 2007 , 2010 ) .
the 12-page written report for this project has three parts : 1 ) case description , 2 ) literature review , and 3 ) research proposal ( cf .
meil , 2007 ) . in part one , students are asked to describe the most salient aspects of the case .
for example , who was the person and what happened to them ? how did their condition influence their life ? why did you choose this case in order to deepen your study of neuroscience ?
in part two , the literature review should include articles that help to answer questions such as what are the typical symptoms and what are the neurobiological or neuropsychological basis of the condition ? how is the condition treated and what is the treatment prognosis ? in this section
the students are asked to evaluate and synthesize the results of at least eight empirical articles and to discuss how the literature review is relevant to course topics and readings . in the final section of the paper ,
students identify at least one unanswered question about the condition described in the case ( causes , symptoms , or treatment ) and then propose an experiment to address this question .
the research proposal includes rationale , hypotheses , participants , materials and apparatus , procedures , and statistical analyses .
finally , students are also asked to describe the potential significance of the proposed research for the field of neuroscience .
the final three class meetings of the semester are devoted to oral presentations of the case studies .
each presentation is 12 minutes in length and can include powerpoint slides . in the presentation , students describe the very most salient aspects of the case in terms of the underlying neurological issue .
they also explain their decision to choose the case and how the case is important for the study of neuroscience .
students briefly present the key research goals , methods , results , and conclusions from only two of the research articles selected from the literature review .
the students also explain why or how these two articles have relevance to an understanding of the case study .
finally , each student describes how the case study project has enhanced their study of neuroscience and the neuroscience topics that have been examined in the course .
the topics of the case studies chosen by students have included creutzfeld - jakob disease , capgras syndrome , post - traumatic stress disorder , autism , tourette s syndrome , amnesia , epilepsy , dissociative identity disorder , charles bonnet syndrome , and auditory and visual hallucinations .
students are strongly encouraged to provide written feedback and rating scale responses for the course as part of the end - of - semester course evaluation program in place at denison .
recent efforts on our campus have resulted in increased student participation in the course evaluation process by carving out class time during the last week of classes for this purpose . in neur 400 , participation rates for course evaluations are high , typically at 93% or better .
students have evaluated the course quite favorably . in the past few offerings , the majority of students numeric ratings consisted of very good to
excellent evaluations on items such as the course is challenging , their interest in neuroscience increased , their knowledge of the subject increased , and that faculty were clear , well - prepared , provided useful feedback , and were effective in their teaching .
for example , one student commented that if i could i would take ( the class ) over again there has not been a single week that i did not learn something completely new .
the depth of the material was something that i had not been exposed to previously ; this gave me an extreme increase in knowledge of the subject matter .
we were particularly interested in student feedback regarding the team taught nature of the course , as this is something that the majority of undergraduate students have little or no experience with .
generally , this was viewed as a positive aspect of the course . as one student noted ,
i like the variety of instructors and the variety of topics covered ; while another wrote the use of professors from different backgrounds presenting their primary focus of work and research to us was excellent , while still another student commented i really liked the idea of having different faculty come in to teach on a subject that was an area of expertise for them .
we had a wonderful group of faculty who were really passionate about what they were teaching . finally , one student stated emphatically , the best part about this course has been the exposure to different professors through the rotation schedule .
some students , however , found that having multiple instructors for the course was confusing and challenging , as noted by the following student comment , sometimes the switching of instructors can feel a little sporadic .
the class felt scattered because we jumped from one topic to another so quickly .
denison s required course evaluation form only contains a short list of course and instructor questions . in the future
, we plan on administering an additional evaluation form in order to obtain information from specific open - ended questions about course content , organization , learning activities , and team - teaching .
an integrative capstone course in neuroscience can provide a valuable culminating experience for students of the discipline ( wiertelak and ramirez , 2008 ) . we feel satisfied that our course challenges students to think about some of the larger contemporary issues and questions in neuroscience , to read and digest primary literature in the field , and to engage in multiple learning opportunities that enable integration of concepts and ideas acquired in previous courses .
however , we do recognize that there are important challenges that our neuroscience faculty continue to discuss regarding ways to improve the course as our neuroscience program evolves .
first and foremost , as a highly interdisciplinary field , neuroscience requires collaboration from individuals across departments .
we feel that a successful team - taught capstone course in neuroscience , therefore , depends on faculty who are committed to an interdisciplinary neuroscience program , and who are willing to commit several hours out of their already busy schedules to the preparation that is necessary in order to meet with the class over two or three sessions with no monetary compensation or teaching credit provided .
we have been quite fortunate over the years to have colleagues from across disciples eager to engage with our students in the capstone course .
most recently , eight faculty participated and represented the departments of biology , chemistry , computer science , philosophy , and psychology .
in addition , guest lecturers from denison s library and ohio state university also presented research . of course , when faculty are on leave or unable to participate for other reasons , the course content must be changed or modified in order to accommodate this change , or other faculty representing the same or some different area must be asked to participate in the course . also , it is our hope to have more faculty from other disciplines including the social sciences , humanities , and the arts participate in neur 400 in future semesters in order to highlight the importance of neuroscience in the interdisciplinary focus of a liberal arts college ( ramirez , 2007 ) .
in addition to the importance of a core group of interested and willing faculty , the instructor of record also has a critical role in the success of such a course .
s / he presents three to four lectures on neuroscience research and throughout the semester must keep the class on track , provide linkages and continuity between topics and speakers and bridge the topics coherently , particularly when faculty speakers change .
the instructor also is responsible for developing all of the learning activities and assignments described in this paper .
however , our neuroscience faculty have contributed ideas and support for these assignments which share a focus on integration and application of neuroscience research .
these learning objectives address the goals of a capstone course within the structure of our neuroscience curriculum but we recognize that there are other learning goals and assignments that could be present in a capstone course .
the course instructor also is solely responsible for grading all of the assignments , exams , and student participation .
therefore , the instructor does have a challenge in ensuring that students will develop their integrative knowledge of neuroscience from the ongoing flow of topics , presenters , and learning activities that occur during the semester .
student comments regarding course flow reinforce the importance of this role , and we continue to work on this in our discussions of our curriculum , particularly when the instructor of record changes between departments .
another important consideration in planning for a neuroscience capstone course is that enrolled students should be near the end of their college career , preferably seniors or second - semester juniors .
this is essential for the types of class discussions and thoughtful conversations that add to the success of such a course .
the course as we design it assumes that students have mastered the basics of neuroscience and have completed adequate coursework that enables them to think critically , ask probing questions and offer meaningful comments in the classroom . for this to be the case
, neuroscience curricula must be carefully structured around common core courses as well as prerequisite courses necessary for subsequent electives . finally , the success of an interdisciplinary and team - taught course depends , in part , upon multiple levels of support .
there must be support from faculty across disciplines as we have already discussed , but also support from multiple departments and the administration .
because the instructor of record earns one teaching credit for designing and coordinating neur 400 , this may very well put some strain on the home department of the instructor in terms of course offerings , enrollments , etc .
having one of his / her courses a neuroscience course means that one fewer course in the home department ( psychology , biology , for example ) can be taught during that semester .
clearly , then , it is imperative that there be departmental support for the program , transparency in what the commitment to neur courses will be , how frequently they will be taught ( every year ? every other year ? ) , and how many members of the department will be contributing regularly to the neuroscience courses and other activities associated with the concentration . on our campus , we had several departmental ( primarily within the psychology and biology departments ) discussions as the neuroscience program was in its early stages , and we continue to discuss particular aspects of the neur concentration and courses in departmental staff meetings as necessary ; in addition , neuroscience faculty meet regularly to plan future neur course offerings , discuss staffing issues , enrollments , and consider other important agenda items relevant to our neuroscience program .
because our concentration and the capstone course require the involvement of multiple faculty representing multiple departments , it also is essential to have the support of a campus administration that encourages and rewards interdisciplinary efforts from faculty ( flint and dorr , 2010 ) .
careful planning and communication with colleagues both within and across departments --- are key when creating an integrative and team - taught course for students of neuroscience . | capstone courses are becoming increasingly visible on college and university campuses . in this paper
, we describe a capstone experience for undergraduate students pursuing our neuroscience concentration .
the course is intended to provide an in - depth and interdisciplinary examination of contemporary topics in the field of neuroscience , and is designed for students who have completed the majority of requirements for the concentration .
we describe the evolution of such a course , the goals and objectives of the course , and offer a workable model for similar courses in the context of a liberal arts institution .
we summarize the positive aspects of such a course , describe the challenges involved in creating a course of this nature , and offer suggestions for successful similar capstone courses in neuroscience . | OVERVIEW OF THE NEUROSCIENCE CONCENTRATION
THE CAPSTONE COURSE: ADVANCED NEUROSCIENCE
LEARNING ACTIVITIES AND ASSIGNMENTS
Neuroscience in the News
Brain Briefings
Neuroscience Case Studies
STUDENT EVALUATIONS OF THE COURSE
REFLECTIONS ON THE CAPSTONE: STRENGTHS, CHALLENGES AND SUGGESTIONS |
cobalamins ( figure 1 ) consist of a six - coordinate low - spin co(iii ) ion
that is ligated equatorially by the four nitrogens of a tetrapyrrole
macrocycle , known as the corrin ring .
the
lower axial position is occupied by a nitrogen from
the 5,6-dimethylbenzimidazole ( dmb ) base that is part of an intramolecular
nucleotide loop bound to the corrin ring at c17 . at low
ph ,
the coordinating nitrogen of the dmb group becomes protonated ,
which converts the cobalamins from their base - on state
to their base - off form in which a solvent - derived
water molecule now serves as the lower ligand .
an excellent model of these base - off species at neutral ph is provided
by cobinamides , which are naturally occurring cobalamin precursors
that lack the terminal dmb group .
chemical structure
of nitrosylcobalamin ( nocbl ) , along with the numbering scheme used
for the atoms in the corrin ring . colored in red is the pendant 5,6-dimethylbenzimidazole
( dmb ) group , which is absent in nitrosylcobinamide ( nocbi ) .
the biologically active forms
of cobalamin differ with respect to the identity of the variable upper
axial ligand , with the best characterized forms being methylcobalamin
( mecbl ) and adenosylcobalamin ( adocbl ) .
these molecules feature an organometallic bond between the co(iii )
ion and either a methyl group or an atp - derived 5-deoxyadenosyl
group . in humans
, mecbl serves as the cofactor of methionine synthase
( meth ) , involved in the synthesis of methionine from homocysteine , while adocbl is required by methylmalonyl - coa
mutase ( mmcm ) for the isomerization of methylmalonyl - coa to succinyl - coa .
experimental studies have revealed that both
meth and mmcm are inhibited by nitric oxide ( no ) . while
these enzymes are chemically unreactive toward no in their resting
states , the co(ii)cbl intermediates formed
during catalysis ( in the case of mmcm ) or cofactor reactivation ( meth )
are believed to be susceptible to reactions with this neutral radical
species . in support of this assumption , in vitro studies
have indicated that no reacts with co(ii)cbl on a microsecond time
scale to yield nitrosylcobalamin ( nocbl ) , while in vivo studies in animals revealed that
hydroxycobalamin supplementation can inhibit the physiological response
to no , due to efficient no scavenging
by the resulting co(ii)cbl formed in the cell . because nocbl does
not support the catalytic activities of meth and mmcm , high cellular
levels of no are expected to result in a buildup of homocysteine and
methylmalonyl - coa , thereby causing disruption of the homocysteine
metabolism and , possibly , the induction of methylmalonic aciduria . despite the wealth of experimental evidence
supporting the formation of nocbl in vivo , the electronic
structure of this species remains incompletely understood , in part
because no is a redox - active ligand , thus making an oxidation state
assignment for co ambiguous . using the
enemark feltham formalism for metal nitrosyls , nocbl can be described as a { m no } type species , where the metal no fragment is treated
as a single unit and is characterized by the total number of metal
d and no * electrons . from a comparison to { m no } metalloporphyrin species , the
co no fragment of nocbl is expected to adopt a bent geometry ,
with the no ligand exerting a strong trans influence .
indeed , the
crystal structure of nocbl obtained at 100 k shows a very long co
n(dmb )
bond ( 2.322.35 ) , a short co no bond ( 1.911.94
) , and a co
n o bond angle of 120. these axial bond distances are consistent with
structures reported for co(iii)cbl species featuring a strongly -donating
upper ligand , thus supporting a co(iii)no description .
additionally , the visible
region of the electronic absorption ( abs ) spectrum of nocbl in aqueous
solution at neutral ph is dominated by a broad asymmetric feature
centered at 480 nm , which is characteristic of co(iii)cbl
species .
the dominant contributors
to this abs feature of co(iii)cbl species are the so - called /
bands that have been attributed to an electronic transition between
corrin /*-based molecular orbitals .
the energies of these bands have been shown to be sensitive
to the electron - donating properties of the axial ligands .
notably , in the case of nocbl , the peak position
of the / bands ( 480 nm ) is similar to that of
base - off alkylcobalamin species in which the lower axial dmb ligand
is replaced by a water molecule ( e.g. , max
460 nm for mecbl at ph 2 ) .
these
observations suggest that the strongly -donating no ligand could promote complete dissociation of the dmb group under
physiological conditions . however , detailed ph titration and nmr studies
by van eldik and co - workers led to the identification of a pka value of 5.1 for the protonation and consequent
dissociation of the dmb nitrogen , indicating that base - on nocbl is
favored at neutral ph . yet , this pka value is only 0.5 ph units lower than
that of the free nucleotide base ( pka of
5.56 in aqueous solution ) , suggesting that the co
additional nmr studies by hassanin et
al . led to the suggestion that at neutral ph , 33% of nocbl
is present in the base - off form , with the remaining 67% being
in the base - on form . to improve
the current understanding of the electronic structures of nocbl in
its base - on and base - off forms
, we carried out abs , circular dichroism
( cd ) , magnetic cd ( mcd ) , and resonance raman ( rr ) spectroscopic studies
of nocbl and nitrosylcobinamide ( nocbi ) .
the spectroscopic
data were analyzed within the framework of density functional theory
( dft ) and time - dependent - dft ( tddft ) calculations , employed previously
with great success in computational studies of other co(iii)cbl and
co(iii)cbi species . to identify the principal co ligand bonding
interactions
collectively , our results provide significant new insights into the
spectral and electronic properties of nocbl and nocbi .
additionally , by carrying out an analogous computational characterization
of superoxocobalamin ( o2cbl ) , intriguing electronic structure
differences between this { co o2 } species and nocbl were identified .
aquacobalamin ( h2ocbl ) , dicyanocobinamide
( ( cn)2cbi ) , sodium borohydride ( nabh4 ) , potassium
formate ( hcook ) , sodium nitrite ( nano2 ) , ascorbic acid ,
and copper tetrachloride ( cucl4 ) were purchased from sigma - aldrich
and used as obtained .
gaseous nitric oxide ( no ) was generated by the
reaction of nano2 with ascorbic acid and aqueous cu(ii )
chloride under an argon ( ar ) atmosphere .
nocbl was prepared by chemical reduction of 2 mm h2ocbl with a saturated solution of hcook under anaerobic
conditions to yield co(ii)cbl , which was subsequently exposed to freshly
prepared no gas for 2 h. to halt the reaction , the solution vials
were purged with ar .
nocbi was prepared according to the
same procedure , except that in this case 2 mm diaquocobinamide
( ( h2o)2cbi ) was used as a precursor .
( h2o)2cbi was synthesized by the
addition of nabh4 to an aqueous solution of ( cn)2cbi , as described in previous reports .
the ph of
the sample solutions was 7 unless otherwise indicated . a comparison
of the electronic abs spectra of the resulting species to published
spectra for nocbl and
nocbi confirmed that the reactions
went to completion ( see supporting information figure s1 for complete details ) .
an
electron paramagnetic resonance ( epr ) characterization of these samples
revealed that less than 3% of co(ii)cbl remained in solution after
no exposure ( see supporting information , figure
s2 ) . up to 60% ( v / v ) degassed glycerol was added under anaerobic
conditions to all samples used for low - temperature abs and mcd experiments
to ensure glass formation upon freezing .
isotopically enriched
samples for rr experiments were prepared by the methods described
above , except with the use of n - labeled nano2 ( 99% pure , cambridge isotope laboratories , inc . ) .
frozen pellets
were prepared by injecting small aliquots of fluid sample into a liquid
n2 bath under an argon atmosphere .
additional samples for
rr studies were prepared by the addition of hcl to nocbl to reach
a final ph value of < 2 .
the room - temperature abs spectra of this
low - ph nocbl species and nocbi were found to be superimposable ,
indicating that the former species was cleanly converted to its base - off
form , as expected on the basis of the pka value of 5.1 reported for the ligating dmb nitrogen of nocbl .
room - temperature cd
and low - temperature abs , cd , and mcd spectra were acquired using a
jasco j-715 spectropolarimeter in conjunction with an oxford instruments
spectromag-4000 8 t magnetocryostat .
all mcd spectra were obtained
by taking the difference between spectra collected with the magnetic
field aligned parallel and antiparallel to the light propagation axis
to remove contributions from the natural cd and glass strain .
room - temperature
abs spectra were obtained with a cary 5e uv / vis spectrophotometer .
the rr spectra were obtained upon excitation at 488.0 nm with a
coherent i-305 ar ion laser and 40 mw of laser
power at the sample .
the scattered light was collected using a 135
backscattering arrangement , dispersed by an acton research triple
monochromator ( equipped with 300 , 1200 , and 2400 grooves / mm gratings ) ,
and analyzed with a princeton instrument spec x : 100br deep depletion ,
back - thinned ccd camera .
spectra were accumulated at 77 k by placing
frozen pellets into a quartz finger dewar filled with liquid n2 .
spectra of fluid solution samples , contained in sealed epr
tubes under an argon atmosphere , were obtained with the same setup
but by filling the finger dewar with an ice / water mixture .
no spectroscopic
changes attributable to photolytic or chemical degradation were observed
during data collection .
all rr spectra were baseline corrected using
a piecewise line function to remove the broad nonresonant fluorescence
contributions , and the intensities were normalized with respect to
the features in the region between 1100 and 1400 cm .
peak positions were calibrated against the 984 cm peak of a potassium sulfate standard as well as the ice peak at
228 cm for frozen samples or the water feature
at 1637 cm for fluid solution samples .
initial coordinates for the model of nocbl were obtained
from the highest - resolution x - ray crystal structure reported by hassanin
et al . in 2010 .
these coordinates were
also used as the basis for generating an initial model of nocbi , whereby the nucleotide loop was replaced by an h atom at
the phosphate position and a water molecule was placed in the lower
axial coordination site originally occupied by the dmb base ( see figure 1 ) . coordinates for the o2cbl model
were
obtained from the structure reported by hohenester et al . and used
as is .
because of the large number of
atoms present in these species , smaller models were prepared for tddft
excited - state and dft frequency calculations by removal of atoms that
were not expected to contribute to the spectroscopic features observed
experimentally .
specifically , the corrin ring substituents were replaced
by hydrogen atoms , except for the four methyl groups at the c1 , c2 , c5 , and c15 positions
( see figure 1 for the atom numbering scheme
used ) .
additionally , the two propionamide groups at c2 and
c18 , along with the nucleotide loop at c7 in
the case of nocbl , were replaced by methyl groups .
this truncation
scheme was adopted because the methyl groups at the c5 and
c15 positions were shown to be crucial for an accurate
treatment of the vibrational modes of the corrin ring , while the others were found to play a role in
preventing excessive flattening of the corrin ring ( vide infra ) .
to further increase the likelihood of obtaining realistic corrin
fold angles , the entire dmb base was included in all nocbl models .
full geometry optimizations of the complete and truncated cofactor
models were performed with the amsterdam density functional ( adf )
2012 suite of programs using the vosko wilk
nusair ( vwn ) local - density
approximation ( lda ) and the perdew , burke ,
and ernzerhof ( pbe ) gradient corrections for exchange and correlation . in each case , the triple with polarization ( tzp ) basis set was chosen , along with an integration constant of 5.0 and a small
frozen core through 1s for c , n , and o and through 2p for co and p.
the optimized models were subjected to frequency calculations to verify
that a true energy minimum was found . in each case , only positive
frequencies were obtained . for the computation of spectroscopic
properties , the orca 2.8 software package developed by dr .
f. neese
was employed . while the optimized truncated
models were used as - is ,
the full models were modified using the truncation
scheme outlined above and subjected to partial geometry optimizations ,
whereby only the added h atoms were allowed to move .
electronic transition
energies and abs intensities were computed with the tddft method within the tamm
the ahlrichs polarized split valence ( svp ) basis set was employed for all atoms , except for cobalt ,
the ligating nitrogens , and the oxygen from no , for which the triple-
valence polarized ( tzvp ) basis was utilized . in each case , 40 excited states were computed by including all one - electron
excitations among molecular orbitals within 3 hartree of the
highest occupied molecular orbital / lowest unoccupied molecular orbital
( homo / lumo ) gap . to increase computational efficiency ,
the resolution
of the identity ( ri ) approximation in conjunction with the sv / j auxiliary basis were employed in the evaluation of the coulomb term
for all calculations .
the tddft results were used as the basis for
simulating abs and cd spectra , assuming that each electronic transition
gives rise to a gaussian - shaped band with a full width at half maximum
( fwhm ) of v1/2 = 1250 cm .
all calculated spectra were uniformly red - shifted by 2200 cm to facilitate a visual comparison with the experimental
data .
finally , off - resonance raman spectra for the fully optimized
truncated models were computed with orca 2.8 , by evaluating the numerical
frequencies and electronic polarizabilities of all normal modes using
dft and the basis sets and functionals described above .
the ground - state
wave functions of the fully optimized models were analyzed further
within the natural bond orbital ( nbo ) framework to assess the major bonding interactions , using the nbo5
interface as implemented in adf via the gennbo and adfnbo programs .
the vwn / lda and the pbe gradient corrections
for exchange and correlation , along with the tzp
basis set ( without frozen core approximation ) , and an integration constant of 5.0 were used to compute
the ground - state electron density .
isosurface plots of all orbitals and electron
density difference maps were generated with pymol using isodensity
values of 0.03 au and 0.003 au , respectively .
the low - temperature ( 4.5 k ) abs spectrum
of nocbl in frozen solution exhibits partially resolved bands in the
lower energy region with an intensity maximum near 20 000 cm , corresponding to the so - called / bands ,
along with a series of broad and more intense bands around 31 000
cm , historically referred to as the region
( figure 2 , top ) .
this spectrum is very similar
to those exhibited by alkyl - co(iii)cbl species , such as mecbl and
adocbl , which were previously classified as unique
abs spectra based primarily on the unusual appearance of the
region . as in those latter spectra ,
the
pair of features at 19 000 and 20 000 cm in the abs spectrum of nocbl also appear to correspond
to the origin and first member of a vibrational progression in a totally
symmetric breathing mode of the corrin ring . in support of these assignments ,
the corresponding features are of the same sign in both the cd and
the mcd spectra of nocbl ( figure 2 , middle
and bottom ) .
the other bands in the visible region of the nocbl abs
spectrum are due to at least one or more additional electronic transitions ,
as indicated by their opposite signs in the cd spectrum .
consistent
with its classification as a unique abs spectrum , several bands that
arise from distinct electronic transitions can be identified in the
region , including two similarly intense features centered
at 31 000 cm with a low - energy shoulder
at 28 000 cm .
abs ( top ) , cd ( center ) ,
and 7 t mcd ( bottom ) spectra at various temperatures of nocbl . upon substitution of the dmb ligand
of nocbl with a more weakly -donating water molecule in nocbi ,
a 1000 cm blue shift of the
/ bands is observed ( figures 3 , top ) .
additionally , a positively signed feature at 18 500
cm appears in the cd spectrum of nocbi that has no counterpart in the nocbl spectrum , while the prominent
negative feature at 19 000 cm decreases
in intensity and shifts to higher energy ( figure 3 , middle ) .
because the mcd spectra of nocbl and nocbi are essentially temperature - independent in the 4.5 to 50
k range ( supporting information , figures s4 and
s5 ) , it can be concluded that both species possess diamagnetic
( s = 0 ) ground states , consistent with a co(iii)/no oxidation state assignment .
previous studies of other
co(iii ) corrinoids have revealed that the spectral changes in the
/ region that occur in response to a dmb h2o lower ligand substitution reflect a stabilization of the
homo relative to the lumo , the extent of which depends on the -donating
strength of the upper axial ligand .
as
the blue shift of the / bands from nocbl to nocbi ( 1000 cm ) is considerably smaller
than the shift observed from mecbl to mecbi ( 2500
cm ) , no may appear
to be a weaker -donating ligand than a methyl group .
however ,
since the / bands of nocbl occur at higher energies
than those of mecbl and all other alkylcobalamins , their small blue shifts from nocbl to nocbi could also be due to the fact that the dmb moiety is only weakly
interacting with the co ion in the former species , as suggested by
the unusually long co
abs ( top ) , cd ( center ) ,
and 7 t mcd ( bottom ) spectra at various temperatures of nocbi , a spectroscopic model of base - off nocbl . intriguingly , while the abs and cd spectra of nocbi obtained at 4.5 and 300 k differ insignificantly with respect
to band positions ( figure 3 , top and middle ) ,
the abs spectrum of nocbl collected in fluid solution at 300 k shows
the / bands blue - shifted by 850 cm from their positions at 4.5 k ( figure 2 ,
top ) .
significant temperature - dependent changes are also observed
in the cd spectrum of nocbl , most notably a drastic decrease in the
intensity of the lowest - energy , negatively signed feature at 18 000
cm and the appearance of a weak , positively signed
feature at even lower energy ( figure 2 , middle ) .
as a result ,
the room - temperature abs and cd spectra of nocbl quite
closely resemble those of nocbi .
collectively , these results
suggest that by increasing the temperature from 4.5 to 300 k , the
base - on to base - off equilibrium for nocbl changes , favoring dissociation
of the n(dmb ) ligand at high temperatures .
however , consistent with
published nmr results , a sizable fraction
of nocbl must remain in the base - on state even under ambient conditions ,
since the 300 k abs spectra of nocbl and nocbi are not
superimposable ( see supporting information , figure
s1 ) . yet , while the nmr data were interpreted to indicate that
at room temperature 67% of the nocbl molecules are in the
base - on state , the traces obtained by adding the 4.5 k abs or cd spectra
of nocbl ( scaled by 0.67 ) and nocbi ( scaled by 0.33 ) differ
from the 300 k abs and cd spectra of nocbl .
n(dmb ) bond of base - on nocbl is longer at 300 k
than it is at 4.5 k. the 77 k rr spectrum of nocbl obtained with laser excitation at 488.0
nm ( 20,490 cm ) shows strong enhancement of four
features between 1450 and 1650 cm . on the basis
of our recent study of the vibrational properties of vitamin b12 and its reduced forms ,
all
of these features can be attributed to corrin - based vibrational modes .
three of them ( termed s1s3 ) arise from totally symmetric modes ( assuming an effective cs symmetry , with the pseudo mirror plane being
oriented along the co and c10 atoms and perpendicular to
the corrin ring plane ) and are thus particularly strongly resonance
enhanced , while the fourth ( as ) is associated with
an asymmetric stretching mode .
a more detailed analysis of these modes
within the framework of our dft frequency calculations is provided
below .
a comparison of the low - energy regions of the rr spectra
of nocbl and its nocbl isotopomer reveals an isotope - sensitive
feature at 532 cm ( figure 4 , top ) , a region where the co no stretching and co
subtraction
of the nocbl from the nocbl trace yields a difference
spectrum that shows an apparent shift of the 532 cm feature to 496 cm upon no no substitution .
an analogous isotope - sensitive feature is
present in the rr spectrum of base - off nocbl ( our model of nocbi , figure 4 , bottom traces ) , though
in this case a much better - resolved difference spectrum is obtained .
closer examination of this difference spectrum clearly discloses the
presence of a shoulder on the low - energy side of the positive feature ,
suggesting that two isotope - sensitive modes actually occur in this
region .
indeed , a gaussian deconvolution of the rr spectra in figure 4 reveals that the vibrational modes of base - on and
base - off nocbl at 515 and 532 cm shift to 500
and 521 cm , respectively , upon no
no substitution ( see supporting
information , figure s9 ) . in a previous rr study of nocbl , a
single isotope - sensitive peak was observed at 514 cm that shifted to 496 cm upon no
no substitution .
however , the spectral resolution
of these published data appears to be relatively low , as only two
high - energy ( > 1,500 cm ) features associated
with corrin - based modes could be identified , compared to the four features that are readily apparent in our spectra
( figure 5 ) . additionally , considering that
mode coupling typically leads to lower isotope shifts than expected ,
the reported isotope shift of 18 cm , which largely
exceeds the 14 cm decrease in vibrational frequency
calculated using a harmonic oscillator model for a localized co
low - energy region of
rr spectra of nocbl and its no - enriched isotopomer in
the base - on ( top ) and base - off ( bottom ) conformations , obtained at
77 k with 488 nm ( 20 491 cm ) laser excitation .
a difference spectrum for each conformation
is included below the
two data sets to highlight the isotope - sensitive vibrational features .
high - energy region of rr spectra of nocbl and its no - enriched
isotopomer in the base - on and base - off conformations , obtained at
77 k with 488 nm ( 20 491 cm ) laser excitation .
a difference spectrum for each conformation
is included below the
two data sets to highlight the isotope - sensitive vibrational features .
the high similarity between the
low - energy regions of the rr spectra of base - on and base - off nocbl
( figure 4 ) indicates that the co no
bonding interaction is largely unperturbed by the dmb h2o lower - ligand substitution .
consistent with this conclusion ,
the rr spectra of fluid solution samples of base - on and base - off nocbl
are very similar to each other as well as to the corresponding 77
k spectra ( see supporting information , figures
s7 and s8 ) , despite the thermochromism exhibited by nocbl .
dft has been used
by us with great success for the study of corrinoids in their co(iii ) ,
co(ii ) , and co(i ) oxidation states . while
different functionals have been shown to provide variable descriptions
of spectroscopic properties , careful
evaluations of the computational results on the basis of experimental
data
have afforded a detailed understanding of the chemical and spectroscopic
properties of a large number of different corrinoid species . from a recent
study of mecbl , kozlowski and co -
workers have suggested that pure
gga functionals , rather than hybrid functionals , provide dft results
more consistent with correlated wave function - based methods .
furthermore , work by our group and others has revealed that
complete cofactor models should be used to obtain accurate geometric
structures of cobalamins , while truncated models can be used to compute
various spectroscopic parameters . in light of these findings , we performed
geometry optimizations of complete nocbl and nocbi species
using the pure pbe functional .
the resulting structures were then
suitably truncated to predict the abs and cd spectra of these species
using tddft , as well as to analyze their vibrational properties ( see methods for details ) .
for comparison , we also carried
out full geometry optimizations of these truncated models and calculated
their abs and cd spectra .
the most
significant differences between the x - ray crystal structure and our
dft - optimized model of nocbl include the co
n(dmb ) bond distance
and the folding of the corrin ring . while the crystal structure of
nocbl shows an unusually long co
n(dmb ) bond of 2.35 ,
our computation predicts this bond to be elongated by an additional
0.16 , to 2.51 ( see table 1 ) .
a further elongation of this bond by 0.1 , along with a tilting
of the dmb ring plane relative to the co
n(dmb ) bond vector ,
occurred during the geometry optimization of our truncated model lacking
the propionamide side chains .
n(dmb ) bonds in our models may also stem from the neglect
of intermolecular interactions that modulate the length of this bond
in the solid state and in solution .
n(dmb ) bond of nocbl is very weak and
can thus be stretched , or potentially even broken , quite readily .
distortions
of the corrin ring can be assessed on the basis of the long - axis and
short - axis fold angles , (la ) and (sa ) , respectively .
(la ) is defined here as the angle between the plane containing
the na , c4 , c5 , c6 , and
nb atoms and the plane containing the nc , c14 , c15 , c16 , and nd atoms ,
while (sa ) corresponds to the angle between the planes containing
the nd , c19 , c1 , and na atoms and the nb , c9 , c10 , c11 , and nc atoms ( see figure 1 for the atom numbering scheme used ) . as such , (la )
correlates
with the amount of butterfly fold of the corrin ring
and is thus particularly sensitive to the positioning or removal of
the bulky dmb ligand .
alternatively , (sa ) reflects the extent
of ring ruffling due to steric constraints imposed
on the propionamide and methyl groups on the a and d rings .
as shown
in table 1 , the (la ) values derived
from the crystal structure and dft - optimized complete model of nocbl
vary quite substantially , as expected from the sizable variation in
the corresponding co
in contrast , the
(sa ) values are nearly identical , suggesting that the corrin
ring conformation observed experimentally is minimally affected by
crystal - packing effects and/or intermolecular h - bonding interactions .
the fact that dft predicts both fold angles to decrease quite substantially
upon removal of the propionamide side chains is consistent with our
previous finding that distortions along the (la ) and (sa )
coordinates require very little energy . despite these modest discrepancies between the experimentally
determined and dft - optimized nocbl structures , the metric parameters
for the co
n(o )
distances and co n o bond angles are identical to within
0.04 and < 1 , respectively .
co
bond distances reported for all other co(iii)cbl species , suggesting that this bond is exceptionally strong .
although the
computation appears to overestimate the n o bond distance by
0.05 , note that the value of 1.14 determined experimentally
is surprisingly small , given that ( i ) the n o bond distance
in nitric oxide is 1.15 and ( ii )
the no ligand in nocbl acquires a partial negative charge and thus
increased -antibonding electron density ( vide infra ) . owing to the overall good agreement between the x - ray crystal
structure and dft - optimized model of nocbl , a closer examination of
the energy - minimized nocbi model is warranted .
as expected ,
replacing the bulky dmb base with a water molecule in the lower axial
position causes sizable changes to both of the corrin fold angles
( table 1 ) .
o(h2 ) distance in our complete nocbi model is 3.58 , suggesting that the co ion resides in an effective
five - coordinate ligand environment .
although this distance shortens
by 0.60 upon removal of the propionamide side chains , the
lone
pairs on oxygen in our truncated model are not actually oriented
properly to engage in a bonding interaction with the co ion .
a closer
inspection of the complete nocbi model reveals that several
of the propionamide side chains form a cage around the h2o molecule .
the fact that removing these side chains causes a large
rearrangement of the water molecule suggests a role of the propionamide
groups in modulating the ligation of the lower ligand by imposing
steric constraints and/or providing a solvent - protected pocket for
the ligand . finally ,
as expected in light of the strong co no bonding interaction
in nocbl , the co no unit is largely unaffected by the dmb
h2o lower - ligand substitution ( table 2 , see nbo analysis for more details ) , with the most notable change
being a small ( 0.02 ) decrease in the co
n(dmb ) bonds in nocbl
and mecbl , the base does play a small role in modulating the upper
axial bonding interactions in these species . the computed abs spectrum for the nocbl model derived
from the complete structure
correctly predicts the major features
observed experimentally , including the presence of two clusters of
intense features near 20 000 cm and 31 000
cm , corresponding to the / region
and the region , respectively ( figure 6 , top ) .
the computed cd spectrum is also in reasonable agreement
with our experimental spectrum , both exhibiting a derivative - shaped
feature associated with two oppositely signed transitions centered
at 20 000 cm and a series of features with
alternating signs in the region ( supporting
information , figure s10 , top ) .
while the computed abs and cd
spectra for the optimized truncated nocbl model are similar to those
obtained with the modified complete model , the / bands
are blue - shifted by 900 cm , and the lowest - energy ,
negatively signed feature in the cd spectrum is considerably weaker ,
which agrees less well with our experimental data ( supporting information , figure s11 ) .
abs spectra of nocbl
( top ) and nocbi ( bottom ) collected at 4.5 k ( dotted lines )
superimposed on the td - dft results .
the td - dft computed transitions
( vertical sticks ) were convoluted with gaussian bands with a constant
fwhm of 1250 cm to obtain the predicted spectra
plotted in dark blue for nocbl and in light blue for nocbi . in each case , the eddm for the -band transition is shown
on the left , where regions of loss and gain of electron density are
shown in gray and gold , respectively .
the calculated spectra were
uniformly red - shifted by 2200 cm to facilitate
a direct comparison with the experimental results .
upon dmb h2o lower - ligand substitution ,
the / bands in the tddft - computed abs and cd spectra
blue shift by 3500 cm , and the negative
cd feature at 20 000 cm decreases in intensity ,
causing an apparent blue shift of the derivative - shaped feature to
21 000 cm ( figure 6 , bottom , supporting information , figure s10 , bottom ) . as expected on the basis of the negligible co
o(h2 ) bonding interactions in the two different nocbi models , the abs and cd spectra predicted for these species are almost
identical ( supporting information , figure s12 ) .
overall , the computed abs spectra for both nocbi models
agree quite well with the experimental spectrum across the entire
spectral range investigated , except for the presence of a small feature
at 15 000 cm that is not observed
experimentally . in contrast , the computed cd spectra only modestly
reproduce the experimental spectrum , which is not unexpected because
magnetic - dipole allowed transitions pose a significant challenge for
tddft computations .
while considerable differences
are observed between the experimental abs , cd , and mcd spectra of
nocbl and nocbi ( cf . figures 2 and 3 ) , the rr spectra obtained for these species show
minimal differences with respect to the frequencies of the major corrin
and co no - based modes ( vide supra ) . for this
reason , and for computational practicality ,
the truncated nocbl and
nocbi models were employed in our dft - assisted vibrational
analysis .
the dft - computed off - resonance raman spectra for these models
exhibit four relatively intense features in the 14501650 cm region ( supporting information ,
figure s13 and s14 ) .
a graphical representation of the corresponding
normal modes of nocbi ( which are very similar to those
of nocbl ) in terms of atomic displacement vectors is provided in figure 7 .
the three modes that retain the approximate cs symmetry of nocbi are expected
to be most strongly resonance - enhanced , because only totally symmetric
modes can couple to electronic transitions and thus gain rr intensity
via the franck
even though
dft methods typically overestimate vibrational frequencies , the agreement
between the experimental and computed frequencies for these modes
is excellent , thus permitting a straightforward assignment of the
relevant vibrational features . specifically , the 1497 cm feature observed experimentally is assigned to the formally totally
symmetric s1 mode predicted at 1506 cm , which involves nuclear motion primarily along the short axis of
the corrin ring .
alternatively , the 1541 and 1603 cm features in the experimental rr spectra are attributed to the s2 and s3 modes predicted at 1554 and 1613
cm , respectively .
n
stretching motion along the long axis of the corrin ring and symmetric
methine stretching motion , respectively .
the remaining feature observed
at 1572 cm is then assigned to the antisymmetric
as mode predicted at 1585 cm . while the large depolarization ratio ( 0.71 ) computed for as suggests that this mode should not be resonance - enhanced ,
it is predicted to carry significant off - resonance raman intensity
and to entail large displacements of corrin ring atoms , thus facilitating
intensity enhancement via herzberg teller coupling .
overall , these assignments are consistent with our vibrational analysis
recently carried out for cncbl and its reduced derivatives .
eigenvector representations of the relevant normal modes
for the truncated nocbi model , as obtained with dft .
the
computed frequency ( ) , isotope shift upon no no substitution ( n15 ) , depolarization
ratio ( ) , off - resonance raman intensity ( i ) , and assignment are shown for each mode . our calculations for nocbi also predict two modes
at 569 and 585 cm with no no isotope shifts of n15 = 7.3
and 6.0 cm , respectively .
these predictions
agree reasonably well with the frequencies ( 515 and 532 cm ) and isotope shifts ( n15 = 15 and
11 cm ) observed experimentally . both of
these modes involve large atomic displacements along the co
bending coordinates and are coupled
out of phase and in phase , respectively , with corrin ring breathing
modes . as such
only one additional no
no isotope - sensitive mode is computed at 1688
cm , corresponding to a relatively pure n
while this mode is predicted to be strongly ir active , it
carries negligible intensity in the computed off - resonance raman spectrum
( supporting information , figures s13 and s14 , respectively ) .
this observation , as well as the lack of mechanical
coupling between the n o and corrin ring stretches and the
weak electronic coupling between the no moiety and corrin
system ( see below ) predicted computationally are consistent with the
absence of a feature attributable to the n o stretch in our
experimental rr spectra of nocbl and nocbi .
the good agreement
between the experimental and computed abs , cd , and vibrational data
presented above indicates that dft successfully models the salient
bonding interactions present in nocbl and nocbi . for both
species ,
the relative energies of the co 3d - based mos reflect the
strongly - and weakly -donating nature of the tetradentate
corrin ligand . among these orbitals ( see figure 8) , the co 3dx y - based mo ( # 158 in the case of nocbl )
is essentially nonbonding and thus lowest in energy , whereas the co
3dxy - based mo ( distributed over mos # 167
and # 168 due to mixing with another , energetically proximate mo ) is
strongly -antibonding with respect to the co
n(corrin )
bonds and therefore highest in energy ( note that the x and y axes are rotated by 45 about the z axis from their usual orientations due to the co
intriguingly , the homo and lumo of
nocbl derive mostly from co 3d and no *orbitals , in contrast
to the case of alkylcobalamins ( e.g. , mecbl ) where these mos are primarily
corrin /*-based .
specifically ,
the homo of nocbl contains 28% co 3dz and 40% no * orbital contributions
( to differentiate between the two no * orbitals , the one with
its lobes roughly parallel to the co
n(o ) bond axis will be
denoted as * and the other as * ) , making it strongly -bonding with respect
to the co
n(o ) bond ( see figure 8 for
mo plots and supporting information , table s1 for compositions ) . alternatively
, the lumo corresponds to the -antibonding
combination of the co 3dyz and the no
* orbitals ( 15% and 78% , respectively ) ,
with the bonding counterpart being considerably lower in energy ( mo
# 162 ) .
the dashed line separates
occupied from empty orbitals . upon replacement of the axial dmb ligand of nocbl with a
more weakly -donating water molecule in nocbi ,
mos with large co 3dz orbital
contributions ( mos # 163 , # 164 , # 167 , and # 168 of nocbl , corresponding
to mos # 128 , # 130 , # 133 , and # 134 of nocbi , see figure 9 ) are stabilized by 0.1 ev relative to the
co 3dx y - based mo , which was chosen as the reference
point because its composition remains essentially unchanged from nocbl
to nocbi ( see supporting information ,
tables s1 and s2 ) . in particular , the co 3dz
/ no *-based homo of nocbi ( # 130 ) loses the -antibonding interaction with the
lower axial ligand and gains contributions from the corrin 7 mo .
this mixing causes a large stabilization of the corrin
7-based mo # 128 relative to the corrin 8-based mo # 132 of nocbi ( corresponding to mos
# 163 and # 166 of nocbl ) .
given the satisfactory agreement between
the experimental and computed spectroscopic data of nocbl and nocbi , it is reasonable to assign the key abs features of these
species within the framework of the dft - based mo descriptions as provided
by the tddft results .
because in the tddft formalism electronic transitions
are described as linear combinations of one - electron excitations between
occupied and virtual mos , it becomes difficult to identify the nature
of a given transition in cases where multiple excitations make significant
contributions .
one approach to overcome this limitation involves computing
electron difference density maps ( eddms ) , which provide a visual representation
of the changes in electron density accompanying an electronic transition .
on the basis of such eddms ( figure 6 )
, the
electronic transitions producing the dominant contributions to the
tddft - computed abs spectra can be assigned as shown in table 3 for nocbl and table 4 for
nocbi . in the case of nocbl ,
the five lowest - energy transitions
carrying significant abs intensity give rise to one broad feature
in region i of the computed abs spectrum ( figure 6 , top ) , with two transitions ( involving excited
states 12 and 15 ) having the largest oscillator strengths .
on the
basis of its eddm , transition 12 primarily entails a one - electron
excitation from the corrin 7-based mo # 163 to the
corrin 8-based mo # 166 , characteristic of the transition
that is generally responsible for the / bands .
the largest
changes in electron density occur within the c9c10c11 fragment of the corrin ring , consistent
with the strong rr enhancement of the s1 mode observed
experimentally ( figure 5 ) .
the eddms for the dominant transitions in regions ii ( excited states 21 , 23 , and 26 ) and iii ( excited state 37 ) display large changes in electron density around
the dmb and no ligands , due to one - electron excitations from mos with
significant orbital contributions from the dmb group .
lastly , regions iv and v are dominated by several intense
corrin * transitions that also cause a moderate
electron density redistribution at the co center as a result of the
sizable co 3d orbital character in the donor and acceptor mos . the
prediction of numerous intense features in the region of the
computed abs spectrum , as opposed to a single , relatively sharp band
as is usually observed for co(iii)cbl species , is consistent with the unique abs spectrum obtained experimentally
for nocbl . from nocbl to nocbi , td - dft predicts
the most intense feature in the visible region of the abs spectrum
to blue shift by 3500 cm , in qualitative agreement
with the 1000 cm shift of the /
bands observed experimentally ( figure 6 ) .
however ,
additional features are present in the near - ir region of the computed
abs spectrum that have no discernible counterparts in the experimental
spectrum . as revealed by their eddms ( supporting
information , figure s15 ) , the transitions associated with the
first two features in regions 0 and ia cause a large
electron redistribution within the co no moiety ( see table 4 ) .
the fact that analogous features are not observed
experimentally indicates that tddft incorrectly predicts the intensities
and/or energies of these charge transfer ( ct)-type transitions of
nocbi ; similar cases where tddft fails to properly describe
ct excited states are well - documented in the literature .
the remaining changes in the computed abs spectra from nocbl to
nocbi pertain to a significant intensity redistribution
among the dominant features in the region , which is again
in good qualitative agreement with our experimental results .
because our tddft computations reproduce the key differences between
the abs spectra of nocbl and nocbi observed experimentally
quite well , the computed mo diagrams can be used as the basis for
exploring the electronic structural origin of these differences .
as
described above , the dmb h2o ligand substitution
causes a stabilization of mos with significant co 3dz orbital contributions relative to the other mos .
of particular importance with respect to the / band
transition
is the stabilization of the corrin 7-based
donor mo relative to the corrin 8-based acceptor
mo from nocbl ( mos # 163 and # 166 ) to nocbi ( mos # 128 and
# 132 ) , which readily explains the blue shift of the /
bands observed experimentally ( figures 2 , 3 , and 6 ) .
interestingly ,
in the case of alkylcobalamins that also possess a strong -donor
ligand in the upper axial position , the blue shift of the /
bands in response to dmb h2o ligand substitution
is considerably larger than it is in the case of nocbl ; for example ,
2500 cm for mecbl versus 1000
cm for nocbl .
this difference can be understood
in terms of the larger -donation from the upper ligand in nocbl
than in alkylcobalamins , which leads to a weaker co
n(dmb )
bond and , thus , to less pronounced changes in the electronic structure
upon lower - ligand substitution in the former species .
in fact , our
computed mo description for nocbl bears some intriguing similarities
to that developed previously for co(ii)cbl , especially with regard
to the energies of the co 3d - based mos relative to the corrin /*-based
mos .
this observation indicates that
the high degree of -donation from the occupied no * orbital into the formally unoccupied co 3dz orbital leads to a significant decrease
in the effective nuclear charge experienced by the co 3d orbitals
in nocbl .
however , the no co charge donation is partially
compensated for by the strong -backbonding interaction involving
the doubly occupied co 3dyz orbital and
formally empty no * orbital . a more detailed
analysis of the salient co no bonding interactions within the
nbo formalism is presented next . while a relatively straightforward
correlation can be established between the dft / tddft - predicted and
experimentally observed properties of nocbl and nocbi ,
a direct evaluation of the key co no bonding interactions is
complicated by the partial delocalization of the relevant mos over
the corrin ring . to overcome this challenge , we resorted to the nbo
formalism within which the calculated electron density is partitioned
into chemically intuitive bonding mos and electron pairs so as to
generate a bonding description closely adhering to the lewis structure
formalism .
n(o )
bonding interactions in nocbl and nocbi are essentially
identical as judged on the basis of our experimental rr data and dft
results ( vide supra ) , the following analysis will
focus on the electronic structure of nocbi .
the relevant
nbos , labeled by their primary orbital contributors and classified
either as bonding ( bd ) or antibonding ( bd * ) with respect to the co no
bond , are shown in figure 10 ( left ) . as expected
on the basis of the dft results described above , the co 3dz bd - nbo is characterized by a highly
covalent co no -bonding interaction .
however , shortcomings
of the lewis structure - like description for nocbi are
evident from the relatively large occupancies of several formally
empty orbitals , most importantly the antibonding co 3dxy bd*-nbo and co 4s bd*-nbo , as well as the occupancies
of considerably less than 2 of some of the formally doubly occupied
orbitals .
these observations indicate that deviations from a single
lewis structure , treated within the nbo framework as donor
acceptor
hyperconjugation interactions , are important
for properly describing the bonding interactions in nocbi .
these interactions can be fully accounted for by a transformation
of the canonical mos to a set of natural localized mos ( nlmos ) , which
are constructed to retain a large amount of nbo character .
these nlmos
possess integer occupancies , like the canonical mos , and thus provide
a useful connection between the canonical mos and the nbos .
isosurface plots of the metal - based nbos
( in blue / gray ) and corresponding nlmos ( in orange / gray ) of nocbi .
the composition
of the nbos and their percent contributions to the nlmos are provided
in table 4 the relevant nlmos for nocbi are shown in figure 10 ( right ) , and their compositions are provided in
table 5 .
inspection of the co 3dz bd - nlmo discloses a very covalent -bonding
interaction between the co ion and no ( 58% co 3dz and 42% n(o ) 2pz orbital
contributions , see table 5 ) , as anticipated
on the basis of the canonical mo and nbo compositions .
intriguingly ,
while the co 3dz bd - nlmo as
well as the nonbonding co 3dx y and 3dxz nlmos are very similar to their corresponding nbos ,
the 3dyz bd - nlmo shows distinct differences .
the composition of this nlmo reveals that it only retains 83.1% of
pure co 3dyz nbo character
( in comparison to > 95% for the other co - based nlmos ) due to a sizable
-backbonding interaction between the co 3dyz orbital and the no * orbital ( figure 10 , center columns ) .
since the co 3dyz orbital only mixes with the no * orbital ( figure 10 ) , the amount of charge
backdonation from co to no can be estimated to be 0.32 electrons .
because of the presence of this -backbonding interaction , the
co ion does not retain a co(ii)-like electronic structure as might
be anticipated on the basis of the large donation from the
no * orbital to the co 3dz orbital .
lastly , the unoccupied co 3dz bd*-nlmo and co 3dxy bd*-nlmo are -antibonding with respect to the corrin
ring and the axial ligand orbitals , as expected , and retain > 95%
of nbo character ( see table 5 and figure 10 , outside columns ) .
na indicates values
not availalable from nlmo composition analysis , but estimated to be
close to 100% .
the nbo / nlmo
results can also be used to estimate the number of electrons in the
co 3d atomic orbitals ( aos ) and thus an effective oxidation state
of the co ion . as the number of d electrons is sensitive to the covalency
of the metal
ligand bonding interactions as well as the delocalization
of the 3d orbitals , the following approach was used to estimate this
value ( see supporting information for additional
details ) .
first , the % nbo composition of each nlmo ( see table 5 ) was used to calculate the fractional occupancy
of the co 3d - based nbo(s ) contributing to this particular nlmo .
this
electron count was then partitioned into co 3d - based and ligand - based
contributions , estimated based on the % co 3d contributions to each
nbo .
finally , the fractional co 3d electron counts for all nlmos obtained
using this procedure were added up to estimate the total number of
co 3d electrons . using this approach ,
an effective number of co 3d
electrons of 6.27 was obtained for nocbi , larger than
a value of 6 expected for a co(iii ) species .
this result corroborates
the conclusion drawn from the energies and compositions of the canonical
mos regarding the relatively high co(ii ) character in the electronic
structures of nocbl and nocbi .
thus , while the large amount
of electron donation from no to co in the co 3dz bd - nlmo is partially offset by the strong -backbonding
interaction in the co 3dyz bd - nlmo , our
nbo / nlmo analysis clearly shows that a co(iii)no description for nocbi , and by analogy nocbl , is inaccurate .
rather , these species are better described as resonance hybrids with
both co(iii)no and co(ii)no limiting structures being major contributors .
given the roles of no as a blood - pressure regulator ,
neurotransmitter , and second messenger , as well as a cytotoxic agent in immunological response , the ability of co(ii)cbl to scavenge this molecule in vivo to form nocbl is of considerable interest .
additionally , nocbl has been shown to be potentially useful for
targeted no delivery as a chemotherapeutic or vasodilating agent , since its co no bond can be broken under physiological conditions
for controlled no release .
thus , elucidating the nature of the bonding
interaction between the no ligand and the co center in nocbl represents
an important step toward the development of an improved understanding
of the chemical and biological properties of this molecule .
previous
reports have highlighted the unique structural properties of nocbl
relative to other biologically relevant cobalamins , most notably the
presence of an unprecedentedly long co
however , because nocbl exhibits similar spectroscopic signatures
as the well - characterized alkyl - co(iii)cbls , such as mecbl and adocbl , it has generally been described as a co(iii)no species . to test this assumption , we developed
experimentally validated electronic structure descriptions for nocbl
in its base - on and base - off states .
while our abs spectrum of nocbl
in fluid solution at 300 k is similar to those reported in the literature , we discovered a previously unobserved thermochromism for this species
( supporting information , figures s3 ) . from
a comparison to the abs data obtained for nocbi , a model
of base - off nocbl , we conclude that an increase in temperature from
4.5 to 300 k causes a shift in the base - on to base - off equilibrium
for nocbl , favoring dissociation of the n(dmb ) ligand at high temperatures .
this change in co coordination environment has , however , negligible
effects on the co no bonding interaction , as evidenced by our
rr data , tddft results , and nbo analysis . the origin and implications
of these findings are discussed below .
collectively , our spectroscopic and computational data reveal that
nocbl is inadequately described as a co(iii)no species , because significant charge donation from the no ligand
notably alters the effective nuclear charge of the metal center .
compared
to the methyl group of mecbl , the no ligand of nocbl is an even stronger
-donor , therefore inducing additional co 3dz orbital character into the homo and further enhancing
the co
our dft - computed mo description indicates that the formally unoccupied
co 3dz orbital contributes
as much as 29% to the homo of nocbl , as compared to 7% in mecbl .
n(dmb ) bond from 2.16 in mecbl
to 2.35 in nocbl as determined by x - ray crystallography
. the high degree of electron donation from no is particularly evident
from an analysis of the nlmos and nbos ( figure 10 ) derived from the canonical mos obtained with dft . on the basis
of this analysis ,
the doubly occupied 3dz bd - nlmo contains 58% co 3d orbital character ( table 5 ) ,
another unique feature of the co no bond in nocbl and
nocbi compared to the axial bonds in other co(iii ) corrinoid
species is the presence of a sizable -backbonding interaction
involving the co 3dyz and no * orbitals .
inspection of the co 3dyz bd - nlmo reveals that the no ligand contribution to this orbital
is 16% , leading to an estimate for the extent of donation
of 0.32 electrons .
this backbonding partially compensates
for the large amount of donation from the no ligand into the
co 3dz orbital , and represents
an unprecedented mechanism by which the electronic properties of the
co(iii ) ion can be modulated by the axial ligands .
the high
degree of no co donation in nocbl weakens the co
n(dmb )
bond to the point that at high temperature , the base - on to base - off
equilibrium favors a unique pentacoordinate species in aqueous solution .
in the case of nocbi ,
an effectively five - coordinate species
is present at all temperatures , suggesting that the strong trans influence
exerted by the no ligand precludes the binding of a weakly donating
water molecule even at 4.5 k. nonetheless , during the geometry optimization
of a complete nocbi model , the lower water ligand did
not fully dissociate .
a comparison of this structure and that obtained
for the truncated model reveals major differences in terms of the
orientation of the water molecule , indicating that the propionamide
side chains of the corrin ligand , which were absent in the truncated
model , may play an important role in modulating the lower axial bonding
interaction in cobalamins .
further experimental support for
our bonding description for nocbl is provided by published nmr data ,
which indicate that the n - resonance of the no ligand
undergoes a 40 ppm upfield shift upon protonation and dissociation
of the dmb ligand in the lower axial position .
although the observed change in electron shielding experienced
by the n nucleus was originally attributed to an increase
in the co
o bond angle from base - on to base - off nocbl ,
our rr and dft results provide compelling evidence that the co no
core structures of these two species are in fact virtually identical .
instead , our computational data suggest that the enhanced shielding
of the n nucleus in base - off nocbl reflects the weaker
-antibonding interaction between co and the lower axial ligand
in that species ( cf .
mos # 164 of nocbl and # 130 of nocbi in figures 8 and 9 , respectively ) , with the consequent increase in n(o ) natural charge
by 4% ( see table 2 ) .
previous crystallographic and epr studies have indicated that
co(ii)cbl binds molecular oxygen reversibly to yield superoxocobalamin
( o2cbl ) as the first step in the autoxidation process that
eventually yields h2ocbl .
o2cbl
may also be of physiological importance as free cobalamin is present
predominantly in the co(ii)cbl state in vivo .
given the similar frontier orbitals of no and
o2 , which has been widely exploited to mimic the binding
of dioxygen to metal centers in proteins by using nitric oxide , it
is interesting to compare the electronic structures of nocbl and o2cbl . while crystallographic data for these species have revealed
that both the no and o2 molecules ligate to the co center
in a bent end - on fashion , the axial co
n(dmb ) bond lengths
differ by 0.29 between nocbl ( 2.35 ) and o2cbl ( 2.06 ) .
n(dmb ) bond length in o2cbl is also considerably shorter than it is in mecbl ( 2.16 ) but very similar to that reported for cncbl
( 2.041 ) , which is considered to
possess a moderately strong donor in the upper axial position
based on computational analyses and the fact that this species exhibits
a typical
the nlmos for o2cbl are shown in figure 11 , and the compositions of the relevant nbos and
nlmos are listed in table 6 ( note that our
electronic structure description for o2cbl is supported
by a previous single - crystal epr characterization of this species , given the good agreement between the computed
and experimental g values and hyperfine coupling
constants , see table 7 ) . because of the spin - unrestricted
formalism used in these calculations , the nlmos have occupancies of
1.00 and are divided into a set of spin - up ( , majority spin .
figure 11 , left ) and spin - down orbitals ( ,
minority spin .
inspection
of the nlmos of o2cbl reveals that these orbitals are very
similar in composition to the nlmos of nocbl , although with some important
differences . for both sets of spin orbitals , the co 3dxz and co 3dx y
nlmos of o2cbl are nonbonding
with respect to the ligand framework , in an analogous manner as the
nlmos of nocbl .
additionally , the compositions of the nlmos
of o2cbl ( table 6 , right ) reflect
the presence of a relatively covalent co o2 bond ,
with a co 3d orbital contribution to the co 3dz(bd ) nlmo of 41% , as compared to 58% predicted
for nocbl ( see table 5 ) .
however , the co contribution
to the spin - up counterpart ( i.e. , the co 3dz(bd ) nlmo ) is merely 24% .
thus , the net
donation to the co(iii ) ion from o2 in o2cbl is smaller than it is from no in nocbl , which is consistent with
the greater oxidizing power of o2 ( e = 0.16 v vs nhe ) versus no ( e = 0.8 v nhe ) .
likewise ,
the effective number of co 3d electrons of o2cbl , computed
as described above for nocbl , is 6.08 , consistent with the classification
of this species as a typical co(iii)cbl species based on its short
co
thus , our computational results indicate that
o2cbl is adequately described as a co(iii)o species .
the orbitals ( majority spin , on left ) are
shown in orange / gray , while orbitals ( minority spin , on right )
are shown in yellow / gray .
nlmos are labeled according to the co - based
nbo providing the largest contribution .
the compositions of the nlmos
in terms of the corresponding nbos are provided in table 5 na indicates values not availalable
from nlmo composition analysis , but estimated to be close to 100% .
biochemical studies have shown that no can inhibit the two b12-dependent enzymes found in humans , namely , meth and mmcm , which could provide an additional pathway for the regulation of
these enzymes .
specifically , it was shown that inhibition of mmcm
in the presence of no donors resulted in the formation of nocbl in
the active site of the enzyme .
for this
and related enzymes , the formation of nocbl in the active site and
consequent inhibition of catalytic activity would require a repair
mechanism involving no dissociation or replacement of nocbl by adocbl .
given the strong co no bond in nocbl as revealed by the present
investigation , nocbl removal from the active site of these enzymes
is expected to be the main pathway for reactivation .
this process
could be facilitated by the strong trans influence exerted by the
no ligand .
conformation , and on the basis of the
results obtained in this study , it can be speculated that no coordination
to the transiently formed co(ii)cbl species in the enzyme active site
will considerably weaken the co
similarly , for
b12-dependent enzymes that bind adocbl in the base - on conformation , the co
n(dmb ) bond elongation and additional
geometric changes in response to no coordination to the co(ii)cbl
intermediate will likely facilitate removal of the nocbl product from
the active site .
although more experimental evidence is needed to
support these hypotheses , the results obtained from this study offer
unprecedented insights into the spectral and electronic properties
of nocbl and provide useful spectroscopic markers for probing the
interaction of this species with b12-dependent enzymes . | nitrosylcobalamin ( nocbl ) is readily
formed when co(ii)balamin reacts with nitric oxide ( no ) gas .
nocbl
has been implicated in the inhibition of various b12-dependent
enzymes , as well as in the modulation of blood pressure and of the immunological response .
previous
studies revealed that among the known biologically relevant cobalamin
species , nocbl possesses the longest bond between the co ion and the
axially bound 5,6-dimethylbenzimidazole base , which was postulated
to result from a strong trans influence exerted by the no ligand .
in this study , various spectroscopic ( electronic absorption , circular
dichroism , magnetic circular dichroism , and resonance raman ) and computational
( density functional theory ( dft ) and time - dependent dft ) techniques
were used to generate experimentally validated electronic structure
descriptions for the base - on and base - off
forms of nocbl .
further insights into the principal co ligand
bonding interactions were obtained by carrying out natural bond orbital
analyses .
collectively , our results indicate that the formally unoccupied
co 3dz2 orbital engages in
a highly covalent bonding interaction with the filled no *
orbital and that the co no bond is strengthened further by
sizable -backbonding interactions that are not present in any
other co(iii)cbl characterized to date . because of the substantial
no to co(iii ) charge donation , nocbl is best described
as a hybrid of co(iii)no and co(ii)no resonance structures .
in contrast , our analogous computational
characterization of a related species , superoxocobalamin , reveals
that in this case a co(iii)o2 description is adequate due to the larger oxidizing power of o2 versus no .
the implications of our results with respect to
the unusual structural features and thermochromism of nocbl and the
proposed inhibition mechanisms of b12-dependent enzymes
by nocbl are discussed . | Introduction
Methods
Results
Discussion |
non - small cell lung cancer ( nsclc ) is the leading cause of cancer mortality in the united states and the world , with a 5-year survival rate of only 15% for all stages combined , despite some recent advances in chemotherapeutic agents.1 , 2 the size and distribution of nsclc makes cytoreductive surgery ineffective . consequently , chemotherapy and/or radiation have been the treatments of choice .
conventional chemotherapy regimens have had limited efficacy , mainly because of resistance of these cancer cells to chemotherapeutic agents.4 , 5 small - molecule inhibitors such as erlotinib and afatinib that target the tyrosine kinase domain of epidermal growth factor receptor ( egfr ) produce responses in approximately 10% of patients with nsclc.6 , 7 , 8 in patients with mutant egfr , responses to egfr - tyrosine kinase inhibitors ( egfr - tkis ) can be dramatic and may last longer than a year .
in contrast , k - ras gene mutation , which occurs in approximately 30% of nsclc , has been associated with a poor response to egfr - tkis.9 , 10 , 11 , 12 , 13 it has been demonstrated previously that somatic mutations in the tyrosine kinase domain of egfr are associated with sensitivity to egfr - tkis , whereas mutations in k - ras , which encodes a guanosine triphosphotase ( gtpase ) downstream of egfr , are associated with primary resistance .
however , there is an unmet need in developing the most appropriate therapy for mutant k - ras nsclc .
several strategies , such as farnesyltransferase inhibitors , have been explored as possible inhibitors of mutant k - ras in lung adenocarcinomas . to date , none of these strategies have proved to be successful , mainly because of lack of specificity .
small interfering rna ( sirna ) inhibition is a very viable alternative given the specificity of this technology . here
we aim to harness the advantages of sirna technology as a therapeutic modality for mutant k - ras in nsclc .
sirna as a therapeutic molecule is very effective at knocking down molecular pathways that are pathogenic .
however , its application in the clinic is often limited by its susceptibility to enzymatic degradation in blood , non - specific uptake by cells , and the difficulty involved in the transfection of sirna to cells because of its relatively large size and polarity.16 , 17 .
clearance by the mononuclear phagocyte system ( mps ) is another limiting factor affecting the possible therapeutic application of sirna.18 , 19 , 20 recently , our group developed a novel hybrid nanoparticle delivery system composed of human immunoglobulin g ( igg ) and poloxamer-188 ( a polyoxyethylene - polyoxypropylene block copolymer ) for stable and efficient sirna delivery to lung adenocarcinoma cells.21 , 22 , 23 we hypothesized that our hybrid nanoparticles would efficiently deliver loaded anti - mutant k - ras sirna into the cytosol of lung adenocarcinoma cells , downregulate g12s - k - ras in a549 cells , and inhibit cancer cell proliferation .
further , in view of the fact that human igg is the main immunoglobulin that protects the body against infection , we hypothesized that these hybrid nanoparticles would not produce the well documented immunogenic / inflammatory reaction experienced with most nanoparticle formulations .
poloxamer- 188 , a nonionic triblock copolymer , is added to the surface of the nanoparticles to help circumvent the mps during systemic circulation .
data obtained in vitro in cell culture supported these hypotheses and demonstrated the effectiveness of these hybrid nanoparticles in accomplishing the set objectives.21 , 23 an anti - proliferation effect of anti - mutant k - ras sirna - loaded hybrid nanoparticles ( akslhns ) in a549 cells was also demonstrated.21 , 22 further , our findings demonstrated that the double - layer protection provided by these nanoparticles helps to protect enclosed sirna from serum nucleases for up to a minimum of 48 hr .
the main objective of the present study was to validate the results obtained from in vitro studies under in vivo conditions using a metastatic orthotopic murine model of nsclc . to achieve this , we hypothesize that akslhns will inhibit the progression of metastatic lung cancer by efficiently downregulating mutant - k - ras in mouse models without eliciting an immune / inflammatory reaction .
further , we hypothesize that akslhns will not cause unnecessary toxicity to normal tissues because of their preferential accumulation in tumors compared with normal tissues .
this is based on the fact that tumors are naturally more permeable to particles between 100800 nm because of an enhanced permeation and retention ( epr ) effect . to test these hypotheses ,
metastatic orthotopic murine models of nsclc were created by injecting a549-luc cells into the tail veins of female severe combined immunodeficiency ( scid ) beige mice .
the efficacy of intraperitoneally injected akslhns at inhibiting or downregulating mutant k - ras and slowing down tumor progression was compared with several control formulations .
akslhns composed of human igg in the inner layer and poloxamer-188 in the outer layer were prepared using our previously reported nanoprecipitation process.21 , 22 , 23
figure 1 shows a schematic of akslhn development .
akslhns were spontaneously produced at ph 7.0 , which is the isoelectric point of human igg .
photon correlation spectroscopy ( pcs ) demonstrated that these particles were 135.4 5.4 nm in diameter , with a polydispersity index ( pi ) of 0.07 0.03 .
these particles demonstrated a zeta potential of + 16.7 0.2 and encapsulation efficiency and loading efficiency of 60% 0.4% and 2.04% 0.06% , respectively .
the maximum tolerated dose ( mtd ) of sig12s in akslhns was determined for in vivo experiments by administering different doses of akslhns ( 0.1 , 0.3 , 0.6 , 0.9 , and 1.2 mg / kg body weight equivalent of sig12s ) progressively by intraperitoneal injection to female nu / nu nude mice .
24 hr after the last dose was injected , the animals were euthanized , and their kidneys , lungs , and livers were harvested .
the results demonstrated that the akslhns had no effect on animal mortality , behavior , or body weight up to 0.9 mg / kg body weight .
apoptosis evaluation demonstrated that the nanoparticle formulation did not cause any significant damage to the liver , lung , and kidney compared with the untreated control up to 0.9 mg / kg ( figure 2 ) .
mice treated with akslhns showed regression in tumor burden over the 4-week experimental period . in contrast , pbs - treated control , erlotinib - treated , and combination - treated ( akslhns and erlotinib ) groups showed lack of sensitivity to their respective treatment , as demonstrated by tumor progression ( figure 3 ) . the tumor burden was quantified by measuring the photon counts for each tumor , which was then plotted against time ( weeks ) to further demonstrate the antitumor effect of injected akslhns , as shown in figure 4a .
the tumor burden in pbs - treated mice continued to progress until metastasis in the colorectal area was observed after 2 weeks of treatment .
metastasis of tumors to both the left and right lumber lymph nodes and the colorectal region could also be conspicuously observed after 3 weeks of monitoring .
we also observed metastasis to the right or left popliteal lymph nodes in some of the mice ( figure s1 ) . to confirm metastasis to other organs , the lungs ,
heart , liver , and kidneys of pbs - treated mice were harvested after the mice were euthanized and imaged ex vivo using xenogen ivis bioluminescence .
metastasis to the liver and heart was demonstrated in these mice , as shown in figure s2 . similarly , the erlotinib - treated and combination - treated groups demonstrated evidence of metastasis to the colorectal region of the mice .
interestingly , tumor metastasis to the brain was observed in one of the five mice in the erlotinib - treated group after 3 weeks of treatment ( figure s2 ) .
apoptosis determination by cell death elisa and tunel showed that akslhns enhanced apoptosis induction in lungs with tumors ( figures 4b and 4c ) .
in contrast , erlotinib- , combination- , and pbs - treated lungs with tumors only demonstrated limited evidence of apoptosis .
expression of mutant k - ras g12s mrna was studied in the tumors using taqman qrt - pcr and western blot analysis ( figures 5a and 5b ) .
the western blot data in figure 5a demonstrate that akslhns downregulated mutant k - ras compared with pbs , combination , and erlotinib .
qrt - pcr showed that akslhns significantly downregulated mutant k - ras mrna by approximately 60% compared with pbs .
further , combination and erlotinib treatments did not show any effect on mutant k - ras expression , as shown in figure 5b .
adenocarcinoma of mice treated with akslhns showed smaller tumors with thin fibrovascular stroma and rare mitotic figures ( figure 6a ) .
mice that received a combination of erlotinib and akslhns showed few mitotic figures on h&e , as shown in figure 6b .
adenocarcinomas from mice treated with erlotinib had numerous mitotic figures ( figure 6c ) .
mice from the control group ( pbs ) had multinucleated tumor cells with eosinophilic cytoplasm , as shown in figure 6d .
toxicity to healthy tissues was evaluated using cell death detection elisa to detect the induction of apoptosis .
figure 7a demonstrates that akslhns did not lead to apoptosis in the lung , kidney , and liver of treated animals after 4 weeks of treatment .
similarly , the combination - treated group did not show any apoptosis in any of these critical organs .
however , the erlotinib - treated group showed signs of apoptosis in the liver , as demonstrated in figure 7a . cytokines interleukin-6 ( il-6 ) and
tumor necrosis factor ( tnf- ) levels were measured in both cultured splenocytes isolated from spleens harvested from treated mice and serum using the respective elisa kits .
figure 7b shows that akslhns did not significantly increase il-6 expression in the spleens of treated mice compared with pbs except for day 2 post - treatment .
similarly , tnf- did not significantly increase in the splenocytes of treated mice throughout the 8-day study period .
expression of both il-6 and tnf- in the sera of treated mice was also evaluated .
figure s3 shows that there was no significant increase in the levels of both il-6 and tnf- throughout the period of study .
akslhns were specially designed to facilitate the delivery of anti - mutant k - ras sirna to mutant k - ras - expressing metastatic lung cancer in an orthotopic mouse model .
it was hypothesized that akslhns will inhibit the progression of metastatic lung cancer by efficiently downregulating mutant - k - ras in mouse models without eliciting immune / inflammatory reactions .
further , we hypothesized that akslhns will not cause unnecessary toxicity to normal tissues because of their preferential accumulation in tumors compared with normal tissues .
it has been reported that an unmet need still exists in finding an appropriate therapy for mutant k - ras - expressing lung cancer because various treatment modalities , including farnesyltransferase inhibitors , have yet to yield positive results .
however , sirnas are known to have the potential to silence critical molecular pathways responsible for pathogenic conditions .
the use of sirna as a therapeutic tool is , however , being compromised by a lack of safe and efficient delivery systems , limiting the efficacy of these molecules in the clinic . in this study ,
akslhns are being evaluated as a potential treatment modality in mutant k - ras - expressing metastatic lung cancer .
akslhns were prepared based on the fact that proteins have minimum solubility but maximum precipitation at the isoelectric point.21 , 22 akslhns were very effective in regressing lung tumors in mice , as shown in figure 3 , after 4 weeks of treatment .
a combination of akslhns and erlotinib was evaluated under the hypothesis that the downregulation of mutant k - ras by akslhns would improve the sensitivity of mutant k - ras - expressing lung cancer to egfr - tkis because mutant k - ras has been implicated in the lack of efficacy of egfr - tkis in lung cancer with mutant k - ras expression.9 , 14 , 26 , 27 surprisingly , this combination treatment produced an inferior antitumor effect compared with akslhns alone .
we previously reported that our hybrid nanoparticles are able to escape endocytic recycling and are successfully delivered to the cytosol of target cells because of the buffering capacity of the hybrid nanoparticles in the endosome .
the solubility of igg and poloxamer-188 at acidic ph levels makes it possible for the dissolved igg in the endosome to activate the proton pump that raises osmotic pressure in the endosome , leading to the swelling and subsequent escape of sirna from endosomes into the cytosol.21 , 27 , 28 however , the presence of erlotinib , a very basic drug , in the endosome at the same time may increase the ph level of the endosome , making it impossible for the nanoparticles to dissolve and release the sirna as it would normally do .
the lack of release of sirna because of the presence of erlotinib is assumed to subsequently lead to an inferior antitumor effect of the combination therapy compared with akslhns alone .
the interaction between a ph - sensitive nucleic acid - loaded nanoparticle system and a concurrently administered basic drug needs to be investigated further for a clear understanding of this phenomenon .
nevertheless , metastasis of the tumor to the brain , colorectal region , right and left lumber lymph nodes , and right popliteal lymph nodes was observed in the other groups of animals , except the akslhn - treated group .
the effective antitumor effect of akslhns was further validated by induction of apoptosis in the tumor - bearing lungs of the akslhn - treated group using both tunel and cell death detection elisa .
further , both mutant k - ras protein and mrna were significantly downregulated in the lungs of akslhn - treated mice compared with the other mouse groups , further validating the effectiveness of this therapy .
previously , we reported the superior and stable downregulation of mutant k - ras by akslhns at the cellular level ( using a549 cells ) over both scramble sirna and lipofectamine - delivered anti - k - ras sirna.21 , 29 toxicity and inflammatory reaction remain the limiting factors preventing the translational application of most nanomedicines.22 , 23 , 30 for any nanoparticle - based therapeutic agent to have a chance of progressing to the clinic , it is important that it is not toxic to healthy tissues and does not elicit a significant immunogenic / inflammatory reaction . on that note , we evaluated the effect of akslhns , erlotinib , and a combination of both on the induction of apoptosis in healthy tissues .
induction of apoptosis in healthy tissues has been reported previously to represent toxicity in such tissues .
akslhns and the combination treatment did not lead to induction of apoptosis in the lung , kidney , and liver of treated mice .
this is not surprising because erlotinib has been implicated previously in acute hepatotoxicity in nsclc patients .
it is , however , interesting to note that the combination therapy involving both akslhns and erlotinib did not lead to hepatotoxicity .
this further corroborates our hypothesis that an interaction occurs between akslhns and erlotinib at the cellular level . to evaluate the ability of akslhns to elicit an immune / inflammatory response , naive balb / c mice were used .
scid beige mice used for development of the metastatic orthotopic murine nsclc model are immunocompromised and would not be suitable for the immunogenic / inflammatory reaction experiment .
scid mice are characterized by the complete inability of the adaptive immune system to mount , coordinate , and sustain an appropriate immune response , usually because of absent or atypical t and b lymphocytes .
il-6 and tnf- expression in isolated splenocytes and sera of akslhn - treated mice was similar to that of pbs - treated ( control ) mice , except for a certain time point where insignificant elevated expression of these cytokines was observed .
this could be attributed to two factors : the presence of poloxamer-188 on the surface of the nanoparticles or the fact that the nanoparticles were fabricated with human igg and tested in mice .
this interspecies variation could contribute to the slight elevation in these cytokines at these time points .
future study will involve the use of mouse igg in the fabrication of the nanoparticles to eliminate interspecies variation as a contributory factor to this observation . in conclusion ,
akslhns are a promising prospect as a treatment modality for mutant k - ras - expressing nsclc as a single therapy .
a further understanding of the cellular - level interaction between akslhns and egfr - tkis during concurrent administration would help in developing an optimal dosage regimen to further harness this treatment modality in nsclc patients .
poloxamer-188 , rnase - free water , and fetal bovine serum were obtained from fisher scientific .
sirna against mutated k - ras g12s was designed by and purchased from thermo scientific ( formerly dharmacon ) . the sig12s sense and antisense sequences were guuggagcuaguggcguagdtdt and cuacgccacuagcuccaacdtdt , respectively .
monoclonal antibodies specific for mutant g12s k - ras proteins was obtained from neweast biosciences .
the tnf- elisa kit was obtained from thermo scientific , and the il-6 elisa kit was obtained from becton dickinson .
human adenocarcinoma cell line a549-luciferase , expressing a k - ras mutation at g12s , was obtained from perkinelmer .
these cells were maintained in 10% fetal bovine serum and 1% antibiotics - supplemented f12k .
female scid beige mice , 78 weeks old and weighing approximately 28 g , and female balb / c mice were supplied by taconic and maintained in the american association for the accreditation of laboratory animal care ( aaalac)-accredited facility at thomas jefferson university .
all animal studies were approved by the institutional animal care and use committee ( iacuc ) of thomas jefferson university .
50 mg of excipient - free human igg was dissolved in 0.01 n hcl containing 20 mg of poloxamer-188 .
187 g of sirna was then added to make a 10-ml total solution in a 50-ml beaker .
the final concentration of human igg in the solution amounted to 5 mg / ml . this solution was then slowly titrated with 0.01 n naoh to bring the ph of the mixture to 7 , which is the isoelectric point ( pi ) of human igg as determined in our laboratory using isoelectric focusing . the nanoparticles were continuously stirred on a magnetic stirrer for 10 min . to isolate the nanoparticles from the precipitation medium ,
the suspension was centrifuged with a microcentrifuge ( eppendorf centrifuge 5418 ) at 2,000 rpm for 5 min .
nanoparticles were then rinsed with double - distilled deionized water before being redispersed in water and snap - frozen using liquid nitrogen .
this was then loaded into a freeze dryer ( labconco freezezone 4.6 ) , and lyophilization was performed for 48 hr .
lung cancer models of nsclc were created by injecting 5 10 a549-luciferase cells in sterile pbs into the tail veins of female scid beige mice .
cancer development and progression were monitored using the xenogen ivis ( perkinelmer ) bioluminescence imaging system .
100 l of 30 mg / ml xenolight rediject d - luciferin ( perkinelmer ) was injected intraperitoneally into the mice approximately 10 min before the animals were imaged .
following the establishment of detectable lung tumors ( 2 weeks from the time of induction ) , mice were divided into four groups of five animals each .
the first group was treated with 20 mg / kg erlotinib daily for 4 weeks by oral gavage ( 100 l ) .
the second group was treated with akslhns twice a week for 4 weeks at a dose of 0.3 mg / kg by intraperitoneal injection ( 100 l ) .
a third group was treated with a combination of akslhns and erlotinib , whereas the fourth group was treated with pbs as a control twice a week by intraperitoneal injection ( 100 l ) .
one part of the tissue was fixed in 10% phosphate - buffered formalin ( fisher scientific ) for paraffin embedding , histology , and immunohistochemistry ( ihc ) analysis , and other three were snap - frozen for real - time pcr , western blot , and cell death detection elisa .
five mice were treated with a single dose of 0.9 mg / kg akslhns by intraperitoneal injection , whereas the remaining four served as controls and were injected with pbs ( 100 l per mouse ) .
blood was collected by retro - orbital puncture under isoflurane anesthesia into edta - covered vacutainers ( becton dickinson ) on days 1 , 2 , 6 , and 8 .
blood was then immediately centrifuged at 1,000 g for 10 min at 4c , and serum was collected and frozen at 80c until ready to be used .
splenocytes were isolated from mice injected with functionalized nanoparticles and pbs on days 1 , 2 , 6 , and 8 .
conditioned medium was collected after 24 hr and frozen at 80c until ready to be used .
the levels of il-6 and tnf- were measured using the respective elisa kits according to the manufacturers instructions .
spleens of mice injected with akslhns and pbs were collected , placed into a tube with rpmi medium , and kept on ice .
the tube was then transferred to the hood , and the spleen was homogenized by two microscope glass slides .
the spleen slurry in 10 ml of rpmi medium was transferred to a 70-m cell strainer ( falcon ) and filtered from clumps . the cell suspension
the cell pellet was resuspended in 3 ml of ammonium - chloride - potassium ( ack ) lysis buffer ( gibco ) to get rid of red blood cells .
the hemolysis was carried out at room temperature with periodic shaking for 5 min , after which the suspension was reconstituted with 10 ml of fresh rpmi medium and centrifuged at 400 g for 5 min at 20c .
the cell pellet was washed with 10 ml of rpmi medium and spun down at 400 g for 5 min .
the resulting cell pellet was dissolved in complete rpmi medium ( rpmi 1640 medium , 10% fbs , 1 penicillin / streptomycin [ pen / strep ] , 10 mm hepes ) , 500,000 cells were seeded in triplicate into wells of a 96-well plate .
the cells were incubated for 24 hr , and the conditioned medium was collected and frozen until ready to be used for determination of the proinflammatory cytokines tnf and il-6 .
tumor - bearing lungs were collected from mice , snap - frozen in liquid nitrogen , and kept at 80c until ready to be used .
tissue lysates were prepared in pierce radioimmunoprecipitation ( ripa ) buffer ( thermo scientific ) with addition of pierce protease inhibitor mini tablets ( thermo scientific ) .
the protein concentration was determined with the coomassie plus ( bradford ) assay kit ( thermo scientific ) .
80 g of total protein was separated on nupage 4%12% bis - tris gels ( life technologies ) with nupage mes sds running buffer ( life technologies ) and subsequently transferred onto a nitrocellulose membrane of 0.45-m pore size ( life technologies ) .
the membrane with proteins was blocked according to the manufacturer s instructions ( invitrogen ) for 1 hr at room temperature and probed with primary antibodies
kras g12s monoclonal antibody ( 1:500 , new east biosciences ) and -actin ( 1:5,000 , sigma - aldrich)overnight at 4c .
the membranes were washed three times for 5 min in wash buffer according to the manufacturer s instructions ( invitrogen ) and incubated with secondary goat anti - mouse antibodies conjugated with horseradish peroxidase ( molecular probes ) at a dilution of 1:1,000 .
immune complexes were detected with chemoluminescent substrate , pierce ecl western blotting substrate ( thermo scientific ) in a dark room on a tabletop processor , srx-101a ( konica minolta ) .
total rna from snap - frozen lung tissues was isolated with the qiagen rnaeasy kit , and cdna was made with the verzo cdna kit ( thermo scientific ) using polya primers .
real - time pcr was carried out in 20-l reaction mixtures containing 25 ng of cdna , 10 l of 2 taqman gene expression master mix ( applied biosystems ) , and 1 l of specific taqman gene expression assay ( 20 , fam dye - labeled ) .
gene expression assays ( obtained from thermo scientific ) contained mutant kras at g12s and -actin primers ( -actin served as a normalizing gene ) .
apoptosis in samples collected from mice with nsclc was detected by tunel assay with the tacs 2tdt - fluor in situ apoptosis detection kit ( trevigen ) according to the manufacturer s instructions .
briefly , lung tissue section slides were deparaffinized in xylene and 100% , 95% , 70% ethanol , followed by two changes of pbs .
next , the samples were digested with 50 l of cytonin solution for 30 min and washed with water and terminal deoxynucleotidyl transferase ( tdt ) labeling buffer .
next , the slides were covered with 50 l of labeling reaction mix ( tdt deoxynucleoside triphosphate [ dntp ] , co , tdt enzyme and tdt labeling buffer ) and incubated for 60 min at 37c in a humidity chamber . at this step ,
positive control was made before the labeling step by incubating the slide with tacs nuclease ( generating dna breaks in every cell ) at room temperature for 40 min .
the labeling reaction was halted by stop buffer , and samples were washed in pbs and covered with 50 l of strep - fluorescein solution for 20 min .
next , the slides were washed with pbs , mounted , and viewed under a fluorescence microscope with a 495-nm filter .
cell death detection elisa was carried out according to the manufacturer s instructions ( roche ) .
sandwich elisa was carried out to determine the amount of mono- and oligonucleosomes in the cytoplasmic fraction of tissue lysates .
2% homogenate of lung tissue was prepared in incubation buffer and centrifuged for 10 min at 15,000 g. the supernatant was collected and kept at 80c until ready to use .
plastic wells were incubated with coating solution ( containing anti - histone antibodies ) overnight at 4c .
the next day , it was substituted for incubation buffer for 30 min and washed three times with washing solution .
100 l of homogenate , diluted 29 times in incubation buffer , was placed into the wells and kept for 90 min at room temperature with mild shaking .
the plate was washed three times and incubated with conjugate solution containing anti - dna - peroxidase antibodies for 90 min .
the wells were washed three times and exposed to abts substrate for 30 min until the color developed .
absorption values were read on a biotek instruments epoch microplate spectrophotometer with gen5 1.10 software .
heat - mediated antigen retrieval was performed in citrate buffer ( ph 6.0 ) at 98c for 20 min .
9664 , cell signaling technology ) , was diluted 1:300 and incubated at room temperature for 30 min ; human tonsil was used as a positive control .
the ihc procedure was done with the dako autostainer plus platform using the vectastain elite abc kit ( standard , catalog no .
statistically significant differences between two groups were determined by two - tailed student s t test .
m.p . carried out most of the experiments and was also involved in data interpretation .
were involved in taking pictures of the histology slides and the interpretation of histopathology data .
were involved in the development and monitoring of the orthotopic metastatic mouse lung cancer models .
s.a.s . initiated the study , planned the experiments , interpreted the data , and wrote the manuscript . | there is an unmet need in the development of an effective therapy for mutant k - ras - expressing non - small - cell lung cancer ( nsclc ) .
although various small molecules have been evaluated , an effective therapy remains a dream .
sirnas have the potential to downregulate mutant k - ras both at the protein and mrna levels .
however , a safe and effective delivery of sirnas to tumors remains a limitation to their translational application in the treatment of this highly debilitating disease .
here we developed a novel hybrid nanoparticle carrier for effective delivery of anti - mutant k - ras to nsclc ( akslhn ) .
the ability of this treatment modality to regress lung tumors in mouse models was evaluated as a monotherapy or as a combination treatment with erlotinib .
further , the toxicity of this treatment modality to healthy tissues was evaluated , along with its ability to elicit immune / inflammatory reactions .
the results suggest that this treatment modality is a promising prospect for the treatment of mutant k - ras - expressing nsclc without any accompanying toxicity .
however , further understanding of the cellular - level interaction between ahslhn and erlotinib needs to be attained before this promising treatment modality can be brought to the bedside . | Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest |
reduction of elevated intraocular pressure is the only established modifiable risk factor shown to reduce the risk of glaucoma - associated optic neuropathy.1,2 topical beta - adrenergic antagonists and carbonic anhydrase inhibitors are well accepted medical treatments for reducing production of aqueous humor.3,4 newer prostaglandin analogs reduce intraocular pressure by promoting uveoscleral aqueous outflow.46 however , the ocular hypertension treatment study showed that almost 40% of patients will require a combination of two or more medications to achieve a 20% reduction in intraocular pressure.1 complex drug regimens requiring the use of numerous bottles can reduce the therapeutic effect , and use of multiple bottles has also been shown to be a barrier to compliance.79 coadministration of drops may introduce a washout effect if not adequately spaced in time.10 similarly , eye drop tolerability will affect compliance and treatment outcomes.7 multiple - drop therapies have been formulated to address some of these compliance issues .
azarga ( alcon laboratories inc , fort worth , tx ) is a fixed combination of brinzolamide 1% + timolol 0.5% , and is effective in reducing intraocular pressure.11 in comparison , a fixed combination of brinzolamide 1% + timolol 0.5% is noninferior to a fixed combination of dorzolamide 2% + timolol 0.5% and achieved lower mean intraocular pressures at nine of 12 study visits.12 tolerability comparisons of these two fixed - combination eye drops seem to show a reduction in eye irritation using the brinzolamide formulation , but an increase in blurred vision.1215 in this study , we directly switched patients requiring multiple - drug treatments from a fixed combination of dorzolamide - timolol to brinzolamide - timolol and investigated the impact of this change on tolerability and compliance .
this study was performed at the glaucoma unit in the royal hallamshire hospital , sheffield , uk .
patients were switched from a fixed preparation of dorzolamide - timolol to brinzolamide - timolol . following the switch ,
patients were contacted by telephone and a number of questions were asked over a period of 426 weeks after changing eye drops .
one independent questioner read a preformed questionnaire to patients over the telephone ( figure 1 ) .
telephone consultations were initiated by confirming the patient details and that correct antiglaucoma treatment was concurrent .
a primary yes / no question was asked to confirm if a specific side effect had been experienced .
if a side effect was noted for either drug , a comparison of the two types of eye drop was made by asking patients to assign a numerical value ( 1 to 9 ) comparing the new ( brinzolamide - timolol ) and old ( dorzolamide - timolol ) drops .
thirty - one consecutive patients whose medication had been altered were contacted and questionnaires were successfully completed .
thirty - one consecutive patients ( 12 males and 19 females aged 4189 years ) successfully completed the questionnaire .
the data were analyzed for significant differences from the assumed mean of 5 using the t - test if the sample set was > 20 .
when the sample set was < 20 the wilcoxon signed - rank test was performed . a test for proportions
was used to compare the numbers of patients experiencing a specific side effect . the population varied in the percentages experiencing side effects , as shown in table 1 .
comparison of the percentages of side effects indicated less stinging but more blurring for brinzolamide - timolol compared with dorzolamide - timolol eye drops ( table 1 ) .
there were no differences in the proportions of patients who experienced altered taste or redness following instillation of the eye drops .
a comparison of the severity and chronicity of the side effects of the two types of fixed - combination eye drops is shown in table 2 .
the fixed combination of brinzolamide - timolol produced significantly less stinging and for a shorter amount of time than the dorzolamide - timolol eye drops ; it also produced less eye redness and for a significantly shorter amount of time .
however , the fixed combination of brinzolamide - timolol produced more blurring , although the length of time this was present was similar to that with dorzolamide - timolol .
no differences between the two eye drops were found for taste , overall impression , and likelihood of compliance .
previous studies have shown the fixed - combination brinzolamide - timolol eye drop to be significantly more effective than its individual components and noninferior to fixed - combination dorzolamide - timolol eye drops in lowering intraocular pressure.11,12 we suggest that although this crossover study was unidirectional and of limited size , it does demonstrate a different side effect profile for the two types of fixed - combination eye drops .
stinging was significantly less and lasted for a shorter period of time with the brinzolamide - timolol eye drops , which is consistent with previous findings.1215 apart from any intrinsic differences in the two molecules which may alter the stinging profile of the two drugs , the ph of the eye drops containing brinzolamide is relatively more neutral than that of dorzolamide ( ph 7.5 and ph 5.6 , respectively ) .
furthermore , eye drops containing dorzolamide use sodium citrate as a buffer whereas none is present in the eye drops containing brinzolamide .
all these differences seem to cause the brinzolamide - timolol eye drop to sting less .
redness can be a problematic side effect of any antiglaucoma medication , and accordingly reduces compliance , but may also be a surrogate of general irritation and stinging .
we found that fixed - combination eye drops containing brinzolamide or dorzolamide show a slight reduction in severity of redness , but of significantly shorter duration . given that this represents another reduction in side effects
, it should have the effect of increasing tolerability.7,9 blurring of vision is a known side effect of brinzolamide,11 but studies have shown conflicting results when compared with dorzolamide .
stewart et al did not find any difference between the two drops.17 however , silver found a higher incidence of visual blurring for brinzolamide than with dorzolamide , as did manni et al and mundorf et al.12,13,16 this increase in blurring of vision with the eye drop containing brinzolamide was also observed in our study , but does not seem to last any longer than the blurring caused by the eye drop containing dorzolamide .
the increase in visual blurring is probably due to the viscosity of the eye drop , with the brinzolamide - timolol combination eye drop being much thicker than the dorzolamide - timolol eye drop . indeed , seven of the 31 patients we interviewed stated that they found the brinzolamide - timolol eye drop difficult to apply due to its thickness .
also , one patient switched back to the dorzolamide preparation because of this application problem , even though she preferred the side effect profile of the brinzolamide eye drop .
unfortunately absolute levels of blurring and the finite time for which the blurring persisted were not assessed in this study .
thus , no assumptions can be made regarding how blurring affects general daily activities using either type of eye drop .
it could be that blurring persists long term and causes a significant reduction in ability to perform daily tasks , or it could be very transient and cause few problems .
an answer to this question would help to make a more meaningful comparison of the side effect profiles of the two combination preparations .
initial studies by silver showed a higher incidence of altered taste sensation with brinzolamide than with dorzolamide,16 although subsequent studies , including our study , showed no difference.12,17,18 there was no statistically significant difference between the overall comparison and likelihood of compliance with the fixed - combination brinzolamide - timolol and dorzolamide - timolol eye drops in this study .
previous studies have found a patient preference for the drop containing brinzolamide over the one containing dorzolamide.13,15 however , this and other studies show a reduction in stinging and redness at the cost of increased blurred vision .
it is possible that the exchange of one side effect for another leaves the eye drops being equivalent overall .
interestingly , jampel et al found that patients would pay more for an eye drop with reduced blurring but would not pay more for a reduction in any other side effect.19 this observation would suggest a preference for the dorzolamide combination overall , but this does not appear to be the case in this study .
our study confirms the finding of other researchers pertaining to the side effect profiles of fixed combinations of brinzolamide - timolol and dorzolamide - timolol , ie , a reduction in stinging and redness but an increase in blurred vision .
the advantage of one eye drop over the other then becomes patient - specific , depending on which side effect they find more tolerable .
we suggest that both types of eye drop are acceptable and interchangeable choices for treating patients with glaucoma , but compliance may be an issue as a result of the specific side effects of one drop or the other . | backgroundthis study aimed to determine the impact of switching patients requiring multiple drug treatment from the dorzolamide - timolol fixed combination to the brinzolamide - timolol fixed combination and potential effects on tolerability and compliance.methodspatients were switched from dorzolamide - timolol to brinzolamide - timolol and questioned within a period of 426 weeks .
questions were asked to confirm if a specific side effect had been experienced , and then a numerical comparison between the two types of eye drop was made.resultsthirty-one consecutive patients ( 12 males and 19 females aged 4189 years ) successfully completed the questionnaire .
comparison of the severity and chronicity of the side effects of the two types of fixed - combination eye drops showed that brinzolamide - timolol caused significantly less stinging for a shorter amount of time than dorzolamide - timolol ; it also produced less eye redness for a significantly shorter amount of time .
brinzolamide - timolol produced more blurring , although the length of time this was present was similar to that for dorzolamide - timolol .
no differences between the two eye drops were found for taste , overall impression , and likelihood of compliance.conclusionour study confirms the findings of other researchers pertaining to the side effect profile of brinzolamide - timolol after switching from dorzolamide - timolol , which is a reduction in stinging but an increase in blurred vision .
the advantage of one eye drop over the other then becomes patient - specific , depending on which side effect they find most tolerable .
we suggest that both eye drops are acceptable choices in treating patients with glaucoma , and are interchangeable if compliance becomes an issue because of a specific side effect of one eye drop or the other . | Introduction
Materials and methods
Results
Discussion
Conclusion |
the advent of rotary nickel titanium ( niti ) instrumentation has brought significant progress to root canal preparation in terms of preserving native root canal morphology and preventing apical transportation . however , separation of instruments during continuous rotary motion is the most common accident in clinical practice .
niti instruments may exhibit premature fatigue failure , thus reducing the life of the instrument or torsional stress failure , which is the result of file binding to the canal walls .
therefore , rotary instrumentation requires manual or mechanical cervical preflaring to mitigate fracture risk . in 2008 , yared used a single rotating instrument ( protaper ) in reciprocating motion for canal preparation and shaping and demonstrated that the technique provides greater speed , good cost - benefit ratio , and reduced fracture rates . a new approach to root canal preparation
was introduced in 2010 with the reciproc ( rc ) system ( vdw , munich , germany ) and later by the waveone ( wo ) system ( dentsply maillefer , ballaigues , switzerland ) .
both use a single niti m - wire alloy instrument driven by reciprocating motion , whereby the instrument cuts the dentin wall of the root canal during the counterclockwise rotation , when taper lock is imminent .
the following clockwise motion then disengages the instrument from dentin , reducing the incidence of separation .
according to manufacturer recommendations , a glidepath must be prepared using small ( # 10 and # 15 ) rotary or hand files , but cervical preflaring is unnecessary . within the context of widespread use of reciprocating systems ,
the present study assessed the influence of multiple reuse ( after resterilization ) and cervical preflaring on fracture strength , plastic deformation , and/or surface cracking in rc and wo reciprocating instruments after root canal instrumentation in extracted human teeth .
the null hypothesis was that both instrument systems would be equally fracture resistant when driven by reciprocating motion , with and without cervical preflaring .
the present study was approved by the local research ethics committee ( caae : 13831213.4.0000.5052 ) . the sample comprised extracted human upper molars ( length 1822 mm ) with fully formed roots , constricted root canals , and root curvature 35. for selection purposes , teeth were identified and imaged by conventional radiography and cone beam computed tomography . only teeth in which the entire root canal system could be visualized were included in the sample for experimental group allocation .
teeth with root curvature > 35 , partially or completely calcified canals , or excessively wide canals were excluded from the study .
after application of the selection criteria , 550 teeth were selected from a set of 1153 extracted human molars .
figure 1 provides a schematic illustration of the study methods . in all selected teeth ,
canal orifices were located with an endodontic explorer and special files under 32 magnification ( mc - m1233 operating microscope , dfv com . and ind .
ltda , valena , rj , brazil ) , and teeth were then randomly allocated across the different study groups .
the presence of a fourth canal was identified in 41.2% of the teeth used in the sample ( n = 200 ) .
forty 25-mm reciprocating instruments were used in this study : 20 reciproc r25 , tip size 25 , 0.08 variable taper ( rc ) ( vdw gmbh , munich , germany ) and 20 waveone primary , tip size 25 , 0.08 variable taper ( wo ) ( dentsply - maillefer , ballaigues , switzerland ) , all powered by a silver reciproc motor and sirona endo 6:1 contra - angle handpiece ( vdw gmbh , munich , germany ) .
rc files were driven by the rc all program and wo files by the wo all program .
four study groups ( n = 10 each ) were defined , according to root canal instrumentation procedure :
g1 : rc;g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc;g3 : wo;g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo .
g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc ; g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo .
procedures were performed by a single operator , who followed the manufacturer - issued instructions for each instrument .
after coronal access , teeth were irrigated with 5 ml sodium hypochlorite ( naocl ) 2.5% ( biodinmica qumica e farmacutica ltda , ibipor , pr , brazil ) and explored with hand files as described elsewhere .
the root canal systems of all teeth were evacuated with a # 10 k - file , which was used to identify patency .
working length determination was performed under operating microscope magnification ( mc - m1233 , dfv com . & ind .
ltda , valena , rj , brazil ) , 1 mm short of the apex . in all teeth
, glidepaths were prepared with a # 13/0.02 mm / mm pathfile ( dentsply - maillefer , ballaigues , switzerland ) , which enabled standardized canal preparation . in groups g1 and g3 , instrumentation was performed with a single file ( rc and wo , respectively ) in reciprocating motion , advancing into each root third in 3-mm increments until the desired length was reached , as recommended by the manufacturers . in groups g2 and g4 , instrumentation was preceded by cervical preflaring , performed using # 2 and # 3 gates - glidden drills ( dentsply - maillefer , ballaigues , switzerland ) , in descending order , and driven by a reciproc silver motor programmed appropriately for these instruments . after this step , rc and
wo instruments were used as in groups g1 and g3 . after each use , any dentin chip adhered to the instruments was removed with moist gauze dressings , and the root canals were irrigated again with 5 ml of naocl and recapitulated with a # 10 k - file to full length .
once instrumentation of each tooth was complete , the instruments were decontaminated and cleaned in an ultrasonic unit , examined under the operating microscope for potential plastic deformation , and autoclaved for later reuse .
the identification ring of each instrument was removed with a scalpel blade so as to allow reinsertion into the sirona contra - angle handpiece .
each instrument was used repeatedly until fracture occurred , at which time the instrument was identified , and the number of uses until separation was recorded .
the surfaces of all instruments were analyzed by scanning electron microscopy ( sem ) , performed with a dsm-940a system ( carl zeiss , jena , germany ) , and checked for surface changes potentially caused by irrigant exposure and for cracks or plastic deformation caused by continued instrumentation .
sem images ( magnification 100 , 250 , 500 , and 1000 ) were obtained at three time points for each instrument : before use [ figure 2 ] ; after first use [ figure 3 ] ; and after fracture [ figure 4 ] .
scanning electron microscope images of reciproc ( a ) and waveone ( b ) instruments before use surface images of waveone instruments after the first use .
note the presence of surface cracks on the waveone instrument ( arrows ) ( scanning electron microscope images magnification : a
250 ; b 500 ; c 1000 ) reciproc ( used in 12 teeth gi ) and waveone ( used in 6 teeth gii ) instruments after separation ( scanning electron microscope images magnification : 250 , 500 , 1000 ) .
note cracks inside the metal mass of reciproc ( arrow ) statistical procedures were performed with the statistical package for the social sciences ( spss 17.0 for windows , spss inc . , chicago , usa ) . because values were normally distributed across all groups , one - way analysis of variance was used for comparative purposes .
the present study was approved by the local research ethics committee ( caae : 13831213.4.0000.5052 ) . the sample comprised extracted human upper molars ( length 1822 mm ) with fully formed roots , constricted root canals , and root curvature 35. for selection purposes , teeth were identified and imaged by conventional radiography and cone beam computed tomography . only teeth in which the entire root canal system could be visualized were included in the sample for experimental group allocation .
teeth with root curvature > 35 , partially or completely calcified canals , or excessively wide canals were excluded from the study .
after application of the selection criteria , 550 teeth were selected from a set of 1153 extracted human molars .
in all selected teeth , endodontic access was established using high - speed diamond burs .
canal orifices were located with an endodontic explorer and special files under 32 magnification ( mc - m1233 operating microscope , dfv com . and ind .
ltda , valena , rj , brazil ) , and teeth were then randomly allocated across the different study groups .
the presence of a fourth canal was identified in 41.2% of the teeth used in the sample ( n = 200 ) .
forty 25-mm reciprocating instruments were used in this study : 20 reciproc r25 , tip size 25 , 0.08 variable taper ( rc ) ( vdw gmbh , munich , germany ) and 20 waveone primary , tip size 25 , 0.08 variable taper ( wo ) ( dentsply - maillefer , ballaigues , switzerland ) , all powered by a silver reciproc motor and sirona endo 6:1 contra - angle handpiece ( vdw gmbh , munich , germany ) .
rc files were driven by the rc all program and wo files by the wo all program .
four study groups ( n = 10 each ) were defined , according to root canal instrumentation procedure :
g1 : rc;g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc;g3 : wo;g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo .
g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc ; g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo .
procedures were performed by a single operator , who followed the manufacturer - issued instructions for each instrument .
after coronal access , teeth were irrigated with 5 ml sodium hypochlorite ( naocl ) 2.5% ( biodinmica qumica e farmacutica ltda , ibipor , pr , brazil ) and explored with hand files as described elsewhere .
the root canal systems of all teeth were evacuated with a # 10 k - file , which was used to identify patency .
working length determination was performed under operating microscope magnification ( mc - m1233 , dfv com .
ltda , valena , rj , brazil ) , 1 mm short of the apex . in all teeth
, glidepaths were prepared with a # 13/0.02 mm / mm pathfile ( dentsply - maillefer , ballaigues , switzerland ) , which enabled standardized canal preparation . in groups g1 and g3 , instrumentation was performed with a single file ( rc and wo , respectively ) in reciprocating motion , advancing into each root third in 3-mm increments until the desired length was reached , as recommended by the manufacturers . in groups g2 and g4 , instrumentation was preceded by cervical preflaring , performed using # 2 and # 3 gates - glidden drills ( dentsply - maillefer , ballaigues , switzerland ) , in descending order , and driven by a reciproc silver motor programmed appropriately for these instruments .
after this step , rc and wo instruments were used as in groups g1 and g3 . after each use
, any dentin chip adhered to the instruments was removed with moist gauze dressings , and the root canals were irrigated again with 5 ml of naocl and recapitulated with a # 10 k - file to full length .
once instrumentation of each tooth was complete , the instruments were decontaminated and cleaned in an ultrasonic unit , examined under the operating microscope for potential plastic deformation , and autoclaved for later reuse .
the identification ring of each instrument was removed with a scalpel blade so as to allow reinsertion into the sirona contra - angle handpiece .
each instrument was used repeatedly until fracture occurred , at which time the instrument was identified , and the number of uses until separation was recorded .
the surfaces of all instruments were analyzed by scanning electron microscopy ( sem ) , performed with a dsm-940a system ( carl zeiss , jena , germany ) , and checked for surface changes potentially caused by irrigant exposure and for cracks or plastic deformation caused by continued instrumentation .
sem images ( magnification 100 , 250 , 500 , and 1000 ) were obtained at three time points for each instrument : before use [ figure 2 ] ; after first use [ figure 3 ] ; and after fracture [ figure 4 ] .
scanning electron microscope images of reciproc ( a ) and waveone ( b ) instruments before use surface images of waveone instruments after the first use .
note the presence of surface cracks on the waveone instrument ( arrows ) ( scanning electron microscope images magnification : a
500 ; c 1000 ) reciproc ( used in 12 teeth gi ) and waveone ( used in 6 teeth gii ) instruments after separation ( scanning electron microscope images magnification : 250 , 500 , 1000 ) .
statistical procedures were performed with the statistical package for the social sciences ( spss 17.0 for windows , spss inc . , chicago , usa ) . because values were normally distributed across all groups , one - way analysis of variance was used for comparative purposes .
rc files exhibited significantly greater fracture strength than wo files ( p < 0.001 ) , under equal conditions .
comparison of groups in which the same instruments were used revealed that cervical preflaring significantly increased the fracture strength ( p < 0.01 ) of both instrument systems . on comparison between different instrument systems with and without preflaring , rc files exhibited the same fracture strength as wo files did after preflaring , providing further evidence of the superiority of the rc system over the wo system . mean and standard deviation number of teeth and root canals instrumented per instrument per study group on sem , surface cracking was only seen in wo instruments [ figure 3 ] .
it should be stressed that although these cracks were already apparent after the first instrument use , the files remained fracture resistant and could be reused at least five times before separation .
major changes in the techniques and principles of endodontics have occurred in the last 20 years .
this is demonstrated clearly by recent advances in instrumentation of root canal systems , such as the development of safer techniques , files made from more elastic and stable alloys , and a lower rate of errors during endodontic instrumentation .
the present study assessed the fracture strength and development of plastic deformations and/or surface cracks on rc r25 and wo primary reciprocating instruments , with and without cervical preflaring , by means of operating microscope and sem images .
the null hypothesis of the study was rejected as the results demonstrated that cervical preflaring had an impact on the fracture strength of the tested instruments .
the choice of instruments for this study was based on the fact that both were made from the same material ( niti m - wire alloy ) , are driven by reciprocating motion , and share similar manufacturing processes .
the method of reusing sterilized instruments on extracted human teeth simulates a real - world clinical setting , particularly in terms of the risk of instrument separation , although not all anatomical features of the pulp cavity can be standardized .
however , the large sample size , random allocation of teeth across study groups , and glidepath standardization with a # 13 pathfile prevented this variable from having any significant influence on results . negotiating the apical foramen and determining apical patency with # 15 or smaller files are essential clinical steps and are firmly enshrined in current practice as they prevent plugging and/or deviation of the root canal . hence , the inclusion of these steps in our methods brings our experiment even closer to the reality of daily clinical practice as clearance of potential interferents from the root canal system and determination of apical patency play a critical role in ensuring safety and improving the prognosis of endodontic treatment .
likewise , the use of cervical preflaring a clinical procedure widely employed in endodontic practice in groups g2 and g4 of this study further demonstrates our concern with making our experimental condition reproduce clinical aspects as closely as possible .
preflaring ensures there is a sufficient space for penetration of the mechanical instrument , prevents coronal binding , and reduces the risk of instrument separation .
in addition , it enhances the range of action of irrigating solution by enabling advancement of the irrigation needle and penetration of the ultrasonic tip during passive ultrasonic irrigation ( pui ) .
although the rc and wo instruments were conceived for single use only , these devices have a high level of embedded technology , which led us to analyze their fracture strength after repeated sterilization before each reuse .
this analysis showed that both instruments remained safe for reuse . according to the current literature
, the advent of reciprocating motion systems has made root canal shaping and cleaning safer .
in addition , the past studies suggest the possibility of using only one instrument to prepare the entire root canal with satisfactory results , particularly because a single instrument has increased fracture strength as compared to full - sequence rotary instrumentation . the influence of cross - section , alloy type , manufacturing process , and type of driving motion on the cyclic fatigue resistance of niti instruments has been the object of several recent studies .
nevertheless , how and why the design of the instrument might influence its behavior under cyclic fatigue stress remains unclear .
cheung and darvell showed , in various instruments , that fatigue resistance does not appear to be affected by instrument design , suggesting that neither the cross - section area of an instrument nor its shape is the primary determinant of fatigue life .
however , other studies have suggested that difference in cross - section appears to be an important determinant of cyclic fatigue resistance across different files . in a study conducted by plotino et al . , rc instruments exhibited significantly greater fatigue strength than wo instruments , which suggests that this difference may be related to the differences in cross - sectional area and reciprocating motion between the two systems .
earlier studies also demonstrated the superiority of rc instrumentation over the wo system in fatigue resistance testing and have shown that the angles employed in instrument motion have a direct influence on cyclic fatigue .
believe another factor that enhances resistance in rc and wo instruments is the type of alloy used in their manufacture .
the authors showed that a synergistic effect between the m - wire alloy and reciprocating motion provides a significant increase in cyclic fatigue resistance .
recently , a new continuous rotary instrument was developed by coltene / whaledent ag ( hyflex edm ) .
manufactured using the electrical discharge machining technique , this instrument has greater resistance to cyclic fatigue when compared with rc and wo . in our sample ,
the rc system was superior to the wo system regardless of cervical preflaring , which suggests that clearance of the root canal system through glidepath preparation before instrumentation is of the utmost clinical importance .
this step is recommended by the manufacturer of wo but deemed unnecessary when using the rc system .
perhaps , choosing not to prepare a glidepath could bias results significantly in favor of the rc system as demonstrated elsewhere in the literature . in a study of discarded niti files , alapati et al . found cracks along the metal body of the instruments . in our sample ,
sem analysis of the instruments after the first use revealed cracks only on the wo files .
we did not observe any plastic deformation , which may be explained by the greater resistance of the m - wire alloy as compared with other niti alloys .
it bears stressing that the back - and - forth motion recommended by manufacturers and followed during specimen preparation in this study is largely intended to reduce torsional strain and , consequently , torsion fatigue , whereas its effect on flexural stresses is probably less evident . to date ,
rc and wo have been marketed for single - file root canal instrumentation and are approved for single use only so as to prevent structural weakening as a result of cyclic fatigue .
means the same instrument may still be used to instrument three or four root canals , each of which may have complex , tortuous anatomy .
therefore , single use reduces but does not altogether eliminate the risk of cumulative fatigue damage and potential instrument separation .
hence , we conclude that cyclic fatigue testing is as valuable for reciprocating instrumentation as for rotary instruments .
it should be noted that the high level of fatigue resistance of reciprocating instruments may play an important role in improving root canal cleaning and shaping procedures as shaping can be done with remarkable speed . within this context
, the principles of endodontics are shifting away from simultaneous cleaning and shaping toward a paradigm , whereby the root canal system is first shaped to enable effective cleaning by means of enhanced irrigation procedures .
evidence suggests that enhanced apical preparation provides a greater reduction of bacterial load and dentin debris , primarily by enabling more effective irrigation , which is made easier by cervical preparation . in the present study , cervical preflaring before instrumentation played a relevant role in increasing the fatigue life of the tested instruments .
another relevant clinical factor is localization , negotiation , and determination of apical patency , especially as endodontic treatment of older adults who often present with constricted pulp chambers filled with dystrophic calcifications
therefore , canal clearance and apical debridement are considered important procedures to reduce instrument stress against the walls of the canal system and thus improve the safety of endodontic treatment . nevertheless , in a recent study by de - deus et al .
, the rc system demonstrated satisfactory fracture strength even in the absence of patent canals .
the present study sought to assess the fracture strength of rc and wo instruments after repeated sterilization and reuse cycles .
our results suggest that both the rc and wo systems can be safely reused several times , preceded by some preliminary manipulation to prevent the instrument from binding and thus reduce the risk of separation .
based on our findings , we suggest that files can be reused safely up to 3 times .
this number also facilitates control of the number of reuses of a single file . on the first use
, the instrument is taken directly from its original packaging and its identifier ring is intact . after the first reprocessing ,
the identifier ring is enlarged and must be removed before the instrument can fit into the handpiece ; this serves as a milestone for the second use of the instrument .
finally , at the time of the third use , the instrument will already be missing its identifier ring , which demonstrates it should be discarded after use . despite these conclusions , we believe that further research is needed to assess the ability of these reciprocating systems to ensure adequate canal antisepsis , as the duration of exposure to naocl irrigating solution tends to be reduced in single - file preparations , and to evaluate the influence of this clinical practice on treatment success over time .
notably , one of the instrument specimens produced dark discoloration of the irrigation solution shortly after the start of its first use .
the canal was irrigated with fresh solution and dark discoloration occurred again . upon removal of the instrument from the root canal , it was found to have been markedly corroded by the 2.5% naocl solution , with loss of material . on sem examination ,
energy dispersive x - ray analysis did not show any difference in alloy properties in relation to the other instruments used in the study [ figure 6 ] .
it bears stressing that no instances of such damage , i.e. , structural corrosion of niti instruments by naocl solution , have ever been reported in the literature .
this event was probably due to a flaw in the manufacturing process as other instruments from the same lot exhibited stable behavior .
scanning electron microscope images ( 100 ) of a reciproc r25 instrument showing corrosion and loss of metal mass energy dispersive x - ray analyses of the surface ( a ) and eroded area ( b ) of the instrument damaged by 2.5% sodium hypochlorite solution
considering the proposed objective of this study , we conclude that both rc and wo instruments exhibited good fracture strength after autoclave sterilization and reuse .
cervical preflaring significantly increased the number of times rc and wo files could be reused safely . | objective : this study sought to assess the influence of cervical preflaring and reuse after resterilization , on fracture strength and plastic deformation and/or surface cracking in reciprocating nickel titanium [ niti ] endodontic instruments after root canal instrumentation of extracted human teeth.materials and methods : forty engine - driven reciprocating niti instruments ( 20 reciproc [ rc ] , vdw ; 20 waveone [ wo ] , dentsply ) were used in root canal instrumentation of extracted human molars . a total of 485 human upper molars with similar anatomical features were selected and randomly distributed across four groups according to the instrumentation procedures performed ( g1 and g3 : rc ; g2 and g4 : wo ) .
reciprocating instruments were used as per manufacturer instructions . in groups g3 and g4 , cervical preflaring was performed prior to instrumentation .
after each use of each instrument , sterilization was repeated and scanning electron microscope ( sem ) images were obtained to check for plastic deformations and/or cracks on instrument surfaces . each instrument was used repeatedly until fracture occurred.results:regardless of flaring , rc files exhibited greater fracture strength than wo instruments ( p < 0.01 ) with and without preflaring .
cervical preflaring significantly reduced the risk of instrument fracture ( p < 0.01 ) .
no plastic deformations were observed before fracturing .
however , cracks were found on wo instruments shortly after their first use .
rc and wo instruments resisted fracturing after autoclave sterilization.conclusions:cervical preflaring allowed a significant increase in the number of times rc and wo files could be reused safely . | INTRODUCTION
MATERIALS AND METHODS
Ethical aspects and sample selection
Preparation of specimens
Division of experimental groups
Root canal preparation
Scanning electron microscopy
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest |
advances in adjuvant and neoadjuvant therapies and accurate preoperative imaging techniques have improved the prognosis of patients with sarcoma .
tumors in the diaphysis are relatively uncommon , and in some patients it may be possible to achieve adequate margins without sacrificing the adjacent articular surface .
available methods include the use of autogeneous extracorporeally irradiated bone [ 13 ] , massive allograft , distraction osteogenesis and intercalary custom - made endoprostheses [ 6 , 7 , 8 ] . previously , long - term results of intercalary endoprosthetic reconstruction are not clear , and we have used extracorporeal radiation and re - implantation in patients with primary or metastatic diaphyseal bone tumors .
custom - made diaphyseal implants allow immediate weight bearing , but the complications include loosening , wear and breakage of the implants [ 6 , 7 ] .
however , intraoperative extracorporeal autogeneous irradiated bone grafting ( iorbg ) after femoral tumor resection also sometimes fails to achieve long - term survival due to non - union or fracture of bones and breakage of implant [ 1 , 2 ] . most especially , a pathologic fracture in a femoral lesion has a marked effect on the patient 's quality of life . here ,
we present a useful salvage surgery to reconstruct femoral diaphyseal defects due to iorbg fracture with custom - made intercalary endoprostheses in two elderly patients .
in 2002 and 2005 , two patients with femoral intercalary resection of malignant tumor underwent iorbg reconstruction with intramedullary nail or plate . at 32 and 96 months after iorbg reconstruction , both patients had iorbg fractures .
we used custom - made endoprostheses in these two patients to reconstruct femoral diaphyseal bone defect after excision of failed iorbg .
the intercalary endoprosthetic system ( k - max , kyocera medical corporation , kyoto , japan ) for femoral and tibial diaphyseal bone tumors is custom made with titanium alloy .
the prosthesis took 3 weeks to manufacture , and the cemented stems were proximally and distally linked by a taper locking system with a bolt passing through parts of the prosthesis .
resection of the graft bone at the appropriate level and removal of the implant were carried out .
active physiotherapy was started on the second postoperative day , wherein , the patient was allowed to partially weight bear with gradual progression to full weight bearing by the time of discharge .
functional outcome was assessed using the musculoskeletal tumor society ( msts ) functional evaluation system for reconstructive procedures after skeletal resection .
the msts score is composed of pain , function , emotional acceptance , walking ability , gait and use of walking aids , with a higher score indicating better functional outcome .
case 1 : a 61-year - old male was treated at the age of 54 for primary soft - tissue sarcoma of the thigh which invaded the femur ( fig .
wide en bloc resection of the tumor with involved femoral diaphysis , isolation of the tumor with involved bone , extracorporeal irradiation with 50 gy as a single bolus dose to the isolated bone and reimplantation of the irradiated bone into the host with intramedullary nail were performed ( fig .
2a ) . at 74 months after primary surgery , graft bone fracture and intramedullary nail breakage occurred at the proximal diaphysis ( fig .
segmental intercalary resection , implant removal and reconstruction with custom - made intercalary prosthesis were performed ( fig .
plain radiograph ( a ) and ct ( b ) showing lytic destructive lesion in left femoral diaphyseal bone . t1- and t2-weighted magnetic resonance imaging ( c ) showing a soft tissue mass ( 20 20 12 cm ) around femoral bone .
figure 2:radiographs showing extracorporeal irradiation after wide resection and stabilization with intramedullary nail ( a ) . at 74 months after operation , radiograph showing fracture of graft bone with broken implant ( b and c ) and revision with intercalary endoprosthesis ( d ) . a 54-year - old male with primary soft tissue sarcoma .
plain radiograph ( a ) and ct ( b ) showing lytic destructive lesion in left femoral diaphyseal bone . t1- and t2-weighted magnetic resonance imaging ( c ) showing a soft tissue mass ( 20 20 12 cm ) around femoral bone .
radiographs showing extracorporeal irradiation after wide resection and stabilization with intramedullary nail ( a ) . at 74 months after operation , radiograph showing fracture of graft bone with broken implant ( b and c ) and revision with intercalary endoprosthesis ( d ) .
case 2 : a 71-year - old female was treated at the age of 68 for metastatic synovial sarcoma of the femur ( fig
wide en bloc resection of the tumor and reconstruction with the use of iorbg were performed ( fig .
distal thigh pain due to fracture of the graft and breakage of plate occurred ( fig .
. failed graft bone resection , implant removal and reconstruction with custom - made intercalary endoprosthesis were performed ( fig .
aseptic loosening occurred at 29 months after the first endoprosthetic replacement which required a revision procedure ( fig .
figure 3:a 68-year - old female with metastatic synovial sarcoma in right femoral diaphyseal bone .
radiograph ( b ) showing wide resection included femoral diaphyseal bone and reconstruction with iorbg and plate . at 32 months after surgery ,
radiograph ( c ) showing graft bone fracture ( arrow ) and reconstruction with intercalary endoprosthesis ( d ) .
aseptic loosening occurred 29 months after surgery ( e ) , and revision surgery was performed ( f ) .
radiograph ( b ) showing wide resection included femoral diaphyseal bone and reconstruction with iorbg and plate . at 32 months after surgery ,
radiograph ( c ) showing graft bone fracture ( arrow ) and reconstruction with intercalary endoprosthesis ( d ) .
aseptic loosening occurred 29 months after surgery ( e ) , and revision surgery was performed ( f ) .
there are several options for reconstruction of defects in the femoral diaphysis after resection of the tumor . however , the optional methods for reconstruction of defects are unclear .
hanna et al . reviewed 23 patients who underwent limb salvage by endoprosthetic replacement of the femoral diaphysis for primary bone tumor with a reconstruction survival of 85% at 5 years and 68% at 10 years .
biological reconstruction is believed to be more time consuming than endoprosthetic replacement with a prolonged period of immobilization after surgery and a significant risk of non - union and/or fracture .
generally , the patients with implant failure and non - union and fracture of graft bone require open reduction and internal fixation with bone grafting . for our two elderly patients with fractures of the graft bones ,
the latter were salvaged using custom - made intercalary prostheses . when the patients were subjected to reconstruction with repeat bone graft for implant and iorbg failure ,
it is difficult for elderly patients to require a lengthy period of non - weight bearing for union .
prolonged immobilization is required for graft union , and there are high rates of failure after biological reconstruction , which makes its use less favorable for these patients , especially those receiving palliative treatment .
endoprosthetic reconstruction of the femoral diaphysis allows patients to recover early weight bearing and function without a lengthy period of no weight bearing .
although both patients resumed daily life early , one patient underwent revision to an intercalary replacement at 29 months for aseptic loosening of the distal stem . as a risk of later failure and subsequent need for surgery , the indication on young patients should be considered .
custom - made intercalary endoprosthesis is a useful surgical treatment for fracture of an iorbg reconstruction in limb salvage surgery especially in elderly patients . | intraoperative extracorporeal autogeneous irradiated bone grafting ( iorbg ) after femoral tumor resection ultimately sometimes will fail in patients achieving long - term survival .
there are several alternative surgical approaches for revision of these reconstructions . in 2002 and 2005 , two patients with femoral intercalary resection of malignant tumor ( synovial sarcoma and mfh ) underwent iorbg reconstruction with intramedullary nail or plate . at 32 and 96
months after iorbg reconstruction , both patients had failed iorbg ( pathological fracture ) in the femur .
we used custom - made endoprostheses in these two patients to reconstruct femoral diaphyseal bone defect after excision of failed iorbg .
follow - up of the patients averaged 40.5 months ( range , 3942 months ) after endoprosthetic revision .
musculoskeletal tumor society scores averaged 75% ( 6683% ) . when used to salvage massive iorbg failure from fractures , intercalary endoprosthetic revision preserves limb function with minimal complications . | INTRODUCTION
CASE REPORT
DISCUSSION |
autism spectrum disorders ( asd ) are a complex group of severe neurodevelopmental disorders that affect over 1% of children in the united states
. typical symptoms of autism include impairments in social interaction , deficits in verbal and nonverbal communication , and repetitive behaviors and restricted interests .
although the exact etiology of the disorder has not been identified , a link between altered immune responses and asd was first recognized nearly 40 years ago .
neurobiological studies in asd have highlighted pathways involved in neural development , synapse plasticity , structural brain abnormalities , cognition , and behavior .
in addition , abnormalities in the cellular immune response have also been reported in children with autism ; in particular , reduced cytotoxic activity and elevated levels of selected proinflammatory cytokines produced by peripheral blood mononuclear cells , such as tumor necrosis factor ( tnf- ) and il1 , have been shown to disrupt neurodevelopment [ 3 , 4 ] .
cytokines are polypeptides that are 825 kda in size and include interleukins , chemokines , interferons , tumor necrosis factors ( tnfs ) , and growth factors .
cytokines have been shown to regulate the growth and cell proliferation of neuronal tissue and to modulate host responses to infection , injury , inflammation , and diseases of uncertain etiology .
the dynamic expression of different cytokines can modify immune system function ; for example , increased levels of interferon - gamma ( ifn- ) and il-12 can induce inflammation , whereas increased production of tgf- can negatively regulate inflammation .
a number of recent studies have demonstrated that the levels of various inflammatory cytokines differ in the blood mononuclear cells , serum , plasma , brain tissue , and cerebrospinal fluid of autistic subjects compared with normal subjects , which might impair immune capacity in the central nervous system ( cns ) and stimulate the production and activation of microglia in the brain [ 7 , 8 ] .
microglia are the unique resident immune cells of the cns ; they act as primary mediators of inflammation , participating in immune surveillance of the cns and synaptic pruning during normal neurodevelopment .
mounting evidence indicates that chronic microglial activation might also contribute to the development and progression of neurodegenerative disorders .
active microglia can induce the production of pro - inflammatory cytokines such as il-1 , il-6 , and tnf- , which are typically intended to prevent further damage to brain tissue
. however , abnormally activated microglia can sometimes be toxic to neurons and other glial cells .
microglia cell activation is a prominent feature of autism , and there is a complex interaction between microglial cells and cytokines .
wei et al . found that il-6 elevation can modulate autism - like behaviors through impairments of synapse formation , dendritic spine development , and neuronal circuit balance . in this review , we focused on several types of proinflammatory and anti - inflammatory cytokines that might affect different cell signal pathways and play a role in the pathogenic mechanism that might be responsible for autism ( table 1 ) .
interleukin-1 ( il-1 ) consists of two different proteins , il-1 and il-1 , which exert their proinflammatory effects by binding to type - i il-1 receptors ( il-1 ri ) , whereas the type ii il-1 receptor ( il-1rii ) is thought to act as a decoy receptor that does not participate in active il-1 signaling [ 9 , 10 ] .
il-1 is produced , processed , and secreted from activated immune cells and plays a major role in the initiation of local and systemic inflammatory processes .
signaling mediated by il-1ri can activate the myeloid differentiation response gene 88 ( myd88 ) and tumor necrosis factor - associated factor 6 ( traf6 ) , leading to the activation of a number of different kinases , including il-1 receptor associated kinases 1 and 4 ( irak1 , irak4 ) , transforming growth factor -activated kinase ( tak ) , and members of the mitogen activated protein kinase ( mapk ) cascade , including p38 , c - jun n - terminal kinases ( jnk ) , and extracellular signal - regulated kinases 1/2 ( erk1/2 ) . in 1991 , singh et al .
first measured the serum levels of soluble il-1 and found that the level did not differ between the subjects with autism and the control subjects .
( 2002 ) demonstrated that there is also no difference in the il-1 receptor between autism and the control subjects .
however , in vitro , jyonouchi and his group observed that stimulated peripheral blood mononuclear cells ( pbmcs ) from children with autism secrete significantly higher amounts of soluble il-1 compared with normal controls . in an animal model , maternal exposure to poly-(i : c ) or lipopolysaccharide ( lps ) can induce a significant increase of il-1 in amniotic fluid , placentas , and fetal brains .
il-1 has also been shown to play a key role in mediating severe placental damage and neurodevelopmental anomalies in offspring [ 15 , 16 ] .
il-1 showed the highest concentration levels in fetal brains and was the only cytokine that was significantly upregulated 24 h after maternal poly ( i : c ) injection , suggesting that il-1 may have a deleterious impact on central nervous system development .
however , these studies did not evaluate the social behavior of offspring , so further study is needed to determine whether there is a link between the development of autism and elevated il-1 levels .
x - linked interleukin-1 receptor accessory protein like-1 ( il1rapl1 ) is a member of the interleukin-1 receptor family and is similar to the interleukin-1 accessory proteins .
( 2008 ) first reported the function of the resulting truncated il1rapl1 protein , which is severely altered in hippocampal neurons , by measuring its effect on neurite outgrowth activity .
the mutations in il1rapl1 cause a spectrum of neurological impairments ranging from mental retardation to high - functioning autism .
pavlowsky and colleagues also reported a novel partner of il1rapl1 , psd-95 , a major scaffold protein of excitatory synapses , and showed that il1rapl1 regulates the dendritic spine number and psd-95 localization to synapses .
further investigation showed that il-1-induced activation of the jnk pathway in neurons is mediated by il1rapl1 .
this finding indicates that a novel pathophysiological mechanism underlying cognitive impairment could be associated with alterations of the jnk pathway in response to il-1 and hence lead to the mislocalization of psd-95 , which subsequently result in abnormal synaptic organization and function .
interleukin-2 ( il-2 ) is a well - known cytokine that plays an important role in multiple immunoregulatory functions related to t - cells in peripheral and cns [ 20 , 21 ] .
the il-2 receptor consists of three subunits , il-2r , ( il-2r ) , and ( il-2r ) , which can deliver biochemical signals to the cell interior .
they affect different signal pathways , such as the mapk pathway , the phosphoinositide 3-kinase ( pi3k ) pathway , and the jak - stat pathway .
il-2 in the brain is potentially produced by neurons and astrocytes and is widely distributed throughout the brain along with its receptors .
there is a large body of literature indicating that il-2 may be involved in cns development , normal brain physiology , and homeostatic repair mechanisms as well as brain dysfunction and neurodegenerative processes .
for instance , it has been shown that prolonged exposure to il-2 in cancer patients can result in cognitive dysfunction , altered behavior , and other negative neuropsychiatric side effects .
in addition , il-2 levels have been found to be elevated in the postmortem brains of patients with alzheimer 's disease compared with normal control subjects .
singh et al . found that serum concentrations of soluble il-2 were significantly higher in autistic children compared with normal controls .
in addition , they showed that stimulated peripheral blood mononuclear cells ( pbmcs ) from children with autism secrete significantly higher amounts of il-2 , whereas soluble il-2 receptor levels did not differ between autistic and control subjects .
however , more recent studies have reported that il-2 levels did not differ significantly between subjects with autism and control groups [ 25 , 26 ] .
vojdani and colleagues measured nk cell activity in 1027 blood samples from autistic children obtained from ten clinics and compared the results to those of 113 healthy controls .
in addition , they cultured lymphocytes from autistic children with low or high nk cell activity and with or without il-2 and determined that the induction of nk cell activity by il-2 was more pronounced in a subgroup with very low nk cell activity . il-6 is characterized as a 26 kda glycoprotein that can trigger cellular responses that mediate inflammation , neurogenesis , gliogenesis , cell growth , cell survival , myelination , and demyelination in the cns [ 28 , 29 ] .
the il-6 receptor protein complex consists of one nonsignaling , membrane - associated subunit ( il-6r ) and two gp130 subunits responsible for signal transduction [ 30 , 31 ] .
ligand binding of this protein complex results in the homodimerization of gp130 and activates multiple signaling mechanisms , including jak / stat , pi3k / akt , and the ras / raf / mapk pathways [ 3134 ] .
recently , the role of maternal immune activation ( mia ) has become a hot topic in autism research .
several groups have found that the developing fetus 's exposure to maternal cytokines precipitates neurological , immunological , and behavioral abnormalities in the offspring [ 35 , 36 ] .
maternal injection with il-6 alone is sufficient to cause abnormal behavior in the offspring following maternal poly-(i : c ) injection or respiratory infection .
conversely , il-6 inhibition was sufficient to attenuate the behavioral deficits caused by mia [ 35 , 37 ] . in our laboratory , we found that mice with elevated il-6 in the brain display many autistic features , including impaired cognitive abilities , learning deficits , abnormal anxiety traits , and habituations and decreased social interactions .
we also examined the development of synapses and found that il-6 elevation altered excitatory and inhibitory synaptic formations , disrupted the balance of excitatory / inhibitory synaptic transmissions , and resulted in an abnormal change in the shape , length and distributing pattern of dendritic spines .
these findings suggest that il-6 elevation in the brain could mediate autistic - like behaviors , possibly through the disruption of the neural circuitry balance and impairments of synaptic plasticity .
hsiao and patterson confirmed that mia elevates il-6 protein and mrna expression in the placenta and found that maternally derived il-6 mediates jak / stat3 pathway activation specifically in the spongiotrophoblast layer , a fetal compartment of the placenta , which results in the expression of acute - phase genes .
furthermore , hsiao and patterson also demonstrated an il-6-dependent dysregulation of the growth hormone / insulin - like growth factor ( gh - igf ) axis in mia placentas that was characterized by decreased levels of gh and igf1 mrna with corresponding decreases in placental igf1 and igf - binding protein 3 ( igfbp3 ) .
singh first examined the plasma levels of il-6 and found no significant differences between subjects with autism and controls .
however , vargas ' group demonstrated that il-6 was increased in autistic brains . in our recent studies , we also found that il-6 was significantly increased in the cerebella of autistic subjects .
the cerebellum was suggested as a main focus of neuroinflammation in autism , and the selective vulnerability of the purkinje cells may play a role in the etiopathogenesis of autism . in further studies
, we investigated how il-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an il-6 viral expression vector .
we demonstrated that il-6 overexpression in granule cells caused impairments in granule cell adhesion and migration but had little effect on the formation of dendritic spines or granule cell apoptosis .
these findings suggest that il-6 not only plays an important role in the etiology of autism , but may provide a potential biological marker that enables the early diagnosis of the disorder and earlier therapeutic intervention ( figure 1 ) . because il-6 can attenuate abnormal behavior caused by mia , it might be a potential therapy target in the future .
the chemokines are a family of small cytokines that play an important role in the development of lymphocytes , including their recruitment and trafficking to specific tissue compartments .
in addition to acting as key chemotactic factors in the immune system , chemokines and their receptors are critical for dictating the movement of leukocytes into the cns in both healthy and diseased individuals .
chemokines have been shown to regulate neuronal cell migration , proliferation , and neuronal cell differentiation and are involved in the communication between neurons and microglia [ 43 , 44 ] .
monocyte chemoattractant protein 1 ( mcp-1/ccl2 ) is known to mediate monocyte and t - cell activation and trafficking into areas of tissue injury . in the cns
, mcp-1 can modulate the recruitment of myeloid cells to injury or inflammation sites , and it is increased in brain in instances of ischemia , alzheimer 's disease , and experimental autoimmune encephalomyelitis .
different groups have measured mcp-1 levels in the brain , csf , and plasma of individuals with autism and found that mcp-1 was elevated in individuals with autism .
elevated mcp-1 production was associated with higher aberrant behavior scores and more impaired developmental and adaptive function [ 8 , 42 ] .
abdallah and colleagues examined the chemokine levels in amniotic fluid ( af ) samples from individuals diagnosed with autism and controls .
in an mia model , mcp-1 was significantly upregulated after the injection of poly-(i : c ) .
mcp-1 can enhance neuronal excitability and synaptic transmission in hippocampal neurons and can modulate neuronal physiology [ 47 , 48 ] . taken together , these findings suggest that elevated mcp-1 in the brain might be linked to microglial activation and to the recruitment of monocytes / macrophages to areas of neurodegeneration .
osteopontin ( opn ) is a 60 kda phosphoprotein that plays different roles , including initiating inflammation , cell adhesion , chemotaxis , immune responses , and protection against apoptosis , depending on its intracellular or extracellular localization .
opn is also involved in regulating chronic inflammatory and autoimmune diseases , including multiple sclerosis ( ms ) , rheumatoid arthritis , inflammatory bowel disease , systemic lupus erythematosus , and type i diabetes [ 5053 ] .
recently , studies have demonstrated that opn also plays a role in the development of neurodegenerative diseases , such as parkinson 's and alzheimer 's disease .
however , opn can trigger neuronal toxicity and death in some contexts and can function as a neuroprotectant in others .
al - ayadhi and mostafa ( 2011 ) measured serum opn levels using elisa in 42 autistic children and 42 matched healthy children .
the authors found that the autistic children had significantly higher serum opn levels compared with the healthy controls , and the autistic children 's serum opn levels had significant positive correlations with their childhood autism rating scale ( cars ) scores .
however , it is difficult to determine the exact roles of specific chemokines during neurodevelopment because of the high degree of interaction between different chemokines and their receptors . at present
however , a number of studies have reported abnormal chemokine production in the brain , csf , and/or plasma of autistic individuals , suggesting that chemokines might be involved in aberrant neuronal development that could lead to altered early brain development and function .
it is a member of a group of cytokines that stimulate the acute phase reaction .
tnf- is produced mainly by activated macrophages ( m1 ) , although it can be produced by other cell types , such as cd4 + lymphocytes and nk cells .
tnf can bind two receptors , tnf receptor type 1 ( tnf - r1 ) and tnf receptor type 2 ( tnf - r2 ) .
binding with ligand , tnf- can activate nf-b , mapk , and the apoptosis signaling pathway . in 1996
, singh demonstrated that plasma tnf- did not significantly differ between autistic subjects and normal controls .
however , jyonouchi and collaborators tested 71 autistic children aged 214 years and compared them with healthy siblings and other controls .
their study showed that pbmcs activated by lps produced higher levels of tnf- , il-1 , and/or il-6 in most autistic children ( 83.1% ) compared with the control group .
the investigators concluded that a majority of the autistic children in the group , especially those with increased tnf- , exhibited excessive or poorly regulated innate immune responses .
( 2007 ) detected elevated tnf- in the cerebrospinal fluid of autistic children . in 2009 , our lab found that tnf- was significantly increased in the brains of autistic subjects .
furthermore , ashwood and colleagues used polyhydroxyalkanoates ( pha ) and tetanus to stimulate pbmcs from autistic subjects and controls to compare group - associated cellular responses .
tnf- was specifically selected to activate the classical nf-b signaling cascade of the innate immune system and to stimulate the nuclear translocation of nf-b in primary cortical neurons .
nf-b dna binding activity was significantly increased in the peripheral blood samples of children with autism .
nf-b has also been shown to be aberrantly expressed in the orbitofrontal cortex in patients with autism , which indicates that nf-b could be part of a putative molecular cascade leading to inflammation in brain regions associated with the behavioral and clinical symptoms of autism .
however , our laboratory results show that the expression of nf-b ( p65 ) and the phosphorylation / activation of nf-b ( p65 ) at ser536 are not significantly changed in the cerebellum and cortex of both autistic subjects and btbr mice in an autism model .
these findings imply that nf-b may be involved in the abnormal inflammatory response processes suggested in autistic brain but do not play an important part .
our results suggest that tnf- might affect the progress of autism through another pathway , such as the mapk / jnk pathway .
ifn- is critical for innate and adaptive immunity against viral and intracellular bacterial infections , and it plays a role in tumor control .
cellular responses to ifn- are activated through its interaction with a heterodimeric receptor consisting of interferon gamma receptor 1 ( ifngr1 ) and interferon gamma receptor 2 ( ifngr2 ) .
aberrant ifn- expression is associated with a number of autoinflammatory and autoimmune diseases . in 1996 , singh found significantly elevated ifn- levels in the plasma of 20 autistic children compared with 20 healthy controls .
recently , our laboratory also demonstrated that the concentration of ifn- was significantly increased in the brains of autistic subjects compared with normal control subjects .
another study detected elevated serum ifn- in women who had given birth to a child who was later diagnosed with autism .
( 2004 ) demonstrated that the plasma levels of ifn- did not differ between the autistic group and control group , but they did correlate positively with plasma nitric oxide measures in the autistic group .
it is known that under normal circumstances , pregnancy shifts the maternal immune system toward a more tolerant stage , causing an overall decrease in proinflammatory cytokine trajectories in the innate and adaptive arms of the immune system and an increase in counter regulatory cytokines .
mothers of children with autism demonstrated increased levels of the inflammatory cytokine ifn- , which may indicate an atypical immune state during gestation .
furthermore , in addition to maternal abnormality of ifn- , there is also elevated ifn- in autistic children .
these findings support the hypothesis that ifn- is involved in mia and plays a role in the pathologic mechanism that may be responsible for autism .
tgf-1 is one of the most important immune regulators that can effectively control diverse aspects of the immune response .
it plays an important role in autoimmune diseases such as myasthenia gravis - related ophthalmoparesis , autoimmune diabetes , and ms [ 6770 ] . in the cns ,
tgf-1 is widely recognized as an injury - related cytokine specifically associated with astrocyte scar formation in response to brain injury . in recent years , emerging data regarding tgf-1 and its signaling molecules have suggested that , in addition to its role in brain injury , tgf-1 might be a crucial regulator of cns development . considering the importance of tgf1 in immune response and nervous system development , a number of studies have investigated whether tgf-1 alters in autistic subjects .
vargas et al . found that tgf-1 was significantly increased in certain cell line specimens from autistic subjects .
however , decreased tgf-1 levels have been observed in the plasma of autistic individuals [ 67 , 72 ] . in addition , there were significant correlations between clinical scores and tgf1 levels ; lower tgf1 levels were associated with reduced adaptive behaviors and worse behavioral symptoms .
these findings suggest that immune responses in autism may be inappropriately regulated because of reductions in tgf1 , and such immune dysregulation may predispose individuals to the development of possible autoimmune responses and/or adverse neuroimmune interactions during critical windows in development . in an animal study ,
depino and colleagues found that overexpression of tgf-1 during postnatal can lead to asd - related behaviors , such as decreased social interaction , increased self - grooming , and depression - related behaviors .
however , adult hippocampal levels of murine tgf-1 were reduced in animals that were injected with adtgf at pd14 compared with adbgal - injected mice .
in addition , the study found that asd - related behaviors correlate with the long - term downregulation of tgf-1 and il-6 expression in the dentate gyrus and with decreases in the mrna levels of the synaptic protein neuroligin 3 and the number of reelin - positive neurons in the dentate gyrus .
conversely , chronic expression of tgf-1 during adulthood leads to transient opposite effects on abnormal social behaviors : it increases social interaction and decreases repetitive behavior .
these results indicate that hippocampal tgf-1 plays an important role in the programming and modulation of social interaction , repetitive behavior , and depression - related behavior .
egf is a growth factor that stimulates cell growth , proliferation , and differentiation by binding to its receptor egfr .
egf can be detected in all regions of the developing and adult brain and in a majority of the neurons and developing astrocytes .
egf has neurotrophic effects on cultured cortical neurons and stimulates neurite outgrowth in dopaminergic neuron cultures .
transgenic mice lacking the egf receptor ( egfr ) develop neurodegenerative disease and die within the first month of life .
researchers examined the association of egf , tgf-1 , and hgf genes with autism in a trio association study using dna samples from families recruited to the autism genetic resource exchange .
their results revealed a significant haplotypic association between egf and autism , but no significant snp or haplotypic associations were observed for tgf-1 or hgf .
these results suggest a possible role of egf in the pathogenesis of autism . in 2007 , a research group measured serum levels of egf in 17 male subjects with high - functioning autism and 18 age - matched healthy male subjects .
they found that the serum egf levels in the subjects with high - functioning autism were significantly lower compared with those of the normal control subjects .
however , there were no correlations between serum egf levels and clinical variables in the subjects with autism .
by contrast , other researchers found that the serum egf levels in subjects with autism were significantly higher than those of normal control subjects .
there were also no correlations between serum egf levels and clinical variables in the subjects with autism [ 79 , 80 ] .
bdnf is a member of the nerve growth factor family ( neurotrophins ) ; it contributes to prenatal and postnatal brain development .
bdnf supports neuronal survival , maintains various neuronal activities , modulates neurotransmitter release , participates in neuronal plasticity , and mediates long - term potentiation and memory fixation .
nelson and miyazaki reported higher bdnf levels in archived samples of neonatal blood and serum obtained from autistic subjects compared with normal controls [ 83 , 84 ] .
however , in a carefully constructed large - sample study , the results demonstrated that the mean levels of bdnf were significantly lower in the serum of 0- to 9-year - old autistic children compared with age - matched healthy controls or teenagers or adults .
our study found that the concentrations of bdnf and bcl2 were reduced in the frontal cerebral cortex of autistic subjects compared with age - matched controls .
we also found that the expression and activation of akt kinase , which regulates bcl2 , are significantly decreased in the autistic brain .
the downregulation of akt may result from a decreased concentration of bdnf , which is the growth factor that modulates akt activities .
we thus reason that the downregulation of the bdnf - akt - bcl2 antiapoptotic signaling pathway in the autistic brain could be one of the underlying mechanisms responsible for the pathogenesis of autism .
granulocyte - macrophage colony - stimulating factor ( gm - csf ) is a cytokine that regulates the survival , proliferation , differentiation , and functional activity of many myeloid hemopoietic cells ; it also regulates dendritic cell and t - cell function .
ashwood and colleagues investigated adaptive cellular immune function in 66 children with a confirmed diagnosis of asd and 73 confirmed typically developing ( td ) controls aged 2 to 5 years . in this study , the in vitro stimulation of pbmc with pha and tetanus was used to compare group - associated cellular responses .
the results showed that gm - csf , tnf- , and il-13 production were significantly increased , whereas il-12p40 was decreased following pha stimulation in autism relative to td controls .
in addition , the study also found that induced cytokine production was associated with altered behaviors in autistic children : increased proinflammatory or t(h)1 cytokines were associated with greater impairments in the core features of autism and more aberrant behaviors . by contrast , the production of gm - csf and t(h)2 cytokines was associated with better cognitive and adaptive function .
our laboratory also found that gm - csf was elevated in the brains of autistic patients compared with controls .
however , a study by manzardo 's group showed that the gm - csf levels were significantly lower in the plasma of children with autism compared with unrelated siblings without autism .
gm - csf can upregulate neuronal receptors on cd34 + hematopoietic stem progenitor cells to enhance responses to neurotransmitters , stimulate neurite growth , and promote axonal regeneration [ 8991 ] .
gm - csf can also promote neuronal differentiation of adult neuronal stem cells and act as a neuronal growth factor in the brain .
by contrast , gm - csf receptors are present on microglia , astrocytes , neurons , and oligodendrocytes .
the blockade of gm - csf receptors with anti - gm - csf antibodies suppresses microglia activity .
these findings suggest that the reduction of microglia activity by gm - csf receptor blockade may have important anti - inflammatory effects and implications for therapy .
further investigation of the role of gm - csf in immune responses and neurodevelopment in autism is warranted .
the studies published to date that have explored possible cytokines abnormalities in autism have been limited by small sample sizes , study design issues , and conflicting results .
although many inconsistencies and controversies exist within these studies , the available data indicate a possible relationship between cytokine alterations and autism . however , systematic investigations of the neuroimmunological factors in autism are needed and will provide a better understanding of the underlying mechanisms responsible for the pathogenesis of autism , thus improving diagnosis and therapy . | autism is a disorder of neurobiological origin characterized by problems in communication and social skills and repetitive behavior . after more than six decades of research ,
the etiology of autism remains unknown , and no biomarkers have been proven to be characteristic of autism .
a number of studies have shown that the cytokine levels in the blood , brain , and cerebrospinal fluid ( csf ) of autistic subjects differ from that of healthy individuals ; for example , a series of studies suggests that interleukin-6 ( il-6 ) , tumor necrosis factor- ( tnf- ) , and interferon- ( ifn- ) are significantly elevated in different tissues in autistic subjects .
however , the expression of some cytokines , such as il-1 , il-2 , transforming growth factor- ( tgf- ) , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) , is controversial , and different studies have found various results in different tissues . in this review , we focused on several types of proinflammatory and anti - inflammatory cytokines that might affect different cell signal pathways and play a role in the pathophysiological mechanism of autistic spectrum disorders . | 1. Introduction
2. Cytokines and Autism
3. Conclusion |
a 33-year - old patient with good health presented to the outpatient department with the complaints of pain and salty discharge in the left upper anterior quadrant of the jaw for the past 6 years .
the left maxillary lateral incisor ( # 22 ) [ figure 1 ] had an intact crown without caries / fracture , with negative vitality testing and positive to percussion .
the mobility of tooth was grade ii ; on examination , there was a 9-mm pocket and purulent exudate from the gingival sulcus of the palatal aspect . on probing ,
a deep groove extending from the cingulum to mid - root area was noted on distopalatal aspect of the tooth [ figure 2 ] .
lateral incisor showing palatoradicular groove and its management lateral incisor showing palatoradicular groove and its management radiographic examination [ figure 3 ] revealed a radiolucent parapulpal line superimposed over the root canal and a pear - shaped radiolucency in the coronal aspect was present .
a circumscribed radiolucent area with irregular borders having crestal bone loss was found in the region of # 22 .
preoperative radiograph after oral prophylaxis , endodontic treatment [ figure 4 ] was done under rubber dam isolation .
full thickness mucoperiosteal flap was elevated ; granulation tissue was curetted in periradicular area [ figure 5 ] .
since the root apex was infected for a long duration , apicectomy was performed [ figure 6 ] .
thorough scaling and root planing were performed over the groove and the diseased granulation tissue was curetted out to leave the soft tissue more conducive to regeneration .
radicular groove was eliminated by means of radiculoplasty using round bur [ figure 7 ] .
a chemical conditioning of the groove was performed by using 10% polyacrylic acid , and glass ionomer cement type 1 was applied into the defect .
the area was kept isolated of blood and tissue fluids during the setting of the cement by using local hemostatic gelatin sponge .
patient was instructed on post - surgery precautions and maintenance protocol , which included rinsing with 0.12% solution of chlorhexidine twice a day for 5 weeks . during this period ,
sutures were removed 14 days after the surgery . at this time , complete soft tissue closure was still demonstrable . probing
six - month [ figure 11a ] and 1-year follow - up radiographs [ figure 11b ] revealed the complete disappearance of radiolucency in relation to the lateral incisor .
post - obturation radiograph elevation of mucoperiosteal flap apicectomy and root end filling radiculoplasty done with round bur platelet - rich plasma to be packed flap approximated with sling sutures follow - up periodontal probing ( 2 months ) ( a ) follow - up radiograph ( 6 months ) ( b ) follow - up radiograph ( 1 year ) a 20-year - old male patient reported with a complaint of painful , discolored left upper front tooth ( # 21 ) with deposits on it .
patient had a history of trauma to his upper front primary teeth at the age of 2 years .
on clinical examination , a horizontal groove with brown discoloration around the cervical region of # 21 was observed , resembling circular enamel hypoplasia [ figure 12a ] .
intraoral periapical radiograph showed loss of enamel around the cervical region of the tooth [ figure 12b ] with no other abnormalities .
a test cavity was prepared in # 21 and the tooth had no response , indicating non - vitality .
the treatment plan included root canal therapy [ figure 13 ] followed by jacket crown .
( a ) clinical picture showing turner 's hypoplasia ( b ) preoperative radiograph postoperative radiograph
a 33-year - old patient with good health presented to the outpatient department with the complaints of pain and salty discharge in the left upper anterior quadrant of the jaw for the past 6 years .
the left maxillary lateral incisor ( # 22 ) [ figure 1 ] had an intact crown without caries / fracture , with negative vitality testing and positive to percussion .
the mobility of tooth was grade ii ; on examination , there was a 9-mm pocket and purulent exudate from the gingival sulcus of the palatal aspect . on probing ,
a deep groove extending from the cingulum to mid - root area was noted on distopalatal aspect of the tooth [ figure 2 ] .
lateral incisor showing palatoradicular groove and its management lateral incisor showing palatoradicular groove and its management radiographic examination [ figure 3 ] revealed a radiolucent parapulpal line superimposed over the root canal and a pear - shaped radiolucency in the coronal aspect was present .
a circumscribed radiolucent area with irregular borders having crestal bone loss was found in the region of # 22 .
preoperative radiograph after oral prophylaxis , endodontic treatment [ figure 4 ] was done under rubber dam isolation .
full thickness mucoperiosteal flap was elevated ; granulation tissue was curetted in periradicular area [ figure 5 ] .
since the root apex was infected for a long duration , apicectomy was performed [ figure 6 ] .
thorough scaling and root planing were performed over the groove and the diseased granulation tissue was curetted out to leave the soft tissue more conducive to regeneration .
radicular groove was eliminated by means of radiculoplasty using round bur [ figure 7 ] .
a chemical conditioning of the groove was performed by using 10% polyacrylic acid , and glass ionomer cement type 1 was applied into the defect .
the area was kept isolated of blood and tissue fluids during the setting of the cement by using local hemostatic gelatin sponge .
patient was instructed on post - surgery precautions and maintenance protocol , which included rinsing with 0.12% solution of chlorhexidine twice a day for 5 weeks . during this period ,
sutures were removed 14 days after the surgery . at this time , complete soft tissue closure was still demonstrable . probing
six - month [ figure 11a ] and 1-year follow - up radiographs [ figure 11b ] revealed the complete disappearance of radiolucency in relation to the lateral incisor .
post - obturation radiograph elevation of mucoperiosteal flap apicectomy and root end filling radiculoplasty done with round bur platelet - rich plasma to be packed flap approximated with sling sutures follow - up periodontal probing ( 2 months ) ( a ) follow - up radiograph ( 6 months ) ( b ) follow - up radiograph ( 1 year )
a 20-year - old male patient reported with a complaint of painful , discolored left upper front tooth ( # 21 ) with deposits on it .
patient had a history of trauma to his upper front primary teeth at the age of 2 years . on clinical examination , a horizontal groove with brown discoloration around the cervical region of # 21
intraoral periapical radiograph showed loss of enamel around the cervical region of the tooth [ figure 12b ] with no other abnormalities .
a test cavity was prepared in # 21 and the tooth had no response , indicating non - vitality .
the treatment plan included root canal therapy [ figure 13 ] followed by jacket crown .
( a ) clinical picture showing turner 's hypoplasia ( b ) preoperative radiograph postoperative radiograph
presence of palatoradicular groove ( case 1 ) is considered to be an important contributing factor for the development of localized periodontitis .
it acts as a plaque trap , facilitating the development of a combined periodontic endodontic lesion .
perio lesion in the present case seems to be a primary periodontic lesion with secondary endodontic involvement .
it was because of the fact that there was no history of trauma and/or discoloration of the tooth .
though the lesion was principally periodontal , the manifestation of the pathology is diagnosed by the vitality tests .
the groove had a funnel - like shape and irregularities on the external surface that promoted the accumulation of bacterial plaque and calculus , resulting in the onset and progression of periodontal disease .
as a result of this breach in the epithelial attachment , the progression of bacterial products through dentinal tubules could secondarily compromise the pulp tissue , causing a primary periodontic / secondary endodontic lesion which necessitated both pulpal and periodontal therapy .
a favorable outcome can be achieved only with the comprehensive treatment approach that effectively manages all local factors contributing to the disease process .
the rationale behind the selected treatment plan was the following :
removal or saucerization of the radicular portion of the groove ( radiculoplasty ) to eliminate bacterial plaque , calculus , and to prevent bacterial recolonization.cleaning and sealing of the coronal portion of the groove to prevent bacterial recolonization .
removal or saucerization of the radicular portion of the groove ( radiculoplasty ) to eliminate bacterial plaque , calculus , and to prevent bacterial recolonization .
although mineral trioxide aggregate sets in the presence of moisture , it might get washed off from the transgingival defect . instead , glass ionomer cement was chosen because it does not have this limitation .
the technique of conditioning and sealing the groove with glass ionomer cement is a very conservative approach for eliminating deep palatoradicular grooves .
conditioning of the groove removes the surface debris , increases the wettability , and increases the bond strength of glass ionomer cement .
glass ionomer cement was used since it has an antibacterial effect , chemical adhesion to the tooth structure , and good sealing ability .
clinical and histological studies have reported that there is an epithelial and connective tissue adherence to glass ionomer cement during the healing process .
the same clinical observation was also found in this case . in the present case , since the groove was extended onto the root surface with substantial periodontal destruction , a flap procedure , including curettage of granulation tissue and root planing , was undertaken . in the present case
although the palatal surgical approach is difficult to access , saving the tooth is a more conservative treatment plan .
moreover , the tooth was in the esthetic zone and the papillary reconstruction would have been difficult after extraction and implant placement .
therefore , an approach for saving the tooth was chosen instead of the more complex and costlier implant procedure .
the brown discoloration occurs due to disturbances in ameloblastic layer , leading to defective matrix formation caused by traumatic injuries , but the stretched inner enamel epithelium continues to induce the differentiation of new odontoblasts , and hence the dentin formation is not affected .
severe pulpal infection of primary teeth results in excessive osteolysis of inter - radicular bone and early exposure of the succedaneous tooth before adequate root length formation .
these structurally defective teeth are not only weak but also provide favorable area for colonization of bacteria .
the hypoplastic permanent teeth are seven times more sensitive to carious attack compared to those without hypoplasia .
the hypoplastic tooth had turned non - vital without any history of trauma or any carious insult .
the tooth would have turned non - vital because of the defective enamel and open dentinal tubules which act as a nidus for bacterial entry into the pulp space , thereby leading to pulp necrosis . delayed treatment on affected deciduous and permanent teeth
the need for a careful examination and early detection of all possible developmental defects in the permanent dentition and the importance of preventive measures should be stressed for maintaining the vitality of the tooth .
the key to achieving favorable results in these types of developmental anomalies is accurate diagnosis and treatment planning.deep radicular grooves can predispose to pulp necrosis and the establishment of combined periodontic
endodontic lesions.it is advisable to detect enamel hypoplasia as early as possible to prevent the teeth from turning non - vital .
the key to achieving favorable results in these types of developmental anomalies is accurate diagnosis and treatment planning .
it is advisable to detect enamel hypoplasia as early as possible to prevent the teeth from turning non - vital . | anatomic aberrations are seen in human dentition .
the maxillary incisor region of the permanent dentition where these anatomical aberrations are commonly seen is considered an area of embryonic hazard .
aberrations affecting the internal and external morphology can at times be the cause of complex pathological conditions involving the pulpal and periodontal tissues and can pose a challenge to the clinician for the diagnosis and clinical management . detecting and treating the anomalies at an early phase is essential as it poses a threat for the loss of vitality of the concerned teeth .
the aim of this paper is to highlight the fact two different developmental anomalies of maxillary incisors , namely palatoradicular groove and turner 's hypoplasia , led to the loss of vitality of the same . | Case Reports
Case 1
Case 2
Discussion
Conclusion
Key Learning Points |
cardiac arrhythmias in patients with epilepsy represent an important area of clinical interest , particularly given their suspected contributions to sudden unexpected death in epilepsy ( sudep ) .
previous literature suggests that left hemispheric epileptogenic substrates are more often associated with ictal bradyarrhythmias and right hemispheric substrates with tachyarrhythmias .
we present two cases of potentially lethal asystole associated with seizures of right , rather than the expected left , hemispheric onset .
during video - eeg monitoring for seizure characterization , a 37-year - old man with nonlesional epilepsy was observed to have a seizure characterized by a feeling of dj vu followed by altered awareness during which bilateral tonic extension of the upper extremities occurred ( video 1 ) .
electrographically , the seizure demonstrated right temporal onset and remained lateralized to the right hemisphere as the seizure progressed .
ten seconds after electrographic onset , concurrent ekg showed a slow trending heart rate that culminated in asystole for 27 s. correspondingly , the eeg revealed diffuse and marked background attenuation during the period of asystole . upon recovery and stabilization of the heart rate
, the eeg background quickly normalized after 9 s ( fig . 1 ) .
subsequently , the patient sustained two more seizures associated with ictal asystole of similar duration . eventually , the patient underwent placement of a dual chamber pacemaker .
a 60-year - old woman presented with two seizures characterized by staring and altered awareness followed by left arm and leg shaking for 30 s. simultaneously , her ekg revealed an initial bradycardia followed by asystole lasting 15 s. she required cardiopulmonary resuscitation for both episodes .
brain mri revealed an enhancing lesion in the right hemisphere involving the frontal and temporal lobes , insula , thalamus , hypothalamus , and pons ( fig . 2 ) .
although her seizures were not corroborated by simultaneous eeg monitoring , the ictal semiology and imaging findings strongly support a right hemispheric epileptogenic zone .
during video - eeg monitoring for seizure characterization , a 37-year - old man with nonlesional epilepsy was observed to have a seizure characterized by a feeling of dj vu followed by altered awareness during which bilateral tonic extension of the upper extremities occurred ( video 1 ) .
electrographically , the seizure demonstrated right temporal onset and remained lateralized to the right hemisphere as the seizure progressed .
ten seconds after electrographic onset , concurrent ekg showed a slow trending heart rate that culminated in asystole for 27 s. correspondingly , the eeg revealed diffuse and marked background attenuation during the period of asystole . upon recovery and stabilization of the heart rate
, the eeg background quickly normalized after 9 s ( fig . 1 ) .
subsequently , the patient sustained two more seizures associated with ictal asystole of similar duration . eventually , the patient underwent placement of a dual chamber pacemaker .
a 60-year - old woman presented with two seizures characterized by staring and altered awareness followed by left arm and leg shaking for 30 s. simultaneously , her ekg revealed an initial bradycardia followed by asystole lasting 15 s. she required cardiopulmonary resuscitation for both episodes .
brain mri revealed an enhancing lesion in the right hemisphere involving the frontal and temporal lobes , insula , thalamus , hypothalamus , and pons ( fig . 2 ) .
although her seizures were not corroborated by simultaneous eeg monitoring , the ictal semiology and imaging findings strongly support a right hemispheric epileptogenic zone .
ictal cardiac rate changes commonly occur , with sinus tachycardia ( pulse > 100 beats per minute ) evident in the majority of patients .
while ictal bradycardia and asystole are documented in only about 2.1% of recorded seizures , many patients who experienced such arrhythmias had potentially fatal asystole and required subsequent permanent pacemaker .
according to the lateralization hypothesis , seizures with a left - sided focus result in bradyarrhythmias , whereas seizures with a right - sided focus result in tachyarrhythmias .
selective activation of parasympathetic or sympathetic centers via the electrical propagation of seizures may explain ictal cardiac changes . in one study , electrical stimulation of
the left insular cortex resulted in bradycardia , implying that a parasympathetic - mediated pathway was involved .
conversely , stimulation of the right insular cortex induced tachycardia , implying a right hemispheric predominance in cardiac sympathetic regulation .
in contrast to this hypothesis , the two cases of ictal asystole presented here occurred with right - sided seizure foci .
other studies have shown that autonomic alterations may be more prominent in patients with right - sided versus left - sided epileptogenic zones .
interestingly , the second case demonstrated an extensive lesion involving the thalamus and cingulate structures which have been shown to influence cardiac rhythms . furthermore , more recent studies involving 13 patients with ictal bradycardia failed to show any consistent lateralization of seizures .
in fact , 9 of the 13 patients showed bilateral electrographic activity at seizure onset . based on these previous studies as well as our present cases ,
ictal asystole may be driven by a mechanism that is more intricate than the strictly lateralized cardiomotor representation of either sympathetic or parasympathetic function .
the mechanisms underlying ictal cardiac arrhythmias remain elusive , and most patients have no underlying cardiac risk factors . frequently , ictal arrhythmias are detected incidentally during simultaneous eeg and ecg monitoring .
this diagnostic elusiveness , in the midst of a potential lethal condition , underscores the importance of clinical vigilance and the need of an enhanced diagnostic tool for early detection of this condition such that appropriate intervention may promptly ensue .
genomic biomarkers are in development , aiming to reliably detect channelopathies which may subserve dual roles in epileptogenesis as well as cardiorespiratory pathophysiology ( i.e. , neurocardiac genes ) .
there are currently no clinical guidelines pertaining to the type of epilepsy syndrome or the extent of ictal bradycardia / asystole and seizure control for which the placement of a permanent pacemaker becomes indicated . however , even in cases of apparently good seizure control , it may be intuitive to place pacemakers in patients with ictal asystole considering the gravity of a potentially lethal alternative outcome .
important investigations on the mechanisms underlying ictal cardiac arrhythmias and their clinical management are ongoing .
the following are the supplementary data related to this article.video 1cardiac asystole associated with focal seizure arising from the right temporal region .
the patient depicted in the supplementary materials provided permission for his face to be shown in the images . | ictal asystole is frequently underrecognized despite being a potentially lethal condition .
we report two cases of ictal asystole with right hemispheric onset .
these cases are unique since previous literature reports that seizures associated with bradyarrhythmias typically arise from left hemispheric foci . these cases further underscore the importance of clinical vigilance and the need of an enhanced diagnostic biomarker . | Introduction
Methods
Cases
Case1
Case2
Discussion
Conflict of interest |
myoclonus is a brief , involuntary twitching of a muscle or a group of muscles .
it has been classified into cortical , subcortical , spinal , and peripheral lesions12 ) .
although many diseases have been reported as a cause of myoclonus8 ) , only one recently reported a case of spinal myoclonus has been attributed to cervical herniated intervertebral disc ( hivd ) .
although spinal myoclonus can be difficult to manage , some drugs such as clonazepam and tetrabenazine hydrochloride may be effective5,8 ) .
many authors have tried to treat myoclonus with spinal or epidural blocks in difficult cases , which would not infrequently cause worse results .
we report a case of propriospinal myoclonus ( psm ) that might be induced by lumbar hivd in a 15-year - old female who has been successfully treated with an epidural steroid injection using a transforaminal approach .
she was suspected to have left lumbar hivd at l5-s1 based on a physical examination , however , her pain was aggravated in spite of conservative management for 3 days .
she also experienced a brief involuntary muscle twitching involving the left lower leg and trunk . a whole spine magnetic resonance imaging ( mri )
routine laboratory , hematologic studies , electrolyte study included calcium , magnesium , copper and ceruloplasmin concentration and cerebrospinal fluid study were unremarkable .
the nerve conduction test showed that the f - wave latency was prolonged and the h - reflex was normal in the left peroneal nerve , which is not a peripheral neuropathic finding .
however , intermittent brief synchronous activity was noted in the left sternocleidomastoid , rectus abdominalis , gluteus medius , and gastrocnemius muscles based on surface electro - myography , which was consistent with psm .
the myoclonus was characterized by the hip and knee joints extending 1 - 2 times per second with the lower back extended .
myoclonus was decreased to 1 - 2 times per 3 - 4 seconds in frequency after administration of clonazepam ( 0.5 mg ) , but there was no further improvement in symptoms . on physical examination , myoclonus and pain were aggravated in the straight leg raising test at approximately 50 degrees hip flexion or ankle dorsiflexion .
myoclonus was intensified during the one - leg standing and spread to the thoracic muscles .
a transforaminal epidural steroid injection was delivered with triamcinolone ( 20 mg ) and 0.3% lidocaine to the left l5 , s1 root on one occasion ( fig .
, the sciatic pain was disappeared immediately and the myoclonus was decreased to 1 - 2 times per 10 seconds in frequency .
we observed her without medication in the outpatient clinic for 6 months after the procedure .
brown and colleagues2 ) recommended that spinal myoclonus would be classified as segmental spinal myoclonus and psm in 1991 .
the affected area may include direct damage of the spinal cord or may cause abnormal changes in the function of the spinal cord .
but psm involves the following : all of the spinal cord , rather than a specific segment , complex muscles jerks ; more extensive simultaneous involvement of muscles , such as the trunk and limbs ; the pattern is usually arrhythmic ; and stimulus - sensitive with a delay due to the slow conducting propriospinal nerve fibers .
according to a psm study , diagnostic clues for psm include widespread rhythmic or arrhythmic myoclonus prominently involving the trunk muscles and sparing the cranially - innervated muscles5 ) . in our case , the myoclonic jerks were arrhythmic , starting from the left leg and extending to the trunk , ascending , and aggravated with nerve stimulation ( e.g. , straight leg raising and one - leg standing ) . furthermore , there are no myoclonic jerks in the cranial - innervated muscles .
spinal myoclonus has been associated with laminectomies , the remote effects of cancer , spinal cord injuries , post - operative pseudomeningoceles , laparotomies , thoracic sympathectomies , poliomyelitis , herpes myelitis , lumbosacral radiculopathies , spinal epidural blocks , myelopathy due to demyelination , electrical injuries , acquired immunodeficiency syndrome , multiple sclerosis and cervical spondylosis8,9 ) . in rare occasions
although various causes have been reported1,6,11,14,15 ) , there has been only one report of a cervical disc as the cause of psm resulting from disc disease .
capelle , et al.,4 ) described a patient who presented with right c6 radiculopathy due to hivd c5 - 6 and myoclonic twitches predominantly affecting the abdominal muscles but spreading to adjacent muscles which was treated with discectomy and cage augmented fusion . to the date , this report might be the first report of psm with lumbar disc disease .
a variety of mechanisms have been proposed for spinal myoclonus , including dysfunction of segmental spinal cord circuitry , deficient inhibitory glycinergic transmission in the spinal cord , subsequent release of synchronous motor neuron oscillations within segments of the cord , and vitamin b12 deficiency causing vacuolar swelling of the myelin layers in the mid - thoracic cord and resulting in degeneration of the descending pyramidal and ascending posterior column traces18 ) .
also , according to campos , et al.,3 ) there is evidence that various possible mechanisms can be involved : loss of inhibitory function of local dorsal horn interneurons , abnormal hyperactivity of local anterior horn neurons , aberrant local axons re - excitations , and loss of inhibition from supra - segmental descending pathways .
ogata , et al.,16 ) experienced that psm due to injury to the l4 nerve root during epidural anesthesia was treated by l4 root block using 1.5ml of 1% lidocaine .
he described that an injury to the left l4 nerve root possibly cause an abnormal transmission of the impulses or ectopic hyperexcitability in the nerve root , which might lead to the disturbance of the spinal inhibitory interneurons and hyperexcitability of the anterior horn cells causing myoclonus .
the authors assumed that local anesthetic agents which was administered to the spinal epidural space via a caudal or interlaminar injection may distribute more in the posterior lateral part than in the anterior part of spinal canal , in which the effect of the local anesthetic drug on inhibitory neurons might have led to a loss of inhibitory function in the spinal cord7,13 ) .
thus , a transforaminal injection has an advantage over a caudal or trans - interlamina block . in the transforaminal injection ,
the injected drugs spread more onto the ventral surface of the spinal cord and can block the lesional root at the same time ( fig .
symptoms of myoclonus have been subsided by avoiding the disturbance of the spinal inhibitory interneurons activity .
this case shows that hivd may be the one of the causes of myoclonus , and transforaminal epidural injection can be option of treatments , in which can approach the ventral surface of the spinal cord and affected root in order to avoid the disturbance of the spinal inhibitory interneurons activity .
we recommend epidural steroid injections via the transforaminal approach as a treatment option for psm . | the cause of propriospinal myoclonus ( psm ) is idiopathic .
cervical trauma , ischemic myelopathy secondary to a spinal dural arteriovenous fistula , syringomyelia , lyme neuroborreliosis , human immunodeficiency virus central nervous system infection , and cervical disc herniation can be the cause of psm , but lumbar herniated intervertebral disc ( hivd ) induced psm has not been reported .
we describe a patient who presented with psm induced by hivd and was treated with an epidural steroid injection using a transforaminal approach . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION |
early reports of an increased risk of cancer in a particular exposed group have led to the identification of several agents as human carcinogens ( 3 ) , but for many other agents the suspicion of a carcinogenic effect has not been confirmed in subsequent studies . because cancer epidemiology has great societal impact , and avoidance of false - positive reports in this field deserves particular attention , we have selected one example from environmental epidemiology and one example of a reported occupational cancer association .
obviously , considerations addressed in these examples are applicable to other areas of epidemiological research as well .
the first example is of an apparently strong positive result for a pollutant in the general environment that was initially suspected to give rise to cancer but for which such an association has not been confirmed in subsequent studies . in 1993 ,
a report of a case control study nested in a new york city based cohort ( 10 ) showed an association between breast cancer risk and serum levels of 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene ( dde ) , the major metabolite of 1,1,1,-trichloro-2,2-bis(p - chlorophenyl)ethane ( ddt ) , an organochlorine pesticide that was used extensively until the early 1970s .
the study involved 58 women who had been diagnosed with breast cancer within 6 months of entry in the prospective cohort study and 171 control subjects from the same cohort , and it reported a relative risk of 3.7 ( 95% confidence interval [ ci ] = 1.0 to 13.5 ) for the highest vs lowest 20% of the dde distribution in serum .
this study was prompted by earlier results of small ( < 50 case patients ) case control studies ( 1114 ) that had suggested a modest association between breast cancer and exposure to dde and polychlorinated biphenyls .
an editorial accompanying the report of the new york city study ( 15 ) stated that these findings may have extraordinary implications for the prevention of breast cancer and that this study should serve as a wake - up call for further urgent research . accordingly , a series of studies were subsequently launched , including a large case
control study of breast cancer in long island , ny ( 16 ) , but they failed to replicate the earlier findings . the results on serum dde level from the prospective studies , including an expanded analysis of women who had been included in the original study from new york city and a combined analysis of five prospective studies , were published between 1994 and 2001 ( 1728 ) ( table 1 ) .
we performed a cumulative meta - analysis of the initial study and seven subsequent studies published in 19942000 ( figure 1 ) .
the final pooled estimate of the relative risk of breast cancer for the highest vs lowest dde category was 0.95 ( 95% ci = 0.7 to 1.3 ) .
results of prospective studies on serum 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene level and risk of breast cancer * dde = 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene ; rr = relative risk ; ci = confidence interval ; hmo = health maintenance organization ; na = not available .
dde serum levels are either not lipid adjusted ( expressed as parts per billion [ ppb ] , equivalent to milligrams per gram serum ) or lipid adjusted ( expressed as nanograms per gram lipid ) .
mean level , control subjects : 1260 ng / g ; case patients : 1230 ng / g .
cumulative meta - analysis of cohort studies of breast cancer and serum level of 1,1-dichloro-2,2-bis(p - chlorophenyl ) ethylene ( highest vs lowest category in each study ) . estimated relative risks ( rrs )
are shown with 95% confidence intervals ( cis ) ( error bars ) by year of publication of subsequent reports .
in parentheses are references of studies included in the cumulative meta - analyses ( see table 1 for details ) .
the reason for the positive findings in the original new york city study is unknown .
the short time that elapsed between blood collection and diagnosis of breast cancer may be another explanation because organochlorine compounds are stored in fat tissue , whose metabolism may be affected by the neoplastic process itself ( 29 ) .
overinterpretation and an apparent lack of skepticism had major roles in the wide acceptance of the result .
although the relative risk estimate of breast cancer among women in the highest quintile of serum dde compared with the lowest was almost 4.0 , the width of the confidence interval and the marginal level of statistical significance should have tempered the enthusiasm for the finding .
similarly , a recently reported relative risk estimate of 5.0 ( 95% ci = 1.7 to 14.8 ) for serum levels of p , p-ddt and breast cancer risk , based on subgroup analyses in a nested case
what is of ongoing concern is that given the history of studies of this exposure disease issue , the results were still interpreted with few reservations and little if any caution .
a second example relates to the results of occupational cohort studies of lung cancer risk among workers who were exposed to acrylonitrile .
acrylonitrile is a chemical used in the manufacture of acrylic fibers , resins , and nitrile rubber , as an intermediate in the chemical industry and , in the past , as a fumigant .
the first study of lung cancer mortality among workers who were exposed to acrylonitrile in a textile factory in the united states , which was reported in 1978 ( 31 ) , showed a fourfold increased risk , based on six deaths from lung cancer .
an international agency for research on cancer ( iarc ) working group cited this study in its review of acrylonitrile in 1979 and concluded that while confirmatory evidence in experimental animals and humans is desirable , acrylonitrile should be regarded as if it were carcinogenic to humans ( 32 ) .
following the initial report , a total of 16 partially overlapping occupational cohort studies have provided results on lung cancer risk among acrylonitrile - exposed workers , including an expanded analysis of the first study , which identified two additional observed and 2.9 additional expected lung cancer deaths ( 33 ) ( table 2 ) .
results of cohort studies on lung cancer risk among workers who were exposed to acrylonitrile * n = number of observed lung cancer deaths ; rr = relative risk ; ci = confidence interval . for studies overlapping with previous reports , only the additional observed lung cancers are reported ( and the relative risk is based on these additional cancers only ) .
when we performed a cumulative meta - analysis of the initial 1978 findings and of 15 subsequent studies of acrylonitrile and lung cancer that were published in 19801998 , we found a steady decrease over time in the reported overall relative risk estimate of lung cancer in acrylonitrile workers ( figure 2 ) .
the final pooled estimate of the relative risk of lung cancer among the workers was 1.1 ( 95% ci = 0.9 to 1.4 ) .
this approach admittedly represents an overly simplified review because , for the sake of comparability , we restricted the meta - analysis to historical cohort studies and did not consider case
control studies [ eg , sclo et al . ( 48 ) ] that may provide additional relevant information , we did not consider the results of internal ( eg , dose response ) analyses available for several cohorts , and we did not consider the evidence for or against bias and confounding in each of the studies
. however , the declining trend in the summary relative risk estimate as further data accumulated provides evidence that the initial finding was a false - positive result . in an updated review of the evidence in 1999 , an iarc working group modified the classification for acrylonitrile from
cumulative meta - analysis of cohort studies of lung cancer and occupational exposure to acrylonitrile . estimated relative risks ( rrs ) are shown with 95% confidence intervals ( cis ) ( error bars ) by year of publication of subsequent reports .
in parentheses are references of studies included in the cumulative meta - analyses ( see table 2 for details ) .
similar examples of false - positive results are relevant to the evidence linking exposure to established occupational carcinogens with cancer of organs that are not the primary target of their carcinogenic effect .
a detailed analysis of results on other suspected occupational carcinogens is beyond the scope of this commentary , but for several agents it would show the same pattern seen for lung cancer among workers who were exposed to acrylonitrile , that is , early positive results were not confirmed , or were shown to be less strong , in subsequent studies .
the issue of multiplicity of exposures and outcomes , and consequent multiple comparisons and subgroup analyses leading to many statistically significant
( yet false ) findings arising by chance , is likely the major contributor to false - positive findings in epidemiology . epidemiological studies , whether cross - sectional , case control , or cohort in design , tend to obtain information on multiple exposures and disease outcomes , so the possibility often exists for examining hundreds or thousands of exposure disease combinations .
a similar situation arises in emerging genetic epidemiological studies in which even larger numbers of traits , up to tens or hundreds of thousands of variants , can be examined . in a quantitative approach to this problem ,
( 8) reviewed 36 genetic disease associations and found that for 25 of them , the first report gave a stronger estimate of genetic association than did subsequent studies ; in 10 of these associations , the difference between the first and the subsequent results was statistically significant .
the same authors subsequently considered the results of 55 meta - analyses ( 50 ) , including 579 study comparisons of genetic associations , and found that for each association the largest study generally yielded more conservative results than the meta - analysis , that in 26% of the meta - analyses the association was statistically significantly stronger in the first study , and that in only 16% was the genetic association found in the first study replicated without evidence of heterogeneity and bias .
statistical techniques aimed at reducing the likelihood of false - positive associations , such as false discovery rate ( 51,52 ) , false - positive report probability ( 53 ) , and bayesian false discovery probability ( 54 ) , are now included in the analysis of databases with large numbers of genetic variants [ eg , wellcome trust case control consortium ( 55 ) ] .
it seems appropriate that similar approaches be applied in other areas of cancer epidemiology that are prone to reporting false - positive results , including studies of potential occupational and environmental carcinogens .
there are multiple reasons besides chance why positive results , particularly initial results , in epidemiological studies may be false ( 2,56 ) . to formally address the determinants of false - positive results in occupational cancer , swaen et al .
( 57 ) scored the main characteristics of studies reporting a positive association with established target ( true positive ) and nontarget ( false positive ) organs of 19 occupational carcinogens .
the main determinants of false - positive findings were the absence of a specific a priori hypothesis , a small magnitude of the association , the absence of a dose effect relationship , and the lack of adjustment for tobacco smoking .
although this analysis can be criticized because of subjective aspects in the definition of target organs and in quality scoring , it highlights the important roles of confounding and bias , together with chance , in the generation of false - positive results .
although chance is a major cause of false - positive results , it is less appreciated that false leads may also be generated by bias .
bias consists of a systematic alteration of the research findings due to factors related to study design , data acquisition , analysis , or reporting of results .
there is a fundamental distinction between false - positive results that are generated by chance and those caused by bias
the former will rarely be replicated in subsequent investigations , whereas bias may operate in a similar fashion in different settings and populations and thus will provide a consistent pattern of independently generated results . even if only a relatively low proportion of results are generated by bias
information bias , or recall bias , is a likely source of false - positive findings .
early studies examining a possible association between induced abortion and breast cancer risk that were based on retrospectively ascertained information generated evidence indicating that women who had undergone an induced abortion were at increased risk of breast cancer ( 58 ) .
a collaborative reanalysis of data from 53 epidemiological studies comparing women with prospectively ascertained records of one or more induced abortions vs women with no such record ( 59 ) reported a relative risk of breast cancer equal to 0.93 ( 95% ci = 0.89 to 0.96 ) .
it is now apparent that information bias had a major role in generating the early false - positive results .
control women were less likely to report their previous induced abortions than were women with breast cancer ( 58,60 ) .
in general , patients have greater motivation ( eg , to explain their disease ) and thus are more likely to remember and/or report certain past exposures than are healthy individuals who are selected as control subjects .
a likely example of selection bias is the 1981 report of increased pancreatic cancer risk associated with coffee consumption in one of the earliest epidemiological studies of this disease ( 61 ) .
many investigators aimed to replicate these findings , but the results of subsequent studies were in general null and by the end of the 1980s a causal association between coffee drinking and pancreatic cancer was considered unlikely ( 62,63 ) . yet for several years the suspicion that coffee drinking was associated with a highly lethal cancer was widespread in the medical community and the general public .
this false - positive result was generated , at least in part , by exclusions from the control patient population , but not from the case patients , of individuals with a history of diseases related to cigarette smoking and alcohol consumption ; because these exposures were highly correlated with coffee consumption , the exclusions likely led to a deficit of coffee consumers in the control group .
another source of bias and false - positive findings in observational studies is confounding resulting either from incomplete statistical adjustment for measured variables or from the inability to adjust for unmeasured distorting variables .
for example , results of early epidemiological studies showed an increased risk of cervical cancer among women who were infected with agents such as herpes simplex virus 2 and chlamydia . when sensitive and specific markers of infection with human papilloma virus became available , however , it was clear that the early positive results for the other sexually transmitted agents were due to confounding by human papilloma virus ( 64 ) .
although the importance of residual confounding and unmeasured confounders as a source of bias in epidemiological studies has been downplayed by many ( 65 ) , a recent statistical simulation study ( 66 ) showed that with plausible assumptions , effect sizes on the order of 1.52.0 , which is a magnitude frequently reported in epidemiology studies , can be generated by residual and/or unmeasured confounding .
the real effect of unmeasured confounders is of course unknown but may explain some of the notorious differences between well - known cohort studies and subsequent randomized trials .
the randomized controlled trial is often presented as the gold standard of epidemiological studies , with the random assignment of treatments mitigating against the potential influences of bias and confounding .
systematic reviews have found evidence of higher relative risks in observational studies as compared with randomized controlled trials addressing the same question ( 67,68 ) . however , even randomized controlled studies are not exempt from reporting of false - positive results that arise by chance . in a review of 39
highly cited ( defined as studies cited 1000 or more times in the literature ) randomized controlled trials that reported an original claim of an effect ( 69 ) , the results of 19 were replicated by subsequent studies , whereas for nine trials subsequent results either contradicted the findings of the original report or provided evidence of a weaker effect ( the remaining 11 trials did not have subsequent attempts of replication ) .
premature conclusions can be avoided by cautious interpretation of initial trials , especially when they are small , and by trial registration , which is a requirement of the international committee of medical journal editors ( 70 ) . moreover
, there is an unfortunate tendency to highlight positive or statistically significant findings in the abstracts of both observational studies and randomized trials , even when the results are doubtful or open to criticism ( 71 ) .
another cause of false - positive reporting in cancer epidemiology is publication or reporting bias .
it originates from the tendency of authors and journal reviewers and editors to report and publish positive or statistically significant results over null or
non statistically significant results , particularly if the findings appear to confirm a previously reported association ( ie , the bandwagon effect ) . as with other forms of bias ,
statistical tests have been proposed to assess the presence of publication bias ( 7275 ) ; unfortunately , these tests tend to have low power and require a large number of independent studies to provide evidence of bias .
there are , however , suspected occupational and environmental carcinogens for which a sufficiently large number of studies are available to allow testing for publication bias .
one group of suspected environmental carcinogens is the polychlorinated dibenzo - para - dioxins , which are by - products of the manufacture of chlorophenols and chlorophenoxy herbicides ; they are also produced during incineration of organic material and in other thermal processes , such as metal processing and paper pulp bleaching .
the most toxic is 2,3,7,8-tetrachlorodibenzo - p - dioxin ( 2,3,7,8-tcdd ) , which has been found to cause tumors in rodents under certain conditions ( 62 ) .
concern about the carcinogenicity of dioxins in general , and 2,3,7,8-tcdd in particular , has led to epidemiological studies that are both occupationally based ( eg , workers in the chemical industry , pesticide applicators ) and community based ( eg , residents in contaminated areas , the general population ) .
non - hodgkin lymphoma ( nhl ) has been among the suspected targets of dioxin carcinogenicity in humans . at the time of the most recent iarc monograph evaluation ( 76 ) ,
results on nhl risk were available from 16 studies of 2,3,7,8-tcdd exposed populations eight cohort studies and eight case control studies ( 76 ) .
although a meta - analysis suggests an increased risk of nhl in these populations ( meta - relative risk = 1.6 , 95% ci = 1.2 to 2.1 ) , there is evidence of publication bias [ figure 3 ; p = .02 , begg test ( 75 ) ] . in the absence of publication bias ,
the results of individual studies plotted against their precision , as in figure 3 , should be distributed in a triangular ( funnel ) pattern , with less precise studies ( on the right side of the graph ) randomly dispersed around the mean .
publication bias is suggested by the absence of results in one of the parts of the funnel ( eg , lower right corner in figure 3 , where small null studies would be plotted ) .
formal testing of publication bias is helpful , but keeping the bandwagon effect concept in mind would go a long way to instilling skepticism when assessing the strength of evidence provided by a new report on a popular hypothesis
. funnel plot of results of studies on dioxin exposure and risk of non - hodgkin lymphoma .
the examples presented in this commentary suggest that false - positive results are a common problem in cancer and other types of epidemiological studies . what can be done within the practice of epidemiology to reduce the problem ?
one of the simplest yet potentially most effective remedies involves increasing emphasis on skepticism when assessing study results , particularly when they are new .
put another way , epidemiologists should practice some epistemological modesty when interpreting and presenting their findings .
epidemiologists by training are most often aware of the methodological limitations of observational studies , particularly those done by others , yet when it comes to practice , and especially the interpretation of their own study results , methodological vigilance is often absent .
this absence of skepticism in reporting results in published papers increases the likelihood that a positive finding will receive unwarranted attention in the media and by the public .
the tendency to emphasize and overinterpret what appear to be new findings is commonplace , perhaps in part because of a belief that the findings provide information that may ultimately improve public health .
although the results may turn out to be wrong , some authors feel it is better to err on the side of overreporting or overstating potentially false - positive results than to miss the identification of a potential new hazard or an opportunity for career advancement .
this tendency to hype new findings increases the likelihood of downplaying inconsistencies within the data or any lack of concordance with other sources of evidence .
furthermore , the clear acknowledgement that the statistically significant findings may arise from multiple comparisons or subgroup analyses is often missing ; results from a single study are often dispersed across multiple publications , sometimes without reference to each other , hindering the detection of the multiple comparisons or subgroup reporting .
strict adherence to the highest epidemiological standards in the design , analysis , reporting , and interpretation of studies would help reduce the likelihood and impact of false - positive results .
these standards include provisions to reduce the opportunity for bias and confounding in study design , adequate statistical power , avoidance ( or at least cautious interpretation ) of data - driven subgroup analyses , and accounting for multiple comparisons .
the strategy for reporting study results should be specified before the results are known , and selective reporting or emphasis of statistically significant results based on ex post facto subgroup analyses should be discouraged .
the interpretation of positive results , especially if they are not supported by additional evidence ( eg , other epidemiological or experimental studies ) , should be careful and cautious .
they are also in line with many of the recommendations in the strengthening the reporting of observational studies in epidemiology ( strobe ) statement , which is aimed at strengthening the reporting of epidemiological studies ( 77,78 ) .
we propose that the policies of some journals , which require the explicit acknowledgement of study limitations up front in the abstract or in a note box , become standard practice .
caution should be applied in the communication of results to the media and the general public , because positive findings tend to attract the media and public attention , whereas findings that do not confirm a previously reported association or do not indicate a new association often receive no attention .
consequently , false - positive results tend to survive longer and have larger long - term consequences in the general public than in the scientific community .
likewise , users of epidemiological results outside the scientific community ( eg , regulatory agencies , stakeholders , media , advocacy groups , trial lawyers , the general public ) should be aware of the fact that statistically significant or positive results are often false , in particular when they are not supported by related studies or other lines of evidence .
the examples we have presented in this commentary were restricted largely to the field of cancer epidemiology .
however , we believe that similar problems affect other areas of epidemiological research , in particular those involving studies with large sets of determinant and outcome variables
. a careful analysis of factors enhancing the likelihood of false - positive reporting should be part of the training of epidemiologists , and these issues should be addressed in the discussion section of every epidemiological paper . in general , epidemiologists should recognize the reporting of false - positive results as a major challenge to the scientific credibility of their discipline and institutionalize a greater level of epistemological modesty in this regard ( 4,79 ) . | false - positive results are inherent in the scientific process of testing hypotheses concerning the determinants of cancer and other human illnesses . although much of what is known about the etiology of human cancers has arisen from well - conducted epidemiological studies , epidemiology has been increasingly criticized for producing findings that are often sensationalized in the media and fail to be upheld in subsequent studies .
herein we describe examples from cancer epidemiology of likely false - positive findings and discuss conditions under which such results may occur .
we suggest general guidelines or principles , including the endorsement of editorial policies requiring the prominent listing of study caveats , which may help reduce the reporting of misleading results .
increased epistemological humility regarding findings in epidemiology would go a long way to diminishing the detrimental effects of false - positive results on the allocation of limited research resources , on the advancement of knowledge of the causes and prevention of cancer , and on the scientific reputation of epidemiology and would help to prevent oversimplified interpretations of results by the media and the public . | Examples of False-Positive Results in Environmental and Occupational Cancer Epidemiology
Sources of False-Positive Results
Publication Bias
Caveats and Conclusions |
budd chiari syndrome ( bcs ) is defined as obstruction of the hepatic venous outflow .
this obstruction might be located anywhere between the small hepatic veins to the suprahepatic inferior vena cava ( ivc ) .
the typical patient is a young woman presenting with abdominal pain , ascites , and hepatomegaly ; however , the clinical presentation varies .
usually patients show an underlying prothrombotic disorder , and in a substantial number of patients several risk factors are found .
the disease is mainly diagnosed by imaging , whereas therapeutic options comprise a step - wise approach with medication ( anticoagulation ) , minimally invasive procedures such as thrombolysis , percutaneous angioplasty , transjugular intrahepatic portosystemic shunting ( tips ) , but also surgical portosystemic shunting , and orthotopic liver transplantation ( ltx ) .
moreover , pregnancy in women with bcs is extremely rare ; pregnancy exerts a detrimental effect on the course of the disease .
here we report the case of a patient with bcs due to essential thrombocythemia ( et ) and tips which ended lethal within 19 months after delivery .
the university bonn medical school ethics committee allows publication without consent in such cases . additionally , we propose a score for risk classification of pregnancy in women with pre - existing bcs .
a stable course of disease could be achieved for 8 years by treatment with hydroxyurea , when acute bcs occurred with occlusion of all liver veins .
treatment consisted of lysis and veno - venous shunt ( palmaz - stent ) placement between left hepatic vein and ivc at a regional hospital ( figure 1 ) in conjunction with anticoagulant treatment using the vitamin k - antagonist ( vka ) phenprocoumon .
she remained stable for the subsequent 12 years with well - controlled hematologic disease and without thrombotic or portal hypertensive complications . at 36 years of age , progressive , recurrent ascites developed which was treated by creating a new tips using a baremetal and a covered stent .
( a ) invasive portography after left - sided tips placement demonstrating patent tips perfusion .
( b , c ) invasive tips - control demonstrating cavernous transformed portal occlusion and restored hepatopetal flow after tips elongation : ( 1 ) main portal vein ; ( 2 ) left portal vein ; ( 3 ) left hepatic vein .
this tips - tract was created from the ostium of the left liver vein , very close to the end of the palmaz - stent and the left portal vein branch .
the pressure gradient dropped > 50% ( portosystemic gradient decrease from 36 to 15 mm hg , see figure 1a ) , as usual during tips insertion . in the follow - up visits , the tips was patent and there was no need for invasive measurements until the patient conceived .
one year after tips revision , at the age of 38 , the patient conceived . during pregnancy ,
vka anticoagulation was replaced by therapeutic dose of the low - molecular - weight heparin ( lmwh ) enoxaparin with regular monitoring of anti - fxa ( axa ) activity ( peak axa - level : 1.0 1.2 u / ml ) , and hydroxyurea was discontinued due to its teratogeneity .
even though the recommendation in patients with high - risk et is to use interferon - alpha ( evidence level iii and grade b ) , we advised against it , as there are no controlled studies which show a clear benefit regarding teratogeneity , and , in particular , there is no experience in the situation of bcs .
tips patency was examined 4-weekly using doppler - ultrasound ; small amounts of ascites were first detected at a gestational age ( ga ) of 12 weeks .
first- and second - trimester ultrasound examinations including echocardiography and doppler examination of the fetal and uterine vessels were unremarkable .
g / l ) . despite axa - adjusted anticoagulation within the therapeutic range ( enoxaparin 60 mg qam , 80 mg qhs ) , thrombosis of the right subclavian vein occurred at ga 25 . therefore ,
testing for heparin - induced thrombocytopenia ( hit ) typical antibodies was performed , and medication was changed to fondaparinux ( 5 mg bd ) , as the anticoagulant and antithrombotic efficacy of fondaparinux at this dosage is superior .
a gradual deterioration of her general condition occurred ; she complained of increasing abdominal distension , pain , and dyspnea .
although tips was patent at every control , recurrent paracenteses ( overall volume 15.4 l ) had to be performed to relieve these symptoms .
serial ultrasound of the uterine cervix revealed progressive shortening , prompting tocolytic therapy with atosiban and administration of corticosteroids for acceleration of fetal pulmonary maturity at 28 weeks of gestation . at ga 28 + 2 , abnormal fetal heart rate tracings and progressive cervical dilatation necessitated cesarean delivery , which was performed under general anesthesia .
percentile , apgar 8/9/10 , arterial ph 7.34 ) and transferred to the neonatal intensive care unit .
operation and postoperative surgical course was unremarkable ; several litres of ascites were drained after abdominal incision .
the further course was uncomplicated , and the baby was discharged home 8 weeks later .
post partum , ascites , and upper abdominal pain worsened despite continuous ascites drainage and medication .
upper gastrointestinal endoscopy was therefore performed on postoperative day 4 , which revealed grade ii esophageal and grade i gastric varices .
invasive tips - control demonstrated a foreshortening of the uncovered stent at the portal segment with associated cavernous transformed portal venous thrombosis .
subsequently , a covered stent elongation to a big portal varice was performed ( portosystemic gradient decreased from 12 to 6 mm hg , see figure 1b and c ) .
thereafter , the patient was stable on treatment with hydroxyurea and fondaparinux ; the latter was switched to rivaroxaban 2 weeks postpartum .
this time a tips revision with an adequate lowering of porto - systemic pressure gradient was not expected ; therefore , a spleno - renal shunt was created in order to treat the complications of portal hypertension ( see figure 3a ) . however
, within the course of the following weeks , the patient developed several flares of et with platelets around 800 g / l despite treatment .
when renal dysfunction was present as hepatorenal syndrome , she was listed for ltx 3 months later ( initial model for end - stage liver disease ( meld)-score 28 plus special request for non - standard exception [ nse ] ) .
bone marrow transplantation to cure et was discussed ; however , the patient did not fulfill the criteria . during this time
, the liver function deteriorated , and magnetic resonance imaging revealed a > 7 cm measuring central tumor , which turned out to be a necrotic bcs - nodule ( see figure 3b ) . an organ allocation for ltx
unfortunately , the postoperative course was complicated by a primary nonfunction ( pnf ) of the first graft , and despite double retransplantation the patient succumbed 2 weeks later .
ct scan demonstrating stent in stent configuration and tips reocclusion . ct = computed tomography , tips = transjugular portosystemic shunt .
( a ) ct scan demonstrating occluded tips ( arrowhead ) , patent splenorenal shunt ( arrow ) , and cirrhotic liver disease with multiple regenerative nodules .
( b ) axial t2-weighted fat - suppressed mr image demonstrating a cirrhotic liver with siderotic nodules ( arrows ) and massive central liver fibrosis with edema ( arrowhead ) .
ct = computed tomography , mr = magnetic resonance , tips = transjugular portosystemic shunt .
with a prevalence of 1 in 100.000 to 500.000 , primary bcs is a rare disorder
. characterized by abdominal pain , hepatomegaly , and ascites , the temporal dynamics , site , and extent of obstruction determine its clinical course .
predisposing factors include , among others , inherited or acquired hypercoagulability and especially myeloproliferative diseases , as in our case .
pregnancy itself is also a risk factor for bcs ; however , usually additional risk factors are present . with a 3-year - lethality of 90%
treatment is based on expert consensus and consists of anticoagulation , interventional angioplasty , or tips stent with the aim to decompress the portal venous compartment .
in addition to the pregnancy - induced hypercoagulability , several changes inherent to pregnancy add to its detrimental effect on bcs .
these include blood volume expansion and hypoproteinemia ; the rise in intraabdominal pressure ; pressure of the gravid uterus on the ivc and other intraabdominal vessels , including the lymphatic system ; and the displacement of intraabdominal organs by the expanding uterus , causing changes in their respective anatomical relationship .
the occurrence of thrombosis despite full - dose and axa - adjusted anticoagulant treatment underscores the high thrombotic risk of et patients during pregnancy .
historical case series on bcs and pregnancy , reporting a 1-year maternal mortality of 50% are not suitable for risk assessment due to the advent of additional treatment modalities , in particular tips .
contemporary reports on pregnancy in bcs ( from 1990 onwards ) are restricted to 1 small series and 3 case reports .
even though there was no maternal mortality during pregnancy , delivery and follow - up , morbidity was considerable , including thrombosis , hemorrhage , and tips occlusion . likewise , fetal - neonatal outcome was poor , with a high rate of pregnancy losses and preterm births .
chiari syndrome we can not prove that the detrimental course of the disease could have been avoided if the patient had abstained from pregnancy .
the long time intervals with stable disease and the rapid downward trend during pregnancy and after delivery , however , point to a relevant contribution of pregnancy to the lethal course . in women with pre - existing cardiac disease , a classification has been developed , which is based on disease severity and anticipated impact of pregnancy on the underlying condition .
it allows the attending physician risk assessment , preconception counseling , and risk - adapted care during pregnancy and childbirth . in analogy , we suggest the classification of bcs into world health organization ( who ) classes i to iv as follows : class 1 : maternal mortality not increased ; no or only slight increase in maternal morbidity.class 2 : maternal mortality slightly increased ; maternal morbidity moderately increased.class 3 : maternal mortality significantly increased ; severe maternal morbidity .
interdisciplinary management before , during , and after pregnancy is mandatory.class 4 : extremely high maternal mortality , high risk of severe morbidity .
pregnancy is contraindicated , termination should be considered . in the case of continuation of pregnancy , management as for class 3 is required .
class 1 : maternal mortality not increased ; no or only slight increase in maternal morbidity .
pregnancy is contraindicated , termination should be considered . in the case of continuation of pregnancy , management as for class 3 is required .
allocation criteria may include the following : general : age ; time between diagnosis and pregnancy.history : presence and type of prothrombotic factors ; number of thrombosed veins ; complications of portal hypertension ; type and number of previous interventions.clinical findings : ascites , hepatosplenomegaly , esophageal varices ; cholangiopathy.laboratory results : liver function , kidney function , infection , coagulation.diagnostic imaging : ivc obstruction , collaterals.rotterdam , international prognostic score of thrombosis in world health organization - essential thrombocythemia ( ipset ) or other scores .
history : presence and type of prothrombotic factors ; number of thrombosed veins ; complications of portal hypertension ; type and number of previous interventions .
rotterdam , international prognostic score of thrombosis in world health organization - essential thrombocythemia ( ipset ) or other scores .
the usefulness of these and additional criteria , however , needs to be established . with numbers of women with
bcs seeking pregnancy expected to rise , the scoring system may help in preconception risk assessment and counseling ; furthermore , it may support in the establishment of treatment algorithms of bcs in pregnancy .
notwithstanding that , a favorable outcome of pregnancy in women with bcs requires an interdisciplinary team of specialists in gastroenterology , radiology , visceral surgery , hemostaseology , fetal medicine , obstetrics , and neonatology . | abstractdue to its rarity , experience with pregnancy in budd chiari syndrome ( bcs ) is limited . with the advent of new treatment modalities , transjugular
intrahepatic portosystemic shunt in particular , numbers of affected women seeking pregnancy with bcs are expected to rise . here
, we use a case that ended lethal within 2 years after delivery to discuss the effect of pregnancy on bcs and vice versa , and to highlight the necessity of a multidisciplinary teamwork .
additionally , a risk classification is proposed which may serve as a framework for preconception counseling and assist in the establishment and evaluation of treatment algorithms ; its criteria need to be defined and assessed for their applicability in further studies . | INTRODUCTION
CASE
DISCUSSION |
the unroofed coronary sinus ( cs ) syndrome is a rare entity ( 1 - 5 ) .
approximately , 75% of cases with the unroofed cs syndrome are associated with a persistent left superior vena cava ( svc ) ( 1 ) .
there is no reported case of an unroofed cs syndrome accompanied by the cs stenosis in the english literature .
the clinical presentation of the unroofed cs is generally determined by the size of the defect between the cs and the left atrium ( i.e. the degree of a left - to - right shunt ) and associated abnormalities ( 2 ) .
we present a case of the complete unroofed cs syndrome with rare associated abnormalities ( i.e. the cs stenosis and a collateral pathway through the small cardiac vein and thebesian vein , but the absence of a persistent left svc ) incidentally identified on coronary computed tomography ( ct ) angiography .
we also propose an explanation of the asymptomatic presentation in this case in spite of the large defect between the cs and the left atrium .
a 43-year - old male presented with intermittent atypical chest pain of one month 's duration .
coronary ct angiography from the tracheal bifurcation to the base of the heart ( lightspeed vct , ge healthcare , milwaukee , wi , usa ) to evaluate the underlying cause of the chest pain demonstrated normal coronary arteries .
however , a large defect ( approximately 2.8 cm in diameter ) between the cs and the left atrium and stenosis of the cs orifice were identified ( figure 1 a and 1b ) . the collateral flow from the cs to the right atrium through the small cardiac vein and thebesian vein
there was no evidence of the enlargement of any cardiac chamber , and nor was there a persistent left svc identified ( figure 1 a and 1b ) .
no abnormality was identified at transthoracic echocardiography , although the ct reading was available to the cardiologist performing echocardiography .
transesophageal echocardiography was not performed because the patient was asymptomatic except for the chest pain .
a diagnosis of the unroofed cs syndrome with the cs stenosis in the absence of a persistent left svc was made .
the unroofed cs syndrome is a rare congenital anomaly that is often associated with a persistent left svc ( 1 - 5 ) .
the classification of the unroofed cs syndrome varies in the literature ( 1 - 5 ) .
it can be classified simply complete or partial depending on the size of the defect between the cs and the left atrium .
no previous report exists of a complete unroofed cs syndrome and absence of a persistent left svc in combination with the cs stenosis leading to a collateral pathway through the small cardiac vein and thebesian vein identified on coronary ct angiography .
this unusual combination of three congenital anomalies remained asymptomatic into adulthood , when the patient presented for unrelated reasons .
the clinical presentation of the unroofed cs syndrome varies from the absence of symptoms to severe right heart failure , and is mainly determined by the size of the cs defect between the cs and the left atrium ( i.e. the degree of left - to - right shunting ) and associated anomalies such as a persistent left svc ( i.e. brain abscess or infarction caused by a right - to - left shunt ) ( 1 - 5 ) .
the present case had no related symptoms in spite of a large defect between the cs and the left atrium .
this was presumably due to the associated cs stenosis and the restriction of the flow by the formation of a collateral flow through the small cardiac and thebesian veins between the cs and the right atrium .
brain abscess or infarction may be a complication in patients with the unroofed cs syndrome due to paradoxical embolism caused by a right - to - left shunt ( 1 ) .
as there was no associated persistent left svc , the possibility of a right - to - left shunt was eliminated .
although the underlying cause of chest pain in the present case was not delineated clearly , the chest pain did not seem to be directly related to the unroofed cs syndrome as it had developed recently without accompanying symptoms . in this instance , multiple detector computed tomography ( mdct ) with its high spatial resolution provided not only precise anatomic detail of the unroofed cs syndrome and the associated anomalies , but also a possible explanation for the patient s clinical presentation . in conclusion , the presence of the cs stenosis and the absence of a persistent left svc in a patient with an unroofed cs syndrome may result in an asymptomatic presentation , even in the setting of a large cs defect . | we describe a patient with an asymptomatic complete unroofed coronary sinus ( cs ) syndrome associated with the cs stenosis in the absence of a persistent left superior vena cava ( svc ) as identified on coronary computed tomography angiography .
there was a large defect between the cs and the left atrium ( i.e. a large left - to - right shunt ) , but an unusual combination of the absence of a persistent left svc ( i.e. no risk for brain abscess due to the absence of a right - to - left shunt ) and the cs stenosis ( i.e. a markedly reduced degree of a left - to - right shunt ) , resulting in an asymptomatic presentation . | 1. Introduction
2. Case Presentation
3. Discussion |
mr . a.l . is a 27-year old male , who was originally diagnosed at the age of 6 with tourette s syndrome manifested by vocal tics , presented to his family physician with a 4-month history of hoarseness .
is treated for his tourette s syndrome using haloperidol 1 mg twice daily , clonazepam as needed and sertraline . on physical examination , he was found to have a hoarse voice and vocal tics .
direct flexible nasopharyngoscopy revealed a polypoid lesion originating from the free edge of the anterior left true vocal fold ( figure 1 ) .
this lesion was amenable to transoral endoscopic microsurgical resection using a carbon dioxide ( co2 ) laser , and after a discussion with the patient , he agreed to proceed with resection .
was also asked to completed the university of michigan voice - related quality of life ( v - rqol ) questionnaire which he scored 24 overall and considered his voice quality as poor .
no dysplasia or malignancy was identified and the lesion was diagnosed as a vocal cord polyp .
two weeks after endoscopic co2 resection of the left vocal fold lesion , repeat laryngoscopic examination revealed complete resolution of the lesion with no evidence of scarring or web .
repeated the v - rqol questionnaire post - operatively and scored 11 overall and rated his voice quality as very good .
the relationship between vocal trauma , smoking , and gastroesophageal reflux disease and vocal cord polyps is well documented.1 other rare cases of recurrent vocal cord polyps have been documented in relation to autoimmune diseases such as systemic lupus erythematosus and hashimoto s thyroiditis.2 while there is substantial evidence for vocal cord trauma , a review of literature failed to demonstrate previous documentation of a relationship between vocal tics or tourette syndrome and vocal fold lesions.410 the etiology , pathophysiology and clinical manifestations of vocal polyps and nodules are well understood .
traditionally , polyps are associated with intermittent intense vocal abuse and smoking while vocal nodules are thought to arise from chronic minor vocal cord abuse . in the case of mr .
, he has no history of using his voice professionally or episodes of intense trauma .
the cause of this patient s laryngeal lesion then is likely secondary to the history of chronic minor vocal cord abuse suffered from vocal tics in his tourette syndrome . in this way
, we also demonstrate that the minor laryngeal trauma has led to the development of a polyp rather than bilateral nodules .
as it has been shown previously , it can be difficult for a pathologist to distinguish polyps and nodules.11,12 as previously stated , we had treated his hoarseness by excising the causative lesion . on the other hand , should this situation recur in this patient given the continuing vocal tics , definitive treatment for the vocal tics should be pursued . unfortunately , unlike other incidences of vocal trauma , voice coaching would not be beneficial .
more recently , vincent et al.13 has shown the advantages of using botulinum toxin in the treatment of laryngeal tics . despite the possible confounding history of cigarette smoke exposure
moreover , the possibility of vocal cord polyps should not be isolated to those patients who use their voice professionally . with the evidence from this case report , further study into the prevalence of vocal cord lesions in patients with tourette syndrome manifested by vocal tics may be warranted . | hoarseness and dysphonia are often a result of vocal cord polyps which in turn , are linked to vocal trauma .
we report the case of vocal polyps in the setting of a 27-year old male with a history only remarkable for tourette syndrome .
we review the literature regarding etiology and pathophysiology of vocal cord lesions and propose vocal tics in tourette syndrome as an under - recognized etiology . in this way
, we also review therapies that may aid in treating not only the vocal cord lesions but also particularly in the setting of vocal tics . | Case
Discussion |
in june 2003 , a 50-year - old woman presented with a palpable mass in the subareolar region of her right breast with erythematous skin changes . she underwent mammography and sonography .
a 2.5-cm diameter tumor with an irregular shape and high density on mammography was identified in the right breast , and a nonpalpable 1.2 cm irregular isoechogenic mass with an angular margin ( fig .
a core biopsy revealed that the mass in the right breast was an infiltrating ductal carcinoma , and the mass in the left breast was an intraductal papilloma .
the patient subsequently underwent five cycles of neoadjuvant chemotherapy with the adriamycin and taxol regimen followed by a right modified radical mastectomy and a right axillary node dissection in november 2003 ( tnm staging : t2n3am0 ) .
the patient underwent , an additional , three cycles of adjuvant chemotherapy with the taxol regimen and radiation therapy to the right chest wall region .
at the six month follow - up sonography of the left breast showed architectural distortion and a mixed echogenic mass 1.5 cm in diameter at the inner portion of the left breast , with an internal anechoic cystic portion suggestive of fat necrosis ( fig .
the patient underwent two additional ultrasounds during a 12-month period ; the mixed echogenic mass appeared to be shrinking from 15 mm to 10 mm in diameter ( fig .
a mammogram along with a second follow - up ultrasound was unremarkable . on the fourth follow - up ultrasound , performed in october 2005
, the superficial portion of the mass showed a change in the appearance of the mass , which now had an angular margin with penetrating vascularity on doppler imaging ( figs .
coned magnification views , of the left subareolar region , showed a 4 mm mass in the area corresponding to the sonographic abnormality ( fig .
a lumpectomy confirmed the focal presence of infiltrating ductal carcinoma ( maximal dimension : 4 mm ) , associated with marked fibrosis and foreign body reaction including multinuclear giant cells ( fig .
freund et al . proposed several criteria for identification of breast cancer originating from surgical scars ( 7 ) : 1 ) performance of a prior surgery in the affected breast area for an unrelated condition ; 2 ) integrity of the breast prior to surgery ; 3 ) complete healing of the surgical wound in the breast ;
4 ) correspondence between the site of the tumor and the site of the surgical scar ; and 5 ) reasonable time interval between surgery and appearance of the tumor .
our case fulfilled all five of these criteria . the subsequent tumor site corresponded exactly to the site of the surgical scar .
we requested the pathologist to review again the previous specimen obtained from the excisional biopsy of the original mass from the left breast .
the pathologist obtained additional levels from the original block , but nevertheless , again reported that these histopathological findings revealed only an intraductal papilloma .
freund et al . did not define a specific time interval between surgery and the appearance of a newly developed cancer ; the time interval in the freund report ranged from one to 30 years between surgery and the appearance of breast cancer . in our case ,
we suggest that a two year interval is sufficient time interval for the tumor to be regarded as a newly developed lesion . in the literature we reviewed ,
the twelve cases in which breast cancer developed at the site of old surgical scars , six cases had undergone breast biopsies , three drainage of a breast abscess and the remaining three had undergone thoracotomy that involved the breast area ( 7 ) .
however , none of these case reports demonstrated the sequential changes on imaging as the scar progressed to breast cancer . in our case
, we were able to capture serial morphologic changes and observe the appearance of a breast cancer as it developed from a surgical scar .
the thin and delicate scar tissue is also more friable and susceptible to minor trauma ( 8) .
in addition , scar tissue undergoes a repeated repair processes , during which dysplasia may arise ( 1 , 9 ) .
moreover , because there are no lymphatic channels in scar tissue , a tumor may grow in isolation , unrestricted by immunologic resistance ( 9 , 10 ) .
thus , scar tissue may not only initiate malignant transformation , but also may provide an immunologically vulnerable site for growth .
other factors have also been suggested to be important including the implantation of living cells into the dermis as well as local environmental changes and trauma accompanied by potential carcinogenic agents ( 11 ) . both scar tissue and minor trauma appear to play a major role in malignant transformation , with a possible synergetic effect in this group of patients . in our case , the patient also had an increased risk for breast cancer due to a history of breast cancer in the contralateral breast and a previous surgical biopsy in the ipsilateral breast , which revealed a papilloma .
the combination of these factors appears to have contributed to the development of breast cancer .
the fat necrosis - like lesions may have promoted a repeated repair process with inflammatory changes that included infiltrates of histiocytes and lymphocytes ( 12 ) .
the imaging findings of this case were subtle but the mass that had been shrinking developed an angular margin in one region .
these findings , along with the penetrating vascularity revealed on doppler , prompted us to proceed to a biopsy of the lesion .
it was of critical importance to compare the size and the shape of the lesion with previous ultrasounds in order to exclude malignancy in any iatrogenically altered lesion .
based on this case , we conclude that whenever a subtle change is detected , at a surgical site , a biopsy should be performed . | breast cancer developing from a surgical scar is rare ; this type of malignancy has been reported in only 12 cases to date .
herein , we report on a 52-year - old female who developed infiltrating ductal carcinoma in a surgical scar following excision of a benign mass .
two years previously , the patient underwent surgery and radiotherapy for invasive ductal carcinoma of the contralateral breast .
the initial appearance of the scar was similar to fat necrosis ; it was observed to be progressively shrinking on follow - up sonography .
on the two year follow - up ultrasound , the appearance changed , an angular margin and vascularity at the periphery of the scar were noted .
a biopsy and subsequent excision of the scar were performed ; the diagnosis of infiltrating ductal carcinoma of the scar was confirmed . | CASE REPORT
DISCUSSION |
neurotrophins were the first extracellular signals identified as regulators of somatosensory neuron development nerve growth factor ( ngf ) , a founding member of the neurotrophin family , was discovered over 60 years ago for its role in maintaining sensory neuron survival .
numerous in vitro and in vivo studies have shown that neurotrophin ( nt ) signaling is not only essential for the survival of sensory neurons , but also required in many other processes , such as neuronal differentiation and axon outgrowth .
the discovery of programmed cell death pathways provided an opportunity to separate the survival effects of nts from their other functions . in mice with a null mutation in bax ,
a proapoptotic member of the bcl-2 family , naturally occurring neuronal death was eliminated in peripheral ganglia but gross development of the nervous system appeared normal . combining bax knockout with knockout of ngf or its receptor ,
tropomyosin - related kinase a ( trka ) , allowed ngf / trka - dependent drg neurons to survive .
the central projections of these axons extended collateral branches into the dorsal horn of the spinal cord but their axon branches in the dermis and epidermis of the hindlimb were absent .
axon counts performed in the saphenous nerve suggested that peripheral axons either never entered the major cutaneous nerve branches or could not be maintained .
these experiments demonstrated that , for at least a subset of trka - dependent neurons , nt signaling is required at early steps of peripheral axon outgrowth .
not all nts and nt receptors play analogous roles in the early outgrowth of their respective subtypes , but all regulate aspects of axon morphogenesis . once a peripheral axon begins extending from a sensory neuron cell body , it must choose its initial outgrowth trajectory , a particular challenge for these pseudo - unipolar neurons since central and peripheral axons project in different directions , implying that the two axons respond to different cues .
a study of rb neuron axon outgrowth in zebrafish larvae demonstrated that central and peripheral axons respond differently to the guidance cue semaphorin 3d ( sema3d ) .
sema3d is expressed in the roof plate of the spinal cord , between the two rows of rb cell bodies . in sema3d - deficient embryos , fewer peripheral projections exited the spinal cord , suggesting that they might normally be propelled toward the periphery by repulsive semaphorin cues .
live in vivo imaging showed that peripheral , but not central , growth cones were repelled by ectopic sema3d .
conversely , in transient axonal glycoprotein-1 ( tag-1 ) knockdown embryos , central axons were defasciculated and apparently shorter , but peripheral axons were normal . live imaging revealed that the overall advance of central , but not peripheral , growth cones was slower after tag-1 knockdown .
together these experiments indicated that axon guidance of central and peripheral axons can be specified by differential activation of receptors on these neurites .
peripheral sensory axons often travel toward the periphery alongside motor axons before branching from the nerve trunks and approaching the skin . how are sensory peripheral axons guided to skin rather than muscle ? in vitro assays and embryological experiments suggest that cues in the developing skin attract them .
for example , axons from xenopus drg neurons projected toward skin explants in vitro , a phenomenon that appeared to be independent of nts . to test whether the skin regulates sensory axon guidance in vivo , martin and colleagues ablated patches of chick dorsal wing ectoderm with uv irradiation .
this treatment eliminated the cutaneous nerve plexus and its branches that should target the damaged field .
this finding supported the idea that a long - range signal from the ectoderm triggers divergence of cutaneous nerve branches from the deep mixed nerve .
since irradiation of ectoderm also damages underlying dermal tissue , honig and colleagues used a surgical approach to remove patches of ectoderm from the chick hindlimb at various stages and assessed the consequences on peripheral nerve guidance .
these experiments showed that cutaneous nerves failed to form when ectoderm was removed at specific developmental stages .
together these studies suggest that the skin produces a long - range attractant for sensory axons that has yet to be identified .
studies in trigeminal neurons provided another example for how a target - derived attractant might guide axons to the skin , while also contributing to the formation of specific patterns of innervation .
trigeminal neurons segregate into three branches ( ophthalmic , maxillary , and mandibular ) that project to distinct regions of the face .
it has long been known from co - culture experiments that explanted maxillary or mandibular tissues can stimulate the directed outgrowth of trigeminal sensory axons , implying the existence of a target - derived attractant , termed maxillary factor .
more than a decade later , the nts brain - derived neurotrophic factor ( bdnf ) and neurotrophin-3 ( nt-3 ) were identified as the molecular components of maxillary factor in co - culture experiments . however , these factors are expressed by both the target epithelium and the pathway mesenchyme of the maxillary and mandibular processes , arguing that nt-3 and bdnf may act as short - range signals instead of directional cues to instruct initial axon migration into the maxillary process . moreover ,
this finding hinted that multiple , redundant cues likely work together to guide axons , but left open the question of whether a long - range target - derived signal guides sensory axons to the skin .
recent studies of zebrafish rb neurons identified another signaling system that regulates sensory axon guidance to the skin . simultaneously knocking down two members of the leukocyte common antigen - related ( lar ) family of receptor tyrosine phosphatases in rb neurons , or inhibiting their function with dominant negative proteins , disrupted skin innervation by peripheral sensory axons .
time - lapse imaging indicated that peripheral axon guidance , rather than outgrowth or maintenance , was defective in lar deficient neurons .
the identification of lar receptor tyrosine phosphatases as axonal receptors required for peripheral guidance raised the possibility that heparan sulfate proteoglycans ( hspgs ) , which guide axons in other systems via activation of lar family members , might be involved in skin innervation .
indeed , peripheral axons were misrouted in dackel mutants , which are defective in hspg production .
additionally , axons avoided hspg - depleted areas created locally by injecting the enzyme heparinase iii .
together these results support a model in which skin - produced hspgs are attractive ligands for lar receptors on rb neurons . since the expression of lar receptors in somatosensory neurons is conserved , it is possible that they are also involved in innervation of the embryonic skin in other vertebrate animals .
rb peripheral axons navigate a short distance from the cell body to the skin , and hspgs can be membrane - bound or secreted , so it is not clear whether contact - dependent or diffusible hspgs activate lar guidance receptor proteins on peripheral growth cones . identifying the specific hspg core proteins that serve as attractants would help answer this question .
not all skin is the same : once in the periphery , some sensory neurons preferentially innervate specific regions of the skin .
this distinction is most obvious for regions of the periphery innervated by axons that grow in stereotyped patterns , like the three branches of the trigeminal , and regions of skin that are innervated by different classes of neurons , such as hairy and glabrous skin in mice .
regions of skin can also differ in the quantity , rather than the quality , of innervation .
for example , there is a striking difference in the density of sensory fiber innervation between the hand and digit tips of humans , a pattern that correlates with differential sensitivity to mechanical and painful stimuli .
first , long - range or local cues attract or repel growth cones , thus steering sensory axons toward specific regions of the periphery .
second , factors that regulate the degree of axon branching in the skin influence the density of terminals , as well as territorial patterning , since axons that branch more have larger receptive territories .
guidance and branching cues thus together determine the characteristics of sensory innervation in specific regions of skin .
ngf was one of the first extrinsic factors found to stimulate branching of sensory axons .
ngf is expressed in many areas of the skin at early stages of development , when drg and trigeminal sensory neurons are extending axons , and its expression persists into adulthood . in vitro studies
for example , ngf - coated beads triggered directed collateral sprouting from nearby axons of cultured embryonic chick drg neurons . studies of regeneration and collateral sprouting of cutaneous sensory nerves in rats provided early in vivo evidence for ngf s role in regulating terminal sensory axon branching . diamond and colleagues
isolated the receptive field of individual sensory nerves ( emanating from a particular drg ) innervating the dorsal skin of the rat by removing surrounding nerves .
the nociceptive components of isolated nerves frequently expanded their sensory fields by sprouting collaterals into the neighboring , denervated skin .
interestingly , regeneration of isolated sensory nerves following nerve crush was unaffected by blocking ngf signaling , indicating that ngf is essential for stimulating collateral sprouting of sensory axons but not for their guidance to the skin during regeneration .
mouse genetic studies have demonstrated that ngf promotes branching not only after injury , but also during development .
for example , overexpressing ngf in mouse skin during development promoted increased innervation of the mouse mystacial pad , presumably due to more axon sprouting , as increased survival of trigeminal neurons alone could not account for the excess innervation in animals overexpressing ngf .
conversely , as described above , patel and colleagues observed reduced dermal and epidermal sensory innervation in the distal hindlimbs of ngf / bax and trka / bax double knockout mice . however , there were also dramatically fewer axons in the saphenous nerve of double knockout mice , suggesting that reduced sensory innervation may be attributable to fewer axons reaching the skin , as opposed to a deficit in collateral branching at the axon terminals .
a more recent study found that a majority of sensory axons were able to coarse into the limb buds of embryonic ngf / bax double mutant mice , but failed to innervate and branch normally within the target territory .
similar to nts , slit / robo signaling also promotes axon branching in the periphery .
the slit family of secreted proteins has been extensively characterized as repulsive signals for growing axons , most notably commissural interneurons in the developing spinal cords of mammals and ventral nerve cords of flies , but slit proteins appear to also positively regulate somatosensory axon branching .
this function was first demonstrated by a series of biochemical purifications that isolated the n - terminal fragment of slit2 for its collateral branch - promoting activity in dissociated rat drg neuron cultures . in vivo experiments in embryonic zebrafish supported a role for slit in promoting axon branching : global overexpression of slit2 increased the branching and elongation of peripheral axons from trigeminal and rb neurons .
surprisingly , plexina4 , commonly known for its role as a semaphorin receptor , was required for the branch - promoting activity of slit2 in zebrafish sensory neurons .
this function of slit appears to be conserved in mammals , since in slit2/slit3 or robo1/robo2 double mutant mice branching of trigeminal axons surrounding the eye was reduced .
this branching defect was limited to the ophthalmic branch of the trigeminal nerve that innervates skin just above the eye , while peripheral innervation patterns of the maxillary and mandibular branches , as well as drg axons , appeared largely normal .
this finding illustrates the principle that different peripheral targets produce unique molecular signals to stimulate innervation by appropriate sensory fibers .
perhaps a clearer example of a signal that promotes innervation of specific peripheral targets is provided by the neurotrophin bdnf , which is required for innervation of xenopus cement glands and mouse mammary glands . in xenopus tadpoles
, the cement gland expresses bdnf and receives mechanosensory innervation from the mandibular branch of the trigeminal nerve .
mandibular axons projected aberrantly following cement gland ablation and failed to innervate transplanted cement glands with reduced bdnf expression .
moreover , swapping out the cement gland for ectoderm overexpressing bdnf stimulated mandibular fibers to target and arborize within the ectopic tissue .
in contrast to its apparent guidance role in cement gland innervation , bdnf was recently shown to influence sensory innervation of mouse mammary glands by regulating axon survival .
the sensory innervation of mammary glands is sexually dimorphic ; while both male and female mammary glands receive innervation during embryonic development , at later stages it is rapidly lost in male embryos , prior to mammary gland regression .
androgen receptor signaling triggers this sensory pruning , since treatment of male embryos with androgen receptor antagonists preserved sensory innervation , and treatment of female embryos with testosterone caused loss of innervation , similar to that seen in males .
this process was bdnf - dependent , as knocking out bdnf or its receptor trkb similarly reduced sensory innervation in female embryos .
the mechanism underlying this process of sensory denervation requires androgen - dependent overexpression of a truncated form of trkb ( lacking the intracellular tyrosine kinase domain ) in the mammary gland mesenchyme .
this truncated receptor presumably sequesters extracellular bdnf , thereby preventing signaling that maintains sensory axons . indeed , knocking out expression of this truncated form of trkb significantly increased sensory fiber density in male embryonic mammary glands .
molecular signals can also selectively stimulate particular subtypes of somatosensory axons to innervate appropriate targets .
one example of this phenomenon comes from studies of the neurturin ( nrtn ) protein in the development of mouse nociceptive neurons .
nrtn is a member of the glia cell line - derived neurotrophic factor ( gdnf ) family of ligands and binds specifically to a signaling complex composed of the common gdnf receptor tyrosine kinase ret and the coreceptor gdnf family receptor 2 ( gfr2 ) .
nociception is mediated by peptidergic and nonpeptidergic unmyelinated c - fibers that terminate as free nerve endings .
expression of gfr2 is restricted primarily to a subpopulation of nonpeptigeric c - fiber neurons and its ligand nrtn is expressed in the epidermis beginning at embryonic stages .
knocking out gfr2 dramatically reduced the density of nonpeptidergic free nerve endings innervating the footpad but had no effect on peptidergic nerve endings in the same area .
importantly , this effect was not due to decreased survival or axon outgrowth , since the number of neurons in mutant drgs , as well as unmyelinated axons in the saphenous nerve , did not change .
these results indicate that nrtn signaling through the gfr2 receptor complex is important for stimulating terminal innervation by nonpeptidergic nociceptive neurons . further supporting this idea ,
nrtn overexpression in the skin caused a corresponding increase in the density of nonpeptidergic , but not peptidergic , free nerve endings in the epidermis of the mouse footpad .
overexpression of nrtn in the skin also increased expression of sensory ion channels and made animals more sensitive to mechanical pressure , cooling and menthol exposure , demonstrating that peripheral cues can contribute to specifying the functional properties of specific somatosensory neurons .
in addition to positive factors , repulsive cues restricting axon guidance and branching in certain regions of the periphery also contribute to creating patterns of somatosensory innervation .
semaphorins are the most extensively characterized negative regulators of sensory axon development and are perhaps best known for their role as repellents during axon guidance .
seminal experiments characterizing sema1a ( previously known as fasciclin iv ) function in the development of ti1 sensory neuron axons in the grasshopper limb bud showed that semaphorins also regulate branching . blocking sema1a signaling with monoclonal antibodies caused not only axon guidance defects , but also induced ectopic axonal branching of ti1 axons . this role for semaphorins in branching
mutant mice lacking functional sema3a displayed increased branching of peripheral axons from both trigeminal ganglia and drgs .
additional genetic studies demonstrated that sema3a - mediated negative regulation of axon branching requires neuropilin and plexin co - receptors located on growing peripheral axons . knocking out the gene encoding neuropilin-1 , or mutating its semaphorin binding domain , eliminated sema3a - mediated axon repulsion of drg neurons in culture and increased peripheral branching in vivo , similar to sema3a mutants .
in addition to limiting branching , regionally expressed repulsive cues contribute to differential patterning of innervation territories by funneling axons into particular regions of the periphery .
for example , the repulsive semaphorins sema3a and sema3f are expressed in specific patterns in the face . knocking down both of their receptors , plexina3 and a4 , caused the three trigeminal branches to defasciculate and become severely disorganized .
the ophthalmic branch was the most affected , misprojecting in multiple directions and invading regions from which it is normally excluded . at e12.5 ,
heavily branched ophthalmic axons in the double mutant covered the entire face , including the eyes , demonstrating that repulsive cues pattern sensory territories by excluding innervation from certain regions of the skin .
partitioning the skin into discrete sensory receptive fields is critical for animals to accurately detect and localize stimuli along the surface of the body .
each sensory neuron projecting to the periphery must coordinate the location of its peripheral projection with neighboring terminals to achieve an orderly arrangement of sensory fields .
this may be an easier task for those axons that innervate discrete structures in the dermis that are already spaced in an organized manner , such as hair follicles , merkel cells , and various corpuscles , but poses a challenge to axons that invade the epidermis and terminate as free endings . during embryonic stages interactions between growing neurites
appear to play a role in arranging the territories of free endings with respect to one another .
studies in developing frog and fish embryos demonstrated that axon arbors of trigeminal neurons segregate from one another to form a tiled arrangement , promoting comprehensive innervation of the target territory while minimizing redundant innervation by neighboring arbors . in both systems , ablating the trigeminal ganglion on one side of the head allowed sensory axons from the contralateral ganglion to cross the midline , presumably due to removal of contralateral neighbors that compete for innervation territory . although time - lapse imaging in zebrafish suggested that contact - dependent repulsion between growing axons is the main mechanism limiting overlap between neighboring arbors , competition for a limiting positive factor ( such as an nt ) may also contribute to tiling .
the collateral sprouting and expansion of receptive fields observed by diamond and colleagues following peripheral nerve isolation in rats suggests that somatosensory innervation in mammals is also governed by competitive innervation between somatosensory axons in the skin and requires ngf , at least as a permissive factor .
intriguingly , one study of human patients who received trigeminal sensory root section to treat trigeminal neuralgia effectively eliminating sensory innervation to one side of the face observed similar expansion of mechanosensory and nociceptive receptive fields across the facial midline .
this receptive field expansion was presumably due to collateral sprouting of intact sensory arbors from the contralateral side of the face .
together these studies indicate that tiling is a conserved strategy for arranging sensory territories of free nerve endings .
studies of peripheral sensory axon guidance during the past 20 years have collectively identified the key navigational challenges encountered by many peripheral somatosensory axons for example , whether to grow toward the periphery or the central nervous system , when to exit from nerve bundles and grow toward the skin , and where and how much to branch in particular regions of the skin . although there are surely guidance cues still to be discovered , these studies have identified many of the major players , some of which ( most notably nts and semaphorins ) function at multiple stages of axon pathfinding and morphogenesis .
many of these cues affect particular populations of somatosensory axons , but are not limited to a single subtype .
one of the major challenges for the future will be to identify the cues that make the axon morphologies of each kind of somatosensory neuron subtype different from one another .
somatosensory neurons are a diverse group of cells , reflecting the heterogeneity of the chemical , thermal , and mechanical stimuli that they sense .
differential responsiveness to some of the guidance cues discussed in this review helps explain how different populations of sensory neurons adopt distinct trajectories , but responses to those cues alone are unlikely to generate the impressive diversity of somatosensory neuron morphologies .
for example , some free nerve endings , which are often referred to as unspecialized , form intimate structural associations with epidermal cells and can display distinctive , subtype - specific termination patterns at particular strata of the epidermis . the axon endings innervating dermal corpuscles and hair follicles are perhaps even more striking , forming unique , stereotyped terminals onto their targets .
the intricate association between axons and these sensory organs suggests that corpuscles and hair follicles provide instructive molecular cues that sculpt terminal axon morphologies , but virtually nothing is known about the nature of those cues .
the recent creation of genetic tools for visualizing specific classes of axon terminals will make it possible to study their development and identify the molecular interactions that allow them to adopt their elegant morphologies . | the peripheral axons of vertebrate tactile somatosensory neurons travel long distances from ganglia just outside the central nervous system to the skin . once in the skin these axons form elaborate terminals
whose organization must be regionally patterned to detect and accurately localize different kinds of touch stimuli .
this review describes key studies that identified choice points for somatosensory axon growth cones and the extrinsic molecular cues that function at each of those steps .
while much has been learned in the past 20 years about the guidance of these axons , there is still much to be learned about how the peripheral axons of different kinds of somatosensory neurons adopt different trajectories and form specific terminal structures . | Initiation of Outgrowth
Guidance to the Skin
Positive Cues Contribute to Branching and Patterning in the Skin
Negative Cues Contribute to Branching and Patterning at the Target
Tiling of Axon Terminals in the Skin
Diversity in Somatosensory Axon Morphogenesis |
the aim of this study is to describe the use of electroconvulsive therapy ( ect ) in the treatment of methamphetamine - induced withdrawal delirium and craving in a single case .
a 44-year - old male presented to the hospital in fars province , iran , with methamphetamine - induced withdrawal delirium who responded to ect .
the electroconvulsive therapy can be a suitable option for the treatment of methamphetamine withdrawal delirium and craving .
also , it can be usefully employed in these very serious conditions which may represent a risk to life .
we describe a patient with methamphetamine - induced delirium and craving which responded to electroconvulsive therapy ( ect ) .
methamphetamine abuse was a minor problem in iran ( 1 , 2 ) until the last couple of years .
recently , there has been a marked increase in the use of methamphetamine , especially among the young , with an increase in methamphetamine related psychiatric presentations to hospitals .
formerly , methamphetamine was illegally smuggled in from the abroad , but it is now synthesized in iran in underground laboratories .
the methamphetamine synthesized in iran is of higher potency and is commonly associated with psychosis .
a 44-year - old male employee presented to the hospital in fars province , iran , with decreased level of consciousness .
the patient had no personal or family history of medical problems , and no history of head trauma .
the patient began occasional smoking of opium 14 years prior to admission , which increased to daily smoking for the decade leading up to admission .
he had a distant history of occasional use of alcohol ; however , he stopped using it eight years prior to admission .
moreover , the patient had smoked heroine for six years prior to admission ; however , there was no history of cannabis or cocaine use in the past .
eight months prior to admission , the patient commenced smoking methamphetamine , twice weekly , which then increased to daily use in the two months leading up to admission . from the time he commenced daily smoking of methamphetamine , the patient developed persecutory delusions , believing he was being followed by an agent from .
he also experienced persecutory delusions regarding his neighbors and had a visual hallucination of a man on his roof .
in short , when he was brought to hospital by his wife , he had been smoking heroine for six years and methamphetamine daily for two months .
drug screening was positive for methamphetamine and morphine ( consistent with opium and heroin use ) and negative for cannabis , ecstasy , methadone , buprenorphine , benzodiazepine and alcohol .
the patient had taken methamphetamine on the morning of admission . by that evening his level of consciousness was reduced and he was drowsy .
he was given buprenorphine 2 mg sublingually twice daily , to reduce opioid withdrawal , and closely monitored .
on the second day of admission his drowsiness had increased and it was difficult to arouse him to answer questions .
his condition was further deteriorated and could not speak or take food . on the fourth day of admission ,
the patient was considered to be in mortal danger and he was given emergency ect using a thymatron tm ( system iv , company ind , usa ) .
the electrodes were applied bilaterally , he was administered two seizures during the same anesthetic multiple monitored ect .
fourteen hours later he was alert and orientated , although still with some psychotic thinking and hallucinations .
five hours later the patient was alert and orientated in time , place and person , and free of psychotic features .
single ect was administered on 9th , 11th and 13th days and he was discharged on the 14th day .
he received further single ect on the 16th and 18th days ( as an out - patient ) and remained well . within a week or so
, however , he again began to smoke amphetamine and was soon psychotic as before , with hallucinations and delusions .
multiple monitored ect was again administered , and some hours later , he showed partial improvement .
he was administered double ect on two further occasions over the next 5 days and was discharged free of psychiatric symptoms .
this case illustrates that ect may be beneficial in treating methamphetamine - induced psychosis and methamphetamine withdrawal delirium . its use in these conditions
, a systematic prospective trial of ect in delirium is yet to be published , and this report is an important addition to the literature .
it is of interest that mmect continues to be used with good effect in leading centers in iran . | introduction : the aim of this study is to describe the use of electroconvulsive therapy ( ect ) in the treatment of methamphetamine - induced withdrawal delirium and craving in a single case.case presentation : a 44-year - old male presented to the hospital in fars province , iran , with methamphetamine - induced withdrawal delirium who responded to ect.conclusions:the electroconvulsive therapy can be a suitable option for the treatment of methamphetamine withdrawal delirium and craving .
also , it can be usefully employed in these very serious conditions which may represent a risk to life . | Introduction:
Case Presentation:
Conclusions:
1. Introduction
2. Case Presentation
3. Discussion |
a 32-year - old korean woman found that she had visual loss in her right eye when she woke up in the morning and was referred to our clinic for acute crao 16 hours after symptom occurrence .
eight years prior , she had presented with itching and tender violaceous erythematous non - elevated patches with central necrotic vesicle on the dorsum of both the lower legs and feet ( fig .
1a and 1b ) the lesions were improved by colchicines 0.6 mg / day , hydroxyzine , and methylprednisolone 4 to 8 mg / day .
1c ) . a segmental limb pressure test with a bidirectional doppler test showed normal blood pressures in all four extremities
she was intermittently treated with pentoxifylline and mupirocin ointment for skin lesions for three years .
she was in good medical condition except for the skin lesions and had no experience of smoking .
she had one son and no history of spontaneous abortion . at presentation , her visual acuity was hand motion in the right eye and 20 / 20 in the left eye .
fundus examination of the right eye showed whitish edematous retina , a cherry - red spot , and narrowing and segmentation of retinal arteries suggesting acute crao ( fig .
2a ) laboratory test results , including rheumatological tests such as anti - nuclear antibody ; anti - ro and anti - la antibodies ; anti - neutrophil cytoplasmic antibody ; anti - dna antibodies ; rheumatoid factor ; cryoglobulin ; antithrombin activity ; protein c and s activity ; d - dimer ; anti - beta2-glycoprotein ; anticardiolipin antibody ; lupus anticoagulant ; prothrombin gene mutation ; factor v leiden gene mutation ; and homocysteine , were all within normal limits except for slightly elevated c - reactive protein ( 0.62 mg / dl ) and fibrinogen ( 425 mg / dl ) .
the serology tests were positive for hepatitis b antigen while other serology tests were normal .
she consented to receive intra - arterial thrombolysis after being informed of the possible risks and benefits of the intervention .
cerebral angiography showed severe focal vasospasm of the right internal carotid artery as soon as the catheter was placed ( fig .
3a ) , which was subsequently relieved by continuous intra - arterial infusion of nimodipine 5 mg .
selective angiography of the origin of the right ophthalmic artery showed no definite thrombus or steno - occlusive lesion within the ophthalmic artery ( fig .
the ophthalmic artery was infused with a fibrinolytic agent ( 500,000 units of urokinase ) until the maximal dose of fibrinolytics was reached as based on our protocol , but there was no visual improvement during the procedure .
seven hours after intra - arterial thrombolysis , retinal arterial perfusion on fundus fluorescein angiography ( ffa ) did not improve ( arm to retina time , 29 seconds ; arteriovenous transit time : about 4 minutes ) ( fig .
ffa three days after intra - arterial thrombolysis showed nearly complete restoration of retinal arterial perfusion ( arm to retina time , 17 seconds ; arteriovenous transit time , about 30 seconds ) with the exception of the macular area ( no reflow phenomenon ) .
six weeks after thrombolysis , her visual acuity remained hand motion and fundus photography showed severe retinal atrophy in the macula and disc pallor ( fig . 2c ) .
the patient in our case was in the extremely low risk group for crao in that she was very young and without any known risk factors such as diabetes , hypertension , renal disease , ischemic heart disease , cerebrovascular accidents , or smoking . according to a population - based study , crao occurs more often in older persons , with a mean age of 61.9 , but is relatively rare in young patients .
approximately 75% of cases of retinal arterial obstruction ( rao ) in patients over the age of 40 have findings suggestive of emboli originating from the carotid arteries
. however , young patients with rao rarely have artheromatous vascular diseases , but rather have more diverse etiologic factors . according to studies on rao in young patients ,
one or more systemic or ocular findings were identified in 85% and 91% of patients under the ages of 30 and 40 , respectively .
therefore , it is reasonable to conclude that lv was associated with the pathogenic mechanism of crao in our case , as lv is known to cause occlusion of the dermal vessels by intravascular fibrin , segmental hyalinization , and endothelial proliferation . to date , the pathogenesis of lv has not been fully elucidated .
lv has been reported to be associated with hypercoagulable disorders and/or autoimmune diseases , such as hyperhomocysteinemia , activated protein c resistance , factor v leiden mutation , elevated fibrinopeptide a levels , anticardiolipin antibodies , or defective release of tissue plasminogen activator [ 15 - 19 ] . to our knowledge , there have been no crao cases reported to be associated with lv . as crao
can be appropriately considered as a form of cerebrovascular lesion , our case may also be considered a variant of sneddon 's syndrome , which is characterized by generalized livedo reticularis and cerebrovascular lesion
. there have been 5 case reports of crao in sneddon 's syndrome , of which 3 were male and 2 were female , with an average age of 34.6 years ( range , 18 to 50 years ) [ 5 - 9 ] . among them , antiphospholipid antibody was tested in 4 cases , out of which 3 were positive and 1 was negative [ 5 - 9 ] .
antiphospholipid antibodies have been found in up to 80% of individuals with sneddon 's syndrome .
although there were no differences in clinical manifestations between the antibody - positive and negative groups , only the antiphospholipid antibody - positive patients could fulfill the diagnostic criteria for antiphospholipid antibody syndrome .
therefore , our case is the second case of crao in sneddon 's syndrome not associated with antiphospholipid antibodies . in comparison
with previously reported retinal artery occlusion in patients with sneddon 's syndrome , our case is distinguishable by the presence of skin ulceration and the lack of history of cerebrovascular accidents [ 5 - 8 ] as well as the absence of antiphospholipid antibodies . in conclusion , acute crao can occur in association with lv in middle - aged female patients and may be an atypical manifestation of sneddon 's syndrome . | livedoid vasculopathy ( lv ) is characterized by a long history of ulceration of the feet and legs and histopathology indicating a thrombotic process .
we report a case of acute central retinal artery occlusion in a 32-year - old woman who had lv .
she showed no discernible laboratory abnormalities such as antiphospholipid antibodies and no history of cerebrovascular accidents .
attempted intra - arterial thrombolysis showed no effect in restoring retinal arterial perfusion or vision . the central retinal artery occlusion accompanied by lv in this case
could be regarded as a variant form of sneddon 's syndrome , which is characterized by livedo reticularis and cerebrovascular accidents . | Case Report
Discussion |
thyroid gland disease in children are in second place by frequency among all endocrine disorders ( 1 ) . when interpreting the results of the assessment of thyroid function should be taken into account the significant differences in the concentrations of tsh , thyroid hormones , thyroid a binding protein and calcitonin between children of different ages .
also , different content of iodine in the diet has an impact on the results of hormone tests and the results of radio - nucleotide tests of the thyroid gland .
measurement of serum tsh is the most sensitive test for the detection of primary hypothyroidism .
significantly better indicator of thyroid function are the concentrations of free t4 and t3 in relation to their total concentration in the blood . in cases of suspected thyrotoxicosis ,
measuring serum t3 is the most sensitive test for hypersecretion ( or exogenous input ) of thyroid hormone .
confirmation of primary hyperthyroidism today is based on the determination of very low ( previously undetectable ) concentration of tsh levels ( < 0.1 mj
is used the j fixation . within the false hyperthyroidism , caused by exogenous intake of thyroid hormones ,
congenital hypothyroidism ( ch ) includes all conditions in which exist insufficient function of the thyroid gland , regardless of etiology , and is clinically or laboratory detected at birth .
the incidence of ch of 1:1,500 is comparable to reports in other jurisdictions ( 1,2 ) .
hypothyroidism in the newborn period is almost always overlooked and delayed diagnosis leads to the most severe outcome , mental retardation ( emphasizing the importance of neonatal screening ) ( 3 ) .
the development of the central nervous system during the first trimester of pregnancy up to age of 18 - 24 months depends on the presence of normal amounts of thyroid hormones .
given the fact that the early introduction of substitution therapy is essential for the achievement of normal psycho motor development , it needs to start immediately after confirming the diagnosis , or finding high levels of tsh in a repeated sample of blood .
thyroid hormone levels change markedly during childhood , and that adult reference intervals are not universally applicable to children ( 4 ) .
the treatment of choice is oral administration of l - thyroxine . for infant s dose
is 10 - 15 g / kg or at least 37.5 g / day , but the recommended dose is 50 g / day .
the levels of t4 and tsh levels should be regularly monitored and maintained in the upper half of the normal range for the chronological age of the patient .
the only known side effects of sodium - l - thyroxine are determined by an overdose of the drug .
monitoring the rate of growth and bone maturation in older children is an excellent indicator of the therapy adequacy .
to present the age and sex structure of the patients diagnosed with hypothyroidism , evaluate diagnostic methods for making diagnosis , evaluation of etiology of hypothyroidism , with special review of the therapeutic modality .
the study has a retrospective character , and includes all patients who have the diagnosis of hypothyroidism treated at the endocrinology counseling center of the pediatric clinic , university medical centre in sarajevo , and at the moment of data collection were aged 0 - 18 years .
patients were in relation to age divided into three groups ( 0 - 1 months , 1 month to 10 years , and over 10 years of age ) .
the diagnosis of patients was based on physical findings , hormonal findings of the thyroid gland ( ria method ) , with ultrasound findings ( linear probe of 7.5 mhz ) .
distribution of patients on the basis of gender , revealed more significant representation of female patients ( 65.93% ) ( figure 1 ) .
there were no significant differences in the appearance of the disease in relation to patient s age ( p>0.05 ) .
gender and age distribution of patients physical examination of the struma was not detected in the majority of cases ( 74 ; 81.32% , p<0.05 ) among patient suffering from hypothyroidism ( figure 2 ) .
thireomegaly confirmation by physical examination average value of hormones in time when is diagnosis confirmed the maximum deviation from the average baseline hormone levels shows tsh level , while t3 is within the reference values , and t4 values are slightly lower .
the average tsh value at the time of diagnosis was significantly higher compared to the reference value for all age groups except that the largest deviation was noticed in the age group of 0 - 1 months .
ultrasound examination was used in 44 ( 48.35% ) cases , and among patients examined with ultrasound in 22 ( 50% ) cases the struma was confirmed .
ultrasound findings in most cases 14 ( 31.81% ) demonstrated diffuse struma and hashimoto thyroiditis together , followed by normal results in 11 ( 25% ) cases , hypoplastic thyroid gland in 7 ( 15.90% ) cases , the very diffuse struma in 6 ( 13.63% ) of cases , while the nob - like and cystic glands and the hashimoto thyroiditis is registered in 2 ( 4.54% ) cases , the parenchyma was not visualized in 2 ( 4.54% ) cases , of which one ectopia glands and the second state after total thyroidectomy due to ca of the gland . in relation to the etiology of hypothyroidism most patients belong to a group where hypothyroidism is associated with other diseases and conditions ( 27 ; 29.67% ) , and the least with the congenital hypothyroidism with 18 ( 19.78% ) of cases .
most patients with congenital hypothyroidism was female ( 11 ; 61.11% ) , while the overall average age at diagnosis was 12 days .
most patients with hashimoto thyroiditis are female ( 19 ; 90.47% ) , while the overall average age at diagnosis was 11.33 years .
most patients with ordinary hypothyroidism was female ( 16 ; 64.00% ) , while the overall average age at diagnosis was 7.32 years .
distribution of hypothyroidism in relation to sex is approximately equal what is to be expected given that this is a hypothyroidism related with other diseases and conditions , and the average age at diagnosis was 7.21 years .
the average dose of l - thyroxine in the age of 0 - 1 months was 50 g , 1 month10 years and 37.5 g , and the group of patients over 10 years , 65 g .
epidemiological studies suggest a higher incidence of thyroid disease and hypothyroidism especially in female population up to six times .
our data correlate with the general conclusion on the higher incidence of hypothyroidism in girls .
congenital hypothyroidism ( ch ) occurs in approximately 1:2,000 to 1:4,000 newborns ( 6 ) .
prior to the onset of newborn screening programs , the incidence was in the range of 1;7,000 to 1:10,000 ( 6,7 ) . before the diagnostic process is necessary to take high - quality medical history of the patient ( from perinatal age , including gestational and prenatal history ) . at the time of diagnosis
nearly 40% of patients were aged up to 10 years and the same number at age over 10 years .
the incidence of hypothyroidism in our community is twice as high in the pre - teen and children aged over 10 years compared to congenital hypothyroidism .
the assessment of the existence of struma was performed by physical examination which reliably according to the literature is around 60% even for an experienced doctor examiners .
data on the incidence of struma testifies to the fact that hypothyroidism in most of the patients did not last long and the struma has not developed .
the existence of struma without hormone deficiency may be the expression of the status of iodine deficiency in the region .
tsh mostly deviates from the upper reference value and if we take into account the literature data on desirable variability during therapy of tsh from 0.1 to 5.5 miu / l , then it is six times more than the upper limit of the reference value .
tsh is a long - term indicator of thyroid function or is slow to respond to the change of the target hormone and is of interest to observe the dynamics in the period of 4 - 6 months .
the value of t3 hormone is within the reference value , despite extremely high tsh and t4 just below the lower limit value of euthyroid state .
it is obvious that young body puts great effort to synthesize in this time the maximum desirable amount of t3 and t4 , and markedly elevated tsh indicates hypothyroidism durability .
miu / l , which undoubtedly confirm this diagnosis and suggest an urgent initiation of therapy .
this finding of tsh is three times higher than tsh 20 representing by itself evidence of congenital hypothyroidism , and four times higher than the level of tsh 15 in which the newborn is immediately call for retesting after the screening on congenital hypothyroidism
. ultrasound examination of the thyroid gland is used in the process of diagnosis in about half of patients .
this is a useful diagnostic element to display the volume and morphology of the thyroid gland but because of limited opportunities due to prompt admission and review as part of the initial diagnostic is not always performed . a special benefit for the monitoring of these patients would have a portable ( hand held ) ultrasound device and machine that would produce imaging and
continuously monitor the evolution of morphology in relation to the functional status of the thyroid gland .
the frequency of the ultrasound struma in examined patients of about 50% indicates failure of clinical palpation as thyroid volume estimation method , because of that it is an indication for the administration ultrasound in case of suspected struma or the existence of the node in the thyroid gland .
most patients were with diffuse struma and morphological substrate that on ultrasound looks like hashimoto thyroiditis , which is consistent with literature data .
normal ultrasound and both volumetric and morphological finding is found in as many as 25% of the examined patients .
often children with gracile neck or with protruding thyroid cartilage larynx appears magnified as ultrasound refute findings although clinical and laboratory there is verified hypothyroidism . finding hypoplastic thyroid gland is not rare and corresponds to a later stage of hashimoto thyroiditis or congenital hypoplasia of the thyroid .
it is interesting that in two children ultrasound obtained findings did not visualize the thyroid gland and in the case of congenital hypothyroidism and thyroidectomy in children with thyroid cancer .
the incidence of nodes and cyst formation in ultrasound examination of the thyroid gland is not large and it is only 4.54% ( 2 patients ) , which indirectly speaks about fortunately a small number of potential thyroid cancer , which generally always evolve from a cold thyroid nodule .
a quarter of our patients have the so - called ordinary hypothyroidism without elevated antibodies .
this form of thyroid insufficiency begins on average in prepubertal age ( the average age of diagnosis 7.21 years ) and is more common in girls . as a separate entity
, we pointed out the occurrence of hypothyroidism with other diseases and conditions existing at the time of diagnosis .
it occurs with type 1 diabetes mellitus , slowed growth , down syndrome , vitamin d - resistant rickets , panhypopituitrity and other chronic diseases and conditions .
patients with congenital hypothyroidism are usually introduced with higher initial dose of the drug in order to minimize the already possibly resulting consequences of reduced thyroid function .
our therapy is completely in accordance with these rules and is in the first month was 50 g per dose .
as child grows the dose of the drug decreases , and an average of 4 mg / kg of body weight , a further increment of body weight dose is relatively smaller calculated on kg of bw .
the american academy of pediatrics recommends : at two and four weeks after the initiation of l - thyroxine treatment ; every 1 - 2 months during the first 6 months of life ; every 3 - 4 months between 6 months and three years of age ; every 6 - 12 months thereafter until growth is complete ; four weeks after any change in dose ( more frequently if results are abnormal or non - compliance is suspected ) ( 9 ) .
hypothyroidism is the second most common endocrine disease in children s age ( after type 1 dm ) , with a predominance of females .
congenital hypothyroidism has about one - fifth of patients . physical examination revealed in about one fifth of patients the struma of the thyroid gland ; tsh levels is critical for the diagnosis and correction of therapy in pediatric patients with hypothyroidism .
congenital hypothyroidism is diagnosed on average at the age of 12 days , which is optimal period for therapeutic response ; substitution treatment is carried out by l - thyroxine which have relatively reduced doses from neonatal age onwards ; future research should be directed to the assessment of iodine deficiency in the region in which our patients lives , as an important etiological factor for the development of struma and thyroid gland pathology . | introduction : thyroid gland diseases in children are in second place by frequency among all endocrine disorders . when interpreting the results of the thyroid function assessment it should be taken into account the significant differences in the concentrations of tsh , thyroid hormones , thyroid binding proteins and calcitonin among children of different ages.goal:to present the age and sex structure of the patients diagnosed with hypothyroidism , evaluate diagnostic methods for making diagnosis , evaluation of etiology of hypothyroidism , with special review of the therapeutic modality.patients and methods : the study have retrospective character and includes all patients who have the diagnosis of hypothyroidism , but at the moment of data collection were aged 0 - 18 years.results:distribution of patients on the basis of gender , revealed more significant representation of female ( 65.93% ) , and without significant difference in the presence of the disease in relation to age ( p>0.05 ) .
physical examination of the struma was not verified in the majority of cases ( 74 ; 81.32% , p<0.05 ) suffering from hypothyroidism . ultrasound review in 22 ( 50% ) cases confirmed the struma .
ultrasound findings in most cases 14 ( 31.81% ) demonstrated diffuse struma and hashimoto thyroiditis together . in relation to the etiology of hypothyroidism most patients belong to a group where hypothyroidism is associated with other diseases and conditions ( 27 ; 29.67% ) , but the least with congenital hypothyroidism with 18 ( 19.78% ) cases .
the average dose of l - thyroxine in the age of 0 - 1 months was 50 mg , 1 month10 years 37.5 mcg , and the group of patients over 10 years 65 mcg.conclusion:congenital hypothyroidism has about one - fifth of patients .
physical examination revealed in about one fifth of patients the struma of the thyroid gland ; tsh levels is critical for the diagnosis and correction of therapy in pediatric patients with hypothyroidism .
congenital hypothyroidism is diagnosed on average at the age of 12 days , which is optimal for period for therapeutic response ; substitution treatment is carried out with l - thyroxine which is relatively reduced in doses from neonatal age onwards . | 1. INTRODUCTION
2. GOAL
3. PATIENTS AND METHODS
4. RESULTS
5. DISCUSSION
6. CONCLUSION |
antimicrobial pressure , prescribing practices , and cultural factors , such as drug availability and cost , produce significant regional susceptibility differences among certain bacterial pathogens [ 16 ] .
individual countries first - line treatment options differ and may not be appropriate due to variable resistance patterns in local environments .
country - specific surveillance data are available in most regions , but can be confounding based on the variation of results obtained from different investigations and differences in the susceptibility breakpoints applied in these studies . regardless ,
surveillance studies provide useful information to primary care physicians who need to prescribe rational empiric therapy .
community - acquired urinary tract infections ( ca - uti ) and upper respiratory tract infections ( ca - urti ) are the leading causes of outpatient infections and the most empirically treated infections worldwide [ 1 , 6 , 7 ] . increased resistance to antibiotics has complicated the management of both of these outpatient infections . the spread of enterobacteriaceae that carry a chromosomally mediated ampc -lactamase , an extended - spectrum -lactamase ( esbl ) , or a carbapenemase is becoming a significant concern in the community [ 811 ] .
multidrug - resistant streptococcus pneumoniae and -hemolytic streptococci with tolerance to penicillin and resistance to macrolides and fluoroquinolones are increasing in japan and extreme drug - resistant s. pneumoniae have been observed among multidrug - resistant isolates in canada [ 12 , 13 ] .
regional and country variations in pathogen resistance and the susceptibility breakpoint differences that are applied must be considered when determining empiric treatment options for both ca - uti and ca - urti [ 14 , 15 ] .
many countries adopt or design their own recommendations of treatment guidelines based on local surveillance data which can be scarce in some countries [ 16 ] .
the objective of this study was to investigate the contemporary pattern of antimicrobial susceptibility among pathogens causing ca - uti and ca - urti in argentina , mexico , venezuela , russia , and the philippines .
these five countries have limited local surveillance data available for understanding susceptibility patterns among orally prescribed antimicrobial agents for common outpatient infections .
microbiology laboratories in five countries which have scare surveillance data were recruited to collect isolates from community - acquired infections . only the first isolate collected from a patient who was in a clinic , a physician s office , a hospital emergency room , or in a community hospital for < 48 h at the time of collection
patients were to have had no prior antibiotic exposure within the previous 90 days .
species from ca - urti included s. pneumoniae , streptococcus pyogenes , h. influenzae , and m. catarrhalis , and a limited number of enterobacteriaceae .
a total of 1501 strains were collected during 2012 ( 29% ) and 2013 ( 71% ) from 12 medical centers , including two each in argentina , mexico , venezuela , and the philippines and four in russia .
identification of the bacterial isolates at the study site was performed using routine laboratory procedures and confirmed by matrix - assisted laser desorption ionization - time of flight ( maldi - tof ) mass spectrometry ( maldi biotyper , microflex , bruker daltonik gmbh , bremen , germany ) at a reference laboratory ( international health management associates [ ihma ] , schaumburg , il , usa ) .
minimal inhibitory concentrations ( mic ) were determined using the clinical laboratory standards institute ( clsi ) broth microdilution procedure and panels prepared by ihma using clsi guidelines .
susceptibility breakpoints used were those according to the clsi and european committee for antimicrobial susceptibility testing ( eucast ) guidelines [ 16 , 17 ] . applied eucast breakpoints were those utilized for uncomplicated urinary tract infections which included amoxicillin - clavulanic acid , cefuroxime ( oral dosing ) , cefixime , cefpodoxime , ceftibuten , fosfomycin ( oral dosing ) , and nitrofurantoin .
e. coli atcc 25922 , pseudomonas aeruginosa atcc 27853 , staphylococcus aureus atcc 29213 , h. influenzae atcc 49247 , and s. pneumoniae atcc 49619 were used as quality control ( qc ) strains .
isolates with an esbl phenotype were confirmatory tested using cefotaxime clavulanic acid and ceftazidime clavulanic acid .
the confirmatory test was performed on all e. coli , klebsiellapneumoniae , klebsiella oxytoca , and p. mirabilis with resistance to cefpodoxime ( mics > 1
this article does not contain any new studies with human or animal subjects performed by any of the authors .
among the 960 isolates collected from all infection sources with a patient age provided , 331 were from pediatric patients and 629 were from adults ( 18 years old ) .
with respect to patient gender , among the 957 patients with this information recorded , 601 were female and 356 were male .
enterobacteriaceae from ca - uti included 345 isolates of e. coli , 87 k. pneumoniae , 68 p. mirabilis , 27 enterobacter cloacae , and 40 isolates of other species .
isolates were obtained from patients aged 017 ( n = 76 ) , 1839 ( n = 186 ) , 4059 ( n = 110 ) , 6079 years ( n = 150 ) , and 80 years ( n = 45 ) .
further , approximately 75% of the isolates were obtained from women , and 23% of these were from women aged 65 years . among the 407 isolates collected from ca - urti ,
/ l ) was the most potent oral cephalosporin against enterobacteriaceae from ca - uti in each country ( table 1 ) .
the difference between the susceptibility rates of ceftibuten between countries ranged from 75.2% in the philippines to 93.1% in russia using clsi criteria and from 71.7% ( the philippines ) to 91.3% ( russia ) using eucast criteria .
cefuroxime , cefixime , cefpodoxime , and cefaclor were less active with a rank order of potency ( mic50 ) as follows ; cefixime ( 0.5 mg / l ) > cefpodoxime ( 0.51 mg / l ) > cefuroxime ( 48 mg
/ l ) > cefaclor ( 416 mg / l ) and susceptibility rates lower than ceftibuten in all countries .
susceptibility to amoxicillin - clavulanic acid was < 60% in all countries with the exception of russia ( 75.6% ) .
susceptibility to norfloxacin and ciprofloxacin was similar and varied from approximately 50% in argentina and mexico , 60% in the philippines , 70% in venezuela , and 80% in russia .
trimethoprim - sulfamethoxazole susceptibility was highest in russia ( 75.6% ) and lowest in mexico ( 30.6% ) .
however , susceptibility among combined enterobacteriaceae species ranged from 51.5% ( argentina ) to 83.3% ( russia ) using clsi breakpoint criteria .
all enterobacteriaceae isolated from ca - urti were susceptible to fluoroquinolones using clsi but < 80% using eucast breakpoints while susceptibility percentages ranged from 64% to 79% for the other agents tested using either clsi or eucast breakpoints ( data on file , ihma inc.).table 1susceptibility rates and mic values for enterobacteriaceae collected from ca - uticountry ( n)/drugclsi / eucastmic50
mic90
mic range% s% i% rargentina ( 101 ) amoxicillin / clavulanic acid52.5/82.214.9/-32.7/17.88>321>32 ceftibuten83.2/71.33.0/-13.9/28.70.25>160.06>16 cefixime58.4/58.46.9/-34.7/41.60.5>80.12>8 cefpodoxime56.4/53.55.0/-38.6/46.51>80.12>8 cefuroxime43.6/50.513.9/-42.6/49.58>321>32 cefaclor47.5/na6.9/na45.5/na16>320.5>32 ciprofloxacin50.5/49.549.5/1.00.0/49.51>10.002>1 norfloxacin50.5/38.61.98/6.947.5/54.54>80.03>8 fosfomycin91.1/84.21.0/-7.9/15.84640.25>128 nitrofurantoin51.5/64.4 ( 100 )
12.9/-35.6/35.632>1282>128 trimethoprim / sulfamethoxazole49.5/49.50.0/1.050.5/49.54>640.5>64mexico ( 98 ) amoxicillin / clavulanic acid58.2/92.928.6/-13.3/7.18321>32 ceftibuten77.6/66.36.1/-16.3/33.70.25>160.06>16 cefixime58.2/58.26.1/-35.7/41.80.5>80.12>8 cefpodoxime60.2/54.13.1/-36.7/45.91>80.12>8 cefuroxime43.9/58.216.3/-39.8/41.88>321>32 cefaclor45.9/na7.1/na46.9/na16>320.5>32 ciprofloxacin51.0/45.950.0/5.10.0/49.01>10.004>1 norfloxacin48.0/37.86.1/4.145.9/58.28>80.03>8 fosfomycin95.9/90.81.0/-3.1/9.22320.25>128 nitrofurantoin65.3/80.6 ( 94.7 )
15.3/-19.4/19.416>1282>128 trimethoprim / sulfamethoxazole30.6/30.60.0/3.169.4/66.3>64>640.5>64venezuela ( 95 ) amoxicillin / clavulanic acid55.8/88.426.3/-17.9/11.68>321>32 ceftibuten89.5/84.22.1/-8.4/15.80.25160.06>16 cefixime74.7/74.78.4/-16.8/25.30.5>80.12>8 cefpodoxime82.1/76.81.1/-16.8/23.20.5>80.12>8 cefuroxime52.6/77.929.5/-17.9/22.14>321>32 cefaclor64.2/na6.3/na29.5/na4>320.5>32 ciprofloxacin72.6/72.627.4/0.00.0/27.40.015>10.002>1 norfloxacin72.6/63.20.0/4.227.4/32.60.12>80.015>8 fosfomycin92.6/88.43.2/-4.2/11.62640.25>128 nitrofurantoin68.4/81.1 ( 97.0 )
12.6/-19.0/19.0161284>128 trimethoprim / sulfamethoxazole57.9/57.90.0/1.142.1/41.10.5>640.5>64russia ( 160 ) amoxicillin / clavulanic acid75.6/95.615.6/-8.8/4.44161>32 ceftibuten93.1/91.30.6/-6.3/8.70.1210.06>16 cefixime81.3/81.33.1/-15.6/18.70.580.12>8 cefpodoxime81.9/80.01.2/-16.9/20.00.5>80.12>8 cefuroxime63.7/79.416.9/-19.4/20.64>321>32 cefaclor76.2/na1.9/na21.9/na4>320.5>32 ciprofloxacin80.0/79.420.0/0.60.0/20.00.015>10.004>1 norfloxacin81.9/76.90.0/2.518.1/20.60.06>80.03>8 fosfomycin98.1/95.60.6/-1.3/4.41160.25>128 nitrofurantoin83.8/91.9 ( 98.4 )
8.1/-8.1/8.116642>128 trimethoprim / sulfamethoxazole75.6/75.60.0/0.624.4/23.80.5>640.5>64philippines ( 113 ) amoxicillin / clavulanic acid54.0/84.116.8/-29.2/15.98>321>32 ceftibuten75.2/71.77.1/-17.7/28.30.12>160.06>16 cefixime62.8/62.83.5/-33.6/37.20.5>80.12>8 cefpodoxime67.3/62.82.7/-30.1/37.20.5>80.12>8 cefuroxime48.7/59.316.8/-34.5/40.78>321>32 cefaclor54.0/na3.5/na42.5/na4>320.5>32 ciprofloxacin62.0/59.338.1/2.70/38.10.12>10.002>1 norfloxacin63.7/54.98.9/1.827.4/43.40.5>80.015>8 fosfomycin91.2/83.23.5/-5.3/16.8464.00.25>128 nitrofurantoin53.1/73.5 ( 96.3 )
20.4/-26.6/26.632128.02>128 trimethoprim / sulfamethoxazole44.3/44.30/1.855.8/54.0>64>640.5>64number in parentheses is the susceptibility rates when applied to e. coli only ( eucast recommendation )
clsi clinical laboratory standards institute , eucast european committee for antimicrobial susceptibility testing , i intermediate , mic minimal inhibitory concentrations , r resistant , s susceptible , ca - uti community - acquired urinary tract infection , na or a dash = no interpretive breakpoints available susceptibility rates and mic values for enterobacteriaceae collected from ca - uti number in parentheses is the susceptibility rates when applied to e. coli only ( eucast recommendation )
clsi clinical laboratory standards institute , eucast european committee for antimicrobial susceptibility testing , i intermediate , mic minimal inhibitory concentrations , r resistant , s susceptible , ca - uti community - acquired urinary tract infection , na or a dash = no interpretive breakpoints available esbl percentages for e. coli ranged from 9.2% in venezuela to 40.7% in mexico .
similarly , esbl percentages for k. pneumoniae were lowest ( 18.8% ) in venezuela and highest ( 46.4% ) in mexico .
esbl - producing p. mirabilis ranged from 10% to 33% with the highest percentage observed in argentina .
fosfomycin and nitrofurantoin retained > 90% susceptibility against esbl - positive e. coli in each country ( data on file , ihma inc . ) .
the activity of fosfomycin and nitrofurantoin diminished against esbl - positive k. pneumoniae . among the -lactams tested ,
all h. influenzae isolates were susceptible to ceftibuten , cefixime , cefpodoxime , and cefuroxime using clsi breakpoint criteria ( table 2 ) .
intermediate and resistant mic values were observed for each of these oral cephalosporins utilizing eucast breakpoint criteria .
reduced activity of amoxicillin / clavulanic acid was observed among h. influenzae isolates collected in argentina and mexico .
one of three haemophilus parainfluenzae from the philippines had high - level resistant mic values for all tested fluoroquinolones ( data on file , ihma inc . ) .
isolates of m. catarrhalis were only collected in argentina and all demonstrated a common broad spectrum susceptible pattern to the agents tested.table 2susceptibility and mic values for fastidious respiratory tract pathogens from ca - urtiorganismcountry ( n)/drugclsi / eucastmic50
mic90
% s% i% r
s. pneumoniae
argentina ( 19)amoxicillin100/na0.0/na0.0/na0.120.25amoxicillin / clavulanic acid100/na0.0/na0.0/na0.120.25ceftibutenna / nana / nana / na>4>4cefiximena / nana / nana / na0.252cefpodoxime100/94.70.0/5.30.0/0.00.030.25cefuroxime94.7/94.75.3/0.00.0/5.30.030.5cefaclor84.2/0.05.3/73.710.5/26.30.54azithromycin78.9/0.00.0/78.921.1/21.10.5>4clarithromycin78.9/78.90.0/0.021.1/21.10.25>2ciprofloxacinna/0.0na/100na/0.00.5>1levofloxacin94.7/94.75.3/0.00.0/5.322moxifloxacin100/1000.0/0.00.0/0.00.120.12doxycycline84.2/94.710.5/0.05.3/5.30.060.5trimethoprim / sulfamethoxazole73.7/84.210.5/0.015.8/15.80.5>2
s. pneumoniae
mexico ( 14)amoxicillin71.4/na7.1/na21.4/na0.12>16amoxicillin / clavulanic acid71.4/na0.0/na28.6/na0.12>16ceftibutenna / nana / nana / na>4>4cefiximena / nana / nana / na1>8cefpodoxime64.3/57.10.0/7.135.7/35.70.12>4cefuroxime57.1/57.114.3/0.028.6/42.90.25>4cefaclor28.6/0.028.6/21.442.9/78.62>8azithromycin71.4/0.00.0/71.428.6/28.60.5>4clarithromycin71.4/71.40.0/0.028.6/28.60.25>2ciprofloxacinna/0.0na/100na/0.01>1levofloxacin85.7/85.70.0/0.014.3/14.32>16moxifloxacin85.7/85.70.0/0.014.3/14.30.12>8doxycycline50.0/50.00.0/0.050/50.00.12>2trimethoprim / sulfamethoxazole42.9/57.114.3/0.042.9/42.91>2
s. pneumoniae
russia ( 148)amoxicillin83.8/na8.1/na8.1/na0.124amoxicillin / clavulanic acid83.8/na6.1/na10.1/na0.12>4ceftibutenna / nana / nana / na>4>4cefiximena / nana / nana / na0.5>8cefpodoxime69.6/67.60.7/2.029.7/30.40.06>4cefuroxime67.6/65.61.3/2.031.1/32.40.12>4cefaclor61.5/0.03.4/50.735.1/49.30.5>8azithromycin56.1/0.00.7/56.143.2/43.90.5>4clarithromycin56.8/56.80.0/0.043.2/43.20.25>2ciprofloxacinna/1.4na/98.6na/00.51levofloxacin83.8/87.816.2/0.00.0/16.224moxifloxacin100/1000.0/0.00.0/0.00.120.12doxycycline45.3/49.32.7/11.552.0/39.22>2trimethoprim / sulfamethoxazole28.4/38.524.3/14.247.3/47.32>2
s. pyogenes
argentina ( 20)amoxicillinna / nana / nana / na0.120.12amoxicillin / clavulanic acidna / nana / nana / na0.120.12ceftibutenna / nana / na0.0/na0.50.5cefiximena / nana / nana / na0.060.12cefpodoximena / nana / nana / na0.030.03cefuroximena / nana / nana / na0.030.03cefaclorna / nana / nana / na0.120.12azithromycin100/0.00.0/1000.0/0.00.50.5clarithromycin100/1000.0/0.00.0/0.00.250.25ciprofloxacinna / nana / nana / na0.121levofloxacin85.0/75.015.0/10.00.0/15.014moxifloxacinna/100na/0.0na/0.00.060.25doxycyclinena/95.0na/0.0na/5.00.060.06trimethoprim / sulfamethoxazolena/100na/0.0na/0.00.120.12
s. pyogenes
mexico ( 20)amoxicillinna / nana / nana / na0.120.12amoxicillin / clavulanic acidna / nana / nana / na0.120.12ceftibutenna / nana / nana / na0.250.5cefiximena / nana / nana / na0.060.12cefpodoximena / nana / nana / na0.030.03cefuroximena / nana / nana / na0.030.03cefaclorna / nana / nana / na0.120.12azithromycin100/0.00.0/1000.0/0.00.50.5clarithromycin100/1000.0/0.00.0/0.00.250.25ciprofloxacinna / nana / nana / na0.120.25levofloxacin100/95.00.0/5.00.0/0.011moxifloxacinna/100na/0.0na/0.00.060.12doxycyclinena/90.0na/0na/10.00.060.06trimethoprim / sulfamethoxazolena/100na/0.0na/0.00.120.12
s. pyogenes
philippines ( 11)amoxicillinna / nana / nana / na0.120.12amoxicillin / clavulanic acidna / nana / nana / na0.120.12ceftibutenna / nana / nana / na0.50.5cefiximena / nana / nana / na0.060.12cefpodoximena / nana / nana / na0.030.03cefuroximena
/ nana / nana / na0.030.03cefaclorna / nana / nana / na0.120.25azithromycin81.8/0.00.0/81.818.2/18.20.5>4clarithromycin81.8/81.80.0/0.018.2/18.20.25>2ciprofloxacinna / nana / nana / na0.120.25levofloxacin100/90.90/9.10.0/0.011moxifloxacinna/100na/0.0na/0.00.060.12doxycyclinena/81.8na/0.0na/18.20.06>2trimethoprim / sulfamethoxazolena/100na/0.0na/0.00.120.12
h. influenzae
argentina ( 10)amoxicillinna/90.0na/0.0na/10.00.52amoxicillin / clavulanic acid90.0/90.00.0/0.010.0/10.012ceftibuten100/1000.0/0.00.0/0.00.120.25cefixime100/1000.0/0.00.0/0.00.0150.06cefpodoxime100/1000.0/0.00.0/0.00.120.25cefuroxime100/70.00.0/20.00.0/10.00.52cefaclor80.0/0.020.0/0.00.0/100416azithromycin100/0.00.0/1000.0/0.022clarithromycin70.0/10.030.0/90.00.0/0.0816ciprofloxacin100/1000.0/0.00.0/0.00.0080.015levofloxacin100/1000.0/0.00.0/0.00.030.03moxifloxacin100/1000.0/0.00.0/0.00.0150.015doxycyclinena/90.0na/10.0na/0.011trimethoprim / sulfamethoxazole80.0/80.00.0/0.020.0/20.00.25>4
h. influenzae
mexico ( 12)amoxicillinna/58.3na/0na/41.71>4amoxicillin / clavulanic acid100/83.30.0/0.00.0/16.714ceftibuten100/91.70.0/0.00.0/8.30.120.25cefixime100/91.70.0/0.00.0/8.30.030.06cefpodoxime100/83.30.0/0.00.0/16.70.251cefuroxime100/58.30/41.80.0/0.012cefaclor75.0/0.016.7/08.3/100416azithromycin100/0.00.0/1000.0/0.024clarithromycin66.7/0.025/1008.3/0.0816ciprofloxacin100/1000.0/0.00.0/0.00.0080.015levofloxacin100/1000.0/0.00.0/0.00.030.03moxifloxacin100/1000.0/0.00.0/0.00.0150.015doxycycline0.0/1000.0/0.00.0/0.011trimethoprim / sulfamethoxazole0.0/0.00.0/0.0100/100>4>4
h. influenzae
russia ( 36)amoxicillinna/94.4na/0.0na/5.60.250.5amoxicillin / clavulanic acid100/1000.0/0.00.0/0.00.250.5ceftibuten100/1000.0/0.00.0/0.00.030.06cefixime100/1000.0/0.00.0/0.00.0150.03cefpodoxime100/1000.0/0.00.0/0.00.030.06cefuroxime100/1000.0/0.00.0/0.00.250.5cefaclor100/0.00.0/0.00.0/10024azithromycin100/0.00/1000.0/0.022clarithromycin77.8/2.819.4/97.22.8/0.0816ciprofloxacin100/1000.0/0.00.0/0.00.0080.015levofloxacin100/1000.0/0.00.0/0.00.030.03moxifloxacin100/1000.0/0.00.0/0.00.0080.015doxycycline0.0/1000.0/0.00.0/0.011trimethoprim / sulfamethoxazole63.9/63.92.8/0.033.3/36.10.25>4
h. influenzae
philippines ( 19)amoxicillinna/89.5na/0.0na/10.50.54amoxicillin / clavulanic acid100/1000.0/0.00.0/0.00.50.5ceftibuten100/1000.0/0.00.0/0.00.030.06cefixime100/1000.0/0.00.0/0.00.0150.03cefpodoxime100/1000.0/0.00.0/0.00.060.12cefuroxime100/84.20.0/15.80.0/0.00.52cefaclor100/0.00.0/0.00.0/10048azithromycin100/0.00.0/1000.0/0.024clarithromycin31.6/0.068.4/1000.0/0.01616ciprofloxacin100/1000.0/0.00.0/0.00.0080.015levofloxacin100/1000.0/0.00.0/0.00.030.03moxifloxacin100/1000.0/0.00.0/0.00.0150.03doxycyclinena/100na/0.0na/0.011trimethoprim / sulfamethoxazole31.6/31.65.3/0.063.2/68.4>4>4
m. catarrhalis
argentina ( 11)amoxicillinna / nana / nana / na0.120.12amoxicillin / clavulanic acid100/1000.0/0.0na/0.00.120.12ceftibuten100/1000.0/0.0na/0.00.030.03cefiximena / nana / nana / na0.030.03cefpodoximena / nana / nana / na0.50.5cefuroxime100/1000.0/0.0na/0.00.121cefaclorna/100na/0.0na/0.00.060.12azithromycin100/1000.0/0.00.0/0.00.50.5clarithromycinna/100na/0.0na/0.00.060.06ciprofloxacin63.6/9.136.4/9.10.0/81.80.250.25levofloxacinna/100na/0.0na/0.00.060.25moxifloxacin100/1000.0/0.0na/0.00.120.12doxycycline100/1000.0/0.00.0/0.014trimethoprim / sulfamethoxazolena / nana / nana / na0.120.12
clsi clinical laboratory standards institute , eucast european committee for antimicrobial susceptibility testing , i intermediate , mic minimal inhibitory concentrations , r resistant , s susceptible , ca - urti community - acquired upper respiratory tract infection , na no interpretive breakpoints available susceptibility and mic values for fastidious respiratory tract pathogens from ca - urti
clsi clinical laboratory standards institute , eucast european committee for antimicrobial susceptibility testing , i intermediate , mic minimal inhibitory concentrations , r resistant , s susceptible , ca - urti community - acquired upper respiratory tract infection , na no interpretive breakpoints available streptococcus pneumoniae collected from argentina , mexico , and russia were more susceptible to amoxicillin with and without clavulanic acid compared to other -lactams ( table 2 ) . susceptibility oral cephalosporins were higher in argentina compared to russia and mexico .
levofloxacin susceptibility was high in all countries , though fluoroquinolone - resistant s. pneumoniae isolates were observed in all countries .
trimethoprim - sulfamethoxazole susceptibility was considerably lower in russia ( 2838% ) compared to mexico ( 4357% ) and argentina ( 7485% ) dependent upon the applied breakpoint criteria .
azithromycin- and clarithromycin - resistant s. pyogenes was only observed in the philippines and fluoroquinolone non - susceptible isolates were observed in all three countries that submitted s. pyogenes . among the 47 enterobacteriaceae collected from ca - urti using mic50 values , ceftibuten , cefixime , and fluoroquinolones
this study determined the activity of oral antimicrobial agents from countries with limited information on the susceptibility patterns for pathogens which cause ca - uti and ca - urti .
ceftibuten provided the highest and similar susceptibility rates using either clsi or eucast breakpoint criteria against ca - uti pathogens when compared to the oral cephalosporins tested .
amoxicillin - clavulanic acid susceptibility was much lower when using clsi breakpoints compared to that observed using eucast uncomplicated uti breakpoint criteria for this agent .
country variability in susceptibility was also observed , even within the same region with a 20% difference observed for fluoroquinolone susceptibility among the latin american countries examined .
sites which routinely do not collect specimens for culture from outpatients with ca - urti limited the number of pathogens collected for this indication and not all countries provided a representative sample for this investigation .
also the number of countries in the three regions studied would not represent the entire region .
broad spectrum antimicrobial agents including all of those reported in this study should be used selectively .
cephalosporins and fluoroquinolones may not always be the first choice of therapy ; due to the antibiotic pressure , these and other classes have on increasing esbl rates .
however , depending on the source of the infection and the pharmacokinetic and pharmacodynamics of the drug class , the loss of activity among other commonly used oral agents warrants the need to continue to monitor the viability of oral cephalosporins and other drugs used for treating outpatient infections .
it is important that local epidemiology efforts continue to determine the rate of emerging or epidemic clones and the resistance rates among several class agents which vary based on the susceptibility breakpoints applied [ 1921 ] .
it is equally important to follow the variation of prescribing practices and to reduce antibiotic consumption in the ambulatory setting as this affects antimicrobial resistance in the hospital setting [ 22 , 23 ] .
information related to the local and regional susceptibility patterns of pathogens causing ca - uti and ca - urti is essential for physicians treating patients with these very common infections and that was the intent of this study .
continued surveillance of the pathogens causing ca - uti and ca - urti is necessary in this era of increasing antimicrobial resistance .
antimicrobial stewardship should remain a high priority across all countries to promote the best treatment practices to diminish the problem of antimicrobial resistance .
d. j. biedenbach and r. e. badal employees of international health management associates , inc . , schaumburg and
r. s. kozlov and a. d. roman contributed to the study design , collection and analysis of data and receive honoraria for this study .
m. y. huang , m. motyl , p. k. singhal and s. marcella are employees of merck & co. inc . , kenilworth , new jersey , usa and have financial interests .
this article does not contain any new studies with human or animal subjects performed by any of the authors .
this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made . | introductionbacterial infections that cause community - acquired urinary tract infections ( ca - uti ) and upper respiratory tract infections ( ca - urti ) are most frequently treated empirically .
however , an increase in antimicrobial resistance has become a problem when treating outpatients.methodsthis study determined the in vitro activities of oral antibiotics among 1501 pathogens from outpatients with ca - uti and ca - urti in medical centers during 2012 and 2013 from argentina , mexico , venezuela , russia , and the philippines .
minimal inhibitory concentrations ( mics ) were determined using broth microdilution and susceptibility defined by clinical laboratory standards institute ( clsi ) and european committee for antimicrobial susceptibility testing ( eucast ) criteria.resultsceftibuten ( mic50 , 0.25 mg
/ l ) was more potent in vitro compared to other -lactams against enterobacteriaceae from ca - uti .
susceptibility to fluoroquinolones using clsi criteria varied : argentina and mexico ( 50% ) , the philippines ( 60% ) , venezuela ( 70% ) , and russia ( 80% ) .
fosfomycin susceptibility was > 90% against enterobacteriaceae in each country .
susceptibility among enterobacteriaceae to trimethoprim - sulfamethoxazole was 30.675.6% and nitrofurantoin susceptibility also varied among the countries and was higher when eucast breakpoints were applied ( 65>90% ) compared to clsi ( 5284% ) .
all haemophilus influenzae isolates from ca - urti were susceptible to ceftibuten , cefixime , cefpodoxime , and cefuroxime using clsi breakpoint criteria .
eucast criteria produced intermediate and resistant mic values for these oral cephalosporins .
country - specific susceptibility variation for fluoroquinolones , macrolides , and trimethoprim - sulfamethoxazole was observed among streptococcus pneumoniae and streptococcus pyogenes from ca-urti.conclusionthis study demonstrated that antimicrobial susceptibility patterns varied in the five countries investigated among pathogens from ca - uti and ca-urti.fundingmerck & co. inc . ,
kenilworth , new jersey , usa . | Funding
Introduction
Methods
Results
Discussion
Conclusion
Disclosures
Compliance with Ethics Guidelines
Open Access |
osteoporosis ( op ) is a systemic disease characterized by low bone mass , bone microstructure damage and increased bone fragility .
one of the most severe consequences of op is osteoporotic vertebral compression fracture ( ovcf ) , which can increase the disability rate and mortality rate .
percutaneous vertebroplasty ( pvp ) is a new minimally invasive spinal surgical technique that is mainly indicated in ovcf . in this surgery
, the filler material polymethylmethacrylate ( pmma ) is infused into the fracture site to stabilize the vertebral body and eliminate pain .
in contrast to pvp , percutaneous balloon kyphoplasty ( pkp ) can effectively reduce bone cement leakage and correct kyphosis in ovcf,12 therefore is preferred for managing ovcf .
intravertebral cleft is a structural change in ovcf . with the advancement of preoperative radiographic evaluations of vertebral status , in particular with the population of mri and ct reconstruction , more and more ocvfs
this change is the manifestation of ischemic vertebral osteonecrosis complicated with fracture nonunion and pseudoarthrosis , which appears in the late stage of ovcf.3 theoretically , nonunion and pseudoarthrosis can induce instability of the fracture and thus lead to more severe pain than those without intravertebral cleft.3 further , ovcf with the presence of intravertebral cleft is a potential risk factor for bone cement leakage and may predict the trend and type of bone cement leakage.4 despite many studies on ovcf , few are available in literature regarding the distribution , clinicoradiological characteristics and clinical significance of intravertebral cleft in ovcf . in this study ,
the distribution and characteristics of the intravertebral cleft and the effect of pkp in ovcf are evaluated .
183 patients who were subjected to pkp to treat ovcf from december 2009 to december 2011 were enrolled in this study .
ovcf was confirmed preoperatively by bone density measurement , x - ray , computer tomography ( ct ) scans , and magnetic resonance imaging ( mri ) .
patients with previous spine surgery , infection or tumor were excluded . according to the intravertebral cleft manifestation
, the selected patients were divided into two groups : group a ( without intravertebral cleft , n = 139 ) and group b ( with intravertebral cleft , n = 44 ) .
group a comprised of 22 males and 117 females , with an average age of 70.2 years ( range 51 - 91 years ) .
group b comprised of 5 males and 39 females with an average age of 71.6 years ( range 56 - 89 years ) .
the average number of vertebral bodies involved in group a and b was 1.2 ( 172/139 , range , 1 to 2 ) and 1.2 ( 53/44 , range , 1 to 2 ) .
this study was conducted in accordance to the declaration of helsinki and was approved by the ethics committee of our institution .
frequency distribution of affected vertebral bodies the patients were placed prone on a radiolucent table , with both hands fixed on the lateral sides .
lateral and anteroposterior c - arm fluoroscopic images were obtained to confirm the land marks positions of the pedicles bilaterally at the affected level .
after sterilization , 1% lidocaine was used for local infiltration anesthesia . the needle point on the body surface
a transpedicular approach in the lumbar vertebra and through the intermediate part between the rib head and the pedicle in the thoracic vertebra , were used for penetration .
the tip of the puncture needle ( 11 g , shanghai kailitai medical technology company , china ) penetrated the bone 2 to 3 mm laterally outside the 10 oclock point ( left pedicle ) or the 2 oclock point ( right pedicle ) of the pedicle projection .
the needle tip was located outside the inner wall of the pedicle on normotopia projection .
the needle was inserted until it reached one - third of the anterior part into the vertebral body and into the intravertebral cleft in groups a and b , respectively .
kirschner wire was then inserted to the channel produced by the puncture needle and the working cannula was inserted along the kirschner wire .
a balloon dilator was placed for dilatation after reaming . in group a , standard pasty pmma bone cement ( tianjin synthetic materials research institute , china ) was infused into the affected vertebral body using a bone cement injector ( shanghai kailitai medical technology company , shanghai , china ) . in group
b , 0.5 ml to 1.0 ml of sticky phase bone cement was infused into the anterior part of the cleft .
after the eggshell in the cleft was formed , the balloon dilator was withdrawn and the bone cement in the terminal sticky phase was gradually infused along the medial surface of the eggshell .
the vertebral body and cleft were diffusely filled with bone cement as confirmed by c - arm fluoroscopy and the surgical instruments were removed . the surgery was carried out based on a unilateral , bilateral , or para - pedicle approach .
vitamin d calcium chewable tablet ( 600 mg / d ) and alendronate sodium ( 70 mg / w ) were routinely administered to each patient to treat osteoporosis .
the following evaluation indices were used : ( 1 ) distribution of the intravertebral cleft in vertebral bodies ; ( 2 ) operative time and blood loss ; ( 3 ) volume of infused bone cement , bone cement leakage , leakage type and vertebral height ; and ( 4 ) efficacy .
efficacy was evaluated by comparing the visual analog scale ( vas ) scores and oswestry disability index ( odi ) scores before and 2 days after surgery . for imaging evaluation , x
all the images were analyzed by three independent observers , two orthopedic surgeons and one radiologist , experienced in reading musculoskeletal images .
paired sample t - test was used to compare pre and postoperative differences for continuous variables in the same group .
test or fisher 's exact test was performed to compare the distributions of categorical variables .
the patients were placed prone on a radiolucent table , with both hands fixed on the lateral sides .
lateral and anteroposterior c - arm fluoroscopic images were obtained to confirm the land marks positions of the pedicles bilaterally at the affected level .
after sterilization , 1% lidocaine was used for local infiltration anesthesia . the needle point on the body surface
a transpedicular approach in the lumbar vertebra and through the intermediate part between the rib head and the pedicle in the thoracic vertebra , were used for penetration .
the tip of the puncture needle ( 11 g , shanghai kailitai medical technology company , china ) penetrated the bone 2 to 3 mm laterally outside the 10 oclock point ( left pedicle ) or the 2 oclock point ( right pedicle ) of the pedicle projection .
the needle tip was located outside the inner wall of the pedicle on normotopia projection .
the needle was inserted until it reached one - third of the anterior part into the vertebral body and into the intravertebral cleft in groups a and b , respectively .
kirschner wire was then inserted to the channel produced by the puncture needle and the working cannula was inserted along the kirschner wire .
a balloon dilator was placed for dilatation after reaming . in group a , standard pasty pmma bone cement ( tianjin synthetic materials research institute , china ) was infused into the affected vertebral body using a bone cement injector ( shanghai kailitai medical technology company , shanghai , china ) . in group
b , 0.5 ml to 1.0 ml of sticky phase bone cement was infused into the anterior part of the cleft .
after the eggshell in the cleft was formed , the balloon dilator was withdrawn and the bone cement in the terminal sticky phase was gradually infused along the medial surface of the eggshell . the vertebral body and cleft were diffusely filled with bone cement as confirmed by c - arm fluoroscopy and the surgical instruments were removed . the surgery was carried out based on a unilateral , bilateral , or para - pedicle approach .
vitamin d calcium chewable tablet ( 600 mg / d ) and alendronate sodium ( 70 mg / w ) were routinely administered to each patient to treat osteoporosis .
the following evaluation indices were used : ( 1 ) distribution of the intravertebral cleft in vertebral bodies ; ( 2 ) operative time and blood loss ; ( 3 ) volume of infused bone cement , bone cement leakage , leakage type and vertebral height ; and ( 4 ) efficacy .
efficacy was evaluated by comparing the visual analog scale ( vas ) scores and oswestry disability index ( odi ) scores before and 2 days after surgery . for imaging evaluation , x
all the images were analyzed by three independent observers , two orthopedic surgeons and one radiologist , experienced in reading musculoskeletal images .
paired sample t - test was used to compare pre and postoperative differences for continuous variables in the same group .
test or fisher 's exact test was performed to compare the distributions of categorical variables .
pkp was successfully performed on 225 vertebrae in 183 patients with an operative time of 36 min to 61 min ( average , 43 min ) and intraoperative blood loss of 6 to 11 ml ( average , 8 ml ) .
no significant difference was observed in the distribution of the affected vertebral body between the two groups ( p < 0.05 ; figure 1 ) . in group
a , l1 was the most commonly affected vertebra ( 27.2% ) followed by the l2 vertebra ( 17.8% ) ; in group b , commonly the fracture was observed at t12 vertebra ( 28.6% ) .
the volumes of infused bone cement in groups a and b were 3.5 to 5.1 ml ( average 4.3 ml ) and 3.4 to 5.6 ml ( average , 4.5 ml ) , respectively , indicating no significant difference ( p > 0.05 ) . in both groups ,
the bone cement leakage rate was similar ( p > 0.05 ; table 1 ) , but the leakage types were significantly different ( p < 0.05 ) .
the cement leaked along the blood vessels in ovcf without the intravertebral cleft ( 68.1% ) . in the presence of intravertebral cleft ( 79.2% ) , the cement leaked into the perivertebral soft tissues [ table 2 ] .
comparison of bone cement leakage between two groups comparison of bone cement leakage type between two groups in both the groups , the postoperative height of the affected vertebral body significantly improved ( p < 0.05 ; table 3 ) .
restoration of the vertebral body height in group b was more evident than that in group a ( p < 0.05 ; table 4 ) .
the preoperative vas and odi scores in group b were significantly higher than those in group a ; however , the postoperative scores were comparable in the two groups ( p > 0.05 ; table 5 ) .
the postoperative vas and odi scores were significantly better in both groups compared with the preoperative values ( p < 0.05 ; table 5 ) .
the improvement rates of vas and odi scores of the two groups was not significantly different ( p > 0.05 ; table 6 ) .
preoperative and postoperative height of affected vertebral body in two groups ( s , mm ) restoration values vertebral body height in two groups ( s ) vas and odi score in two groups ( s ) improvement rates of vas and odi score in two groups ( s , % )
with the advancement of mri , ct , and other advanced imaging modalities used to assess the vertebral status before pkp , numerous patients were diagnosed with ovcf complicated with the intravertebral cleft .
the intravertebral cleft is considered as a manifestation of vertebral ischemic osteonecrosis.3 this condition has a very clear mechanism of occurrence .
however , the distribution as well as the clinical and radiological features of the intravertebral cleft in ovcf is rarely reported.456 in x - ray imaging , the intravertebral cleft is manifested as a thread - like or cyst - like transparent area4 [ figure 2a ] , but the detection rate of the intravertebral cleft by an anteroposterior and lateral radiograph is low .
abnormal movement of the vertebral cleft caused by different body postures lead to corresponding changes in the affected vertebral body height . under hyperextension and hyperflexion positions ,
the anterior affected vertebral body and the posterior zygapophyseal joint move and perform abnormal movement .
this movement results in evident height variation in the anterior border of the vertebral body ( opening / closing syndrome ) in the lateral projection on a plain radiograph . on the basis of this characteristic ,
the detection rates of the intravertebral cleft in the lateral projection at a supine position5 are significantly higher than those in the upright and the upright extension position .
mckiernan et al.6 studied patients with ovcf and found that the detection rates of the intravertebral cleft in the lateral projection in upright and supine positions are 14% and 64% , respectively .
( a ) x - ray lumbosacral spine anteroposterior and lateral views showing thread like or cyst - like transparent areas for intravertebral cleft ( black arrow ) .
( b ) mri t1 and t2w showing low signal area ( t1w ) and long and narrow high signal area ( liquid , t2w ) for intravertebral cleft ( black arrow ) . ( c ) mri t1 and t2w showing affected vertebral body for ovcf without intravertebral cleft .
( d ) low density image for intravertebral cleft in ct plain scanning with coronal and sagittal reconstruction ( border , high density sclerotin ) mri is a sensitive and effective imaging method to treat ovcf and the intravertebral cleft , which represents a vertebral structural change after trauma.7 in t1 weighted images , the intravertebral cleft is seen as a limited low signal area ; in t2 weighted images , the intravertebral cleft is seen by a limited long and narrow high signal area ( liquid ; figure 2b ) or a low signal area ( gas ) .
the manifestation is determined by the nature of the contents.8 the incidences of osteonecrosis and adjacent vertebral fracture in a gas phase in the intravertebral cleft are higher than those in a liquid phase.9 for ovcf without the intravertebral cleft , t1w and t2w images exhibit diffused low and high signal areas , respectively10 [ figure 2c ] . according to the intravertebral cleft signal characteristics and contents ,
the manifestations of mri are divided into four types : ( 1 ) liquid phase ( 14% ) : low signal on t1 , high signal on t2 and extensive bone necrosis ; ( 2 ) granulomatous type ( 27% ) : low signal on t1 , medium signal on t2 and granulation tissue formation ; ( 3 ) compression type ( 41% ) : medium signal on t2 , significant loss of anterior column height signal , bone necrosis , bone marrow fibrosis , granulation tissue formation and equal amount of reactive new bone proliferation ; and ( 4 ) mixed type ( 18% ) : low signal in all conventional sequences on bilateral borders ( the same with hardening bone in ct ) . in this study ,
the detection rate was 90.9% , which is significantly higher than x - ray imaging .
given the lack of spinal cord injury symptom for vertebral compression fracture , ct is seldom used to diagnose intravertebral cleft .
ct was often used as a plain scan and its sensitivity and detection rate was lower than x - ray and mri11 . in this study , ct scan found typical intravertebral cleft with coronal and sagittal reconstructions [ figure 2d ] . in 44 patients with intravertebral cleft ,
this result indicates that ct scan with coronal and sagittal reconstructions exhibited good application in diagnosing intravertebral cleft .
pkp is a minimally invasive technique to treat ovcf and benign or malignant vertebral tumors . in pkp
, a balloon distraction is conducted to restore the compressed vertebral height for the correction of kyphosis.12 pkp has been widely used in clinical practice and has shown good efficacy.13 for patients with ovcf complicated with the intravertebral cleft , the surgery requirements are higher .
the cleft area is the puncture target ; an accurate entry to the cleft area is a key to a successful operation .
hyperextendible postural reduction was performed in some patients , which can better elevate the vertebral endplate , thus resulting in better restoration and easier balloon distraction.14 given the existence of the cleft , bone cement can be diffuse on both sides along the cleft to form a fracture pattern block with a smooth edge .
thus , good filling effect and supporting mechanical effect can be achieved.15 on the basis of distribution in the vertebral body after pkp,15 bone cement can be divided into trabecular and fracture patterns . in this study
bone cement filled the loose trabecular gap and presented a jagged block [ figure 3a ] .
this bone cement can not further fill the trabecular gap because of the hardened sclerotin .
thus , a fracture pattern block with a smooth edge is formed [ figure 3b ] .
postoperative x - ray dorsolumbar spine ( anteroposterior and lateral views ) showing ( a ) trabecular pattern of bone cement in pkp for ovcf without intravertebral cleft .
( b ) fracture pattern of bone cement in pkp for ovcf with intravertebral cleft bone cement leakage is one of the most common complication of pkp.1718 most of these leakages are asymptomatic but a small portion of symptomatic leakages may cause serious consequences.19 in group a , bone cement was infused under high pressure .
bone cement was further distributed along the trabecular gap and easily entered the vertebral body or paravertebral venous plexus .
this condition resulted in leakage along the blood vessel ( 49/172 , 28.5% ) or leakage to the adjacent intervertebral disc by weak endplate breakage ( 13/172 , 7.6% ) .
the presence of the intravertebral cleft is a risk factor for bone cement leakage.20 in group b , the bone cortex in the anterior border of the vertebral body was incomplete .
bone cement was uniformly distributed in the cleft , followed by leaking through the crevasse in the anterior border , resulting in the highest rate of bone cement leakage to the surrounding soft tissues ( 19/3 , 35.8% ) . therefore
, no significant difference in the bone cement leakage rates was observed between the two groups .
however , the presence of the intravertebral cleft governed the differences in leakage types in both groups .
preoperative mri data can predict the trend and type of bone cement leakage.21 to avoid or reduce bone cement leakage , we should understand the preoperative fracture anatomy and position . the puncture needle
should be placed in the cleft or in the upper or lower vertebral body to avoid the interosseous venous system .
bone cement should be infused in the sticky stage or terminal sticky stage . for the rupture on the peripheral vertebral wall , the eggshell technology can be applied;22 after this procedure , bone cement filling can be performed .
some of the effective methods include intraoperative graded bone cement infusion and continuous monitoring by using a c arm .
after operation , patients should stay in bed for more than 2.5 h to allow bone cement to solidify . in pkp
, balloon dilatation contributes to further restoration of the vertebral body height.23 in this study , x - ray imaging determined the preoperative and postoperative heights of the anterior , central and posterior borders of the vertebral body [ figure 4 ] .
the results showed that the postoperative height of the affected vertebral body in both groups was significantly improved ; group b exhibited a more evident restoration of the vertebral body height than group a. x - ray lateral view of lumbosacral spine showing measurement of anterior border , center , and posterior border of affected vertebral body before ( a ) and after ( b ) pkp the intravertebral cleft is the result of evident vertebral collapse and bone necrosis in the late stage of ovcf.24 in this stage , the fractured end can produce instability caused by a change in the body position , resulting in severe pain .
therefore , the preoperative pain degree in patients with intravertebral cleft is more severe than that in patients without intravertebral cleft .
after pkp , both groups exhibited a stable vertebral body with reduced pain and improved daily activity function ; no statistical differences were observed between the two groups .
further study is need to investigate the longterm effects of intravertebral cleft on the clinical outcomes . to conclude , intravertebral cleft is a specific manifestation of ovcf , exhibiting unique clinical and imaging features .
this technique can be performed to restore the affected vertebral body height , alleviate pain and improve the daily activity of patients . | background : intravertebral cleft is a structural change in osteoporotic vertebral compression fractures ( ovcf ) , which is the manifestation of ischemic vertebral osteonecrosis complicated with fracture nonunion and pseudoarthrosis and appears in the late stage of ovcf . despite numerous studies on ovcf , few aim to evaluate the clinicoradiological characteristics and clinical significance of intravertebral cleft in ovcf .
this study investigates clinicoradiological characteristics of intravertebral cleft in ovcf and the effect on the efficacy of percutaneous balloon kyphoplasty ( pkp).materials and methods : pkp was performed on 139 ovcf patients without intravertebral cleft ( group a ) and 44 ovcf patients with intravertebral cleft ( group b ) .
the frequency distribution of the affected vertebral body , bone cement infusion volume , imaging manifestation , leakage rate and type , preoperative and postoperative height of the affected vertebral body , visual analog scale ( vas ) and oswestry disability index ( odi ) score were evaluated.results:significant differences were found in the frequency distribution of the affected vertebral body and bone cement leakage type between the two groups ( p < 0.05 ) .
however , differences in bone cement infusion volume and leakage rate ( p > 0.05 ) were not detected . in both groups , the postoperative height of the affected vertebral body was significantly improved ( p < 0.05 ) .
the restoration of vertebral body height in group b was more evident than that in group a ( p < 0.05 ) .
the preoperative vas and odi scores in group b were significantly higher than those in group a ( p < 0.05 ) .
after surgical treatment , pain relief and daily activity function in both groups were significantly improved ( p < 0.05 ) , and no significant difference in postoperative scores was detected between the two groups ( p > 0.05).conclusion : intravertebral cleft exhibits specific clinical and imaging as well as bone cement formation characteristics .
pkp can effectively restore the affected vertebral body height , alleviate pain , and improve daily activity function of patients . | I
M
Operative procedure
Statistical analysis
R
D |
angiolipoma ( al ) is a relatively rare tumor of the head and neck region , although it occurs more commonly in the forearm and the trunk regions .
this tumor has been rarely reported in the oral cavity , and to best of our knowledge , it has only been reported twice in the lip .
the histological characteristics of angiolipoma are a mixture of mature adipocytes and interspersed connective tissues with vascular vessels containing fibrin thrombi and mast cell infiltration , which are distinct from usual lipomas .
we present , a rare case of non - infiltrating angiolipoma occurring on the upper lip in a 9-year - old female .
a 9-year - old female presented with an asymptomatic swelling of upper lip since birth .
she complained of a slight increase in size of swelling for the past 1 month .
clinical examination revealed a swelling of 2 2 cm in diameter at the midline of the upper lip [ figure 1 ] .
swelling was well circumscribed , soft in consistency , movable , non - tender , non - fluctuant , and no bruit was present .
the mass was dissected free of the adjacent tissue and the lesion was surgically removed under local anesthesia . on gross examination ,
the mass was multilobulated , grayish white in color , soft in consistency and measuring about 2 2 cm in diameter [ figure 2 ] .
clinical photograph of patient gross specimen showing multilobulated mass histopathologically , h and e stained section under low magnification revealed an encapsulated tumor mass consisting of adipose tissue separated by branched vascular network [ figure 3 ] .
thin walled small capillary like vessels were present at the periphery of the tumor mass [ figure 4 ] . under higher magnification
numerous mature adipocytes with variable sized endothelial lined blood vessels were seen [ figure 5 ] .
toluidine blue staining showed an increased mast cells density in and around blood vessels [ figure 7 ] with degranulation [ figure 8 ] .
. encapsulated tumor mass consisting of adipose tissue separated by branched vascular network ( h and e stain , original magnification , 10 ) numerous mature adipocytes with variable sized endothelial lined blood vessels ( h and e stain , original magnification , 10 ) thin walled capillary like vessels present at the periphery of the tumor mass ( h and e stain , original magnification , 40 ) presence of fibrinous microthrombi ( h and e stain , original magnification , 40 ) tumor tissue showing increased mast cells density in and around blood vessels ( toluidine blue stain , original magnification 40 ) tumor tissue showing degranulating mast cell in and around blood vessels ( toluidine blue stain , original magnification 40 )
it is widely assumed that benign lipomatous tumors represent a common group of neoplasm 's that cause few complaints or complications and present little diagnostic difficulty .
. thirteen percent of all lipomas occur in head and neck , including cheek , tongue , palate , parotid gland , neck and larynx .
they are usually asymptomatic , slow - growing , soft and well - circumscribed submucosal or superficial lesion , mainly located on the buccal mucosa .
the bulk of lipomatous tumors may be grouped into four categories : superficial lipoma , a tumor composed of mature fat and arising in the superficial ( subcutaneous ) soft tissues , represents by far the most common mesenchymal neoplasm .
deep lipomas arise from or are intimately associated with tissues deep to the subcutis or with specific anatomic sites .
the main subdivisions of this group are angiomyolipoma , intramuscular and intermuscular lipoma , lipoma of the tendon sheath , neural fibrolipoma with or without macrodactyly and lumbosacral lipoma .
infiltrating or diffuse neoplastic or non - neoplastic proliferations of mature fat may cause compression of vital structures or may be confused with atypical lipomatous neoplasm / well - differentiated liposarcoma .
this group is composed of six entities : diffuse lipomatosis , pelvic lipomatosis , symmetric lipomatosis , adiposis dolorosa , steroid lipomatosis , and nevus lipomatosus .
variants of lipoma are much less common and differ from ordinary lipoma by characteristic microscopic picture and specific clinical setting .
these include angiolipoma ( al ) , myolipoma , angiomyolipoma , myelolipoma , chondroid lipoma , spindle cell / pleomorphic lipoma , hibernoma and lipoblastoma / lipoblastomatosis .
al is a benign mesenchymal tumor made up of mature lipocytes and proliferating blood vessels .
they are benign subcutaneous lesions most common in young male patients in their second or third decades of life
. they can be multiple in nature , and are most commonly seen on forearm ( two - third of cases ) followed by the trunk and upper arm .
only 38 cases of head and neck als have been reported . to the best of our knowledge
there are only two cases of al of the lip reported in the english literature . when seen in the oral cavity ,
it has been noted to be on the lip , tongue , palatal tissue , cheek being the most common site . in a review of al of head and neck , alvi et al . mentioned certain distinct characteristics which are present between head and neck al and non - head and neck al [ table 1 ] .
characteristics of hand neck versus non - head and neck al differential diagnosis of a soft mobile mass in the head and neck , and especially in the upper lip , should alert the clinician to consider other entities such as canalicular adenoma , basal cell adenoma , pleomorphic adenoma , angioleiomyoma , schwannoma , neurofibroma , other benign mesenchymal tumors and xanthogranuloma .
different theories suggest that a lipoma differentiates because of some unknown stimulus ; the tumor is of neurogenic origin ; and it is congenital .
possible causes include fatty metamorphosis of a central hemangioma , hyperplasia of fat with an associated increase in vascular channels , or a true neoplasm .
there is support for the theory that an al originates as a congenital lipoma , which later undergoes vascular proliferation .
howard and helwig think that embryonic sequestration of multipotential cells become activated at puberty by hormones and differentiate into a simple lipoma .
further stimuli such as trauma can cause vascular infiltration of the lesion , but trauma is not present in many cases of al .
it has been speculated that mast cells might play a role in their increased vascularity .
mast cells around blood vessels strongly express vascular endothelial growth factor ( vegf ) in angiolipoma , which is known to be an essential growth factor for endothelial cells in angiogenesis . in the present case ,
toluidine blue staining showed an increased mast cells density around blood vessels , with degranulation , speculating its role in vasculogenesis .
however , there has been no direct demonstration of molecular mechanisms for angiogenesis participation by mast cells in angiolipoma . based on studies by gonzales - crussi et al .
it presents as painless or tender subcutaneous nodules , generally in pubescent patients and is rare before puberty .
histologically it is encapsulated , and is a mixture of mature adipocytes and a proliferation of thin - walled vascular channels .
they extend into the surrounding tissue and are characterized by a non - encapsulated tumor mass .
the present case was that of non - infiltrating al of 2 2 cm in diameter located in the upper lip of young female .
following are the current histologic guidelines for diagnosis of al :
well encapsulated ( non - infiltrating als ) or poorly encapsulated ( infiltrating als).evidence of 50% mature adipocytes in the tumor.interspersed angiomatous proliferation in the tumor.fibrinous microthrombi.absence of other mesenchymal elements ( smooth muscle ) or pleomorphism .
well encapsulated ( non - infiltrating als ) or poorly encapsulated ( infiltrating als ) . evidence of 50% mature adipocytes in the tumor . interspersed angiomatous proliferation in the tumor .
histopathological differential diagnosis includes hemangioma , lipoma , angiomyolipoma , infiltrating lipoma , angiofibrolipoma , angiomyxolipoma , and liposarcoma .
angiomyolipoma is composed of varying amounts of blood vessels , smooth muscles and fat cells .
infiltrating lipoma consists of lesional fat tissue infiltrating in the deeper tissue , in the form of long thin streaks radiating from the intratumoral mass .
angiofibrolipoma is composed of varying amounts of blood vessels , fibrous tissue and fat cells .
it is the most common benign tumor of the kidney and is strongly associated with tuberous sclerosis .
the hypovascular lesions may be difficult to distinguish from ordinary lipomas , although the identification of microthrombi allows this distinction .
liposarcoma can be confused with al but can usually be differentiated by the presence of embryonal adipose tissue , pleomorphism , increased number of mitosis , and metastasis . the al has not been shown to spontaneously regress and has been shown to continue to enlarge as opposed to other entities such as a hemangioma .
the treatment of choice for a non - infiltrating al is surgical excision , whereas a wide local excision with free margins has been advocated for the infiltrating type . in the case of inadequate resection
the recurrence of al has been reported to be dependent on whether the mass is infiltrating . infiltrating als have been reported to be more common in an older age group .
their recurrence has been reported to be as high as 62.5% in areas outside the head and neck .
noninfiltrating types are seen more commonly in a younger age group and have no tendency to recur . a successful treatment of a giant infiltrating angiolipoma with interferon alfa also has been reported .
in conclusion , lipomas represent about 1%4% of all neoplasm 's of the oral cavity .
they are usually asymptomatic , slow - growing , soft and well - circumscribed submucosal or superficial lesion .
it consists of mature adipocytes and interspersed connective tissues with vascular vessels containing fibrin thrombi and mast cell infiltration .
wide surgical excision with free margins is the treatment of choice , offering a good prognosis and an almost negligible relapse rate . | angiolipoma is a variant of lipoma with a prominent vascular component .
it is the most common tumor of the forearm followed by trunk and extremities of young individuals .
it 's occurrence in head and neck region is rare . in this report
, we present clinical and histopathologic features of non - infiltrating angiolipoma occurring on the upper lip in a 9-year - old female .
toluidine blue stain is used to demonstrate increased mast cell density around blood vessels , speculating its role in vasculogenesis . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION |
it is not uncommon to find children with grossly carious primary teeth . in most of the cases ,
the only treatment possible is extraction of the decayed teeth , resulting in an increased number of children who require prosthetic rehabilitation with either partial or complete dentures . prosthetic rehabilitation at this stage is essential to restore masticatory function , appearance and muscle function ; to maintain and improve phonetics ; prevent development of deleterious oral habits ; and , to minimize possible psychological disturbances .
the most common cause for premature loss of primary teeth is caries , resulting in multiple extractions of primary teeth ; a usual instance is a child with nursing bottle caries .
premature loss of primary teeth can result in unsatisfactory dietary habits and crowding in the buccal segment of the permanent dentition .
this is a case report of three year nine month child whose primary teeth were extracted due to rampant caries and treated with complete dentures .
a child aged three year and nine months old , accompanied by her parents reported to the department of pedodontics and preventive dentistry , karnataka lingayateducation society 's institute of dental sciences , belgaum with the chief complaint of several decayed teeth , and not being able to eat properly .
on examination of the child it was found that all the primary teeth were grossly decayed , leaving only root stumps with multiple sinus openings .
the parents were informed about the diagnosis and were explained about the treatment plan and need for prosthetic rehabilitation .
after motivation of the parents , they were willing for the treatment and ready to keep up with the appointments that was going to take several sittings .
after extraction of all the teeth and following healing of wounds , alveolar ridges were examined for planning prosthetic rehabilitation .
the alveolar ridges were firm and well developed , giving the indication of the presence of underlying permanent tooth buds [ figure 1 ] .
photograph showing alveolar ridges of the patient showing that they were firm and well developed , and giving the indication of the presence of underlying permanent tooth buds it was decided to rehabilitate the child with complete dentures .
impressions were taken with rubber base material to improve the quality of models [ figure 2 ] . during hardening of the material ,
photograph showing impression of alveolar ridges with rubber base material shellac base plates were adapted on the upper and lower casts and wax occlusal rims were prepared .
photograph showing teeth arrangement on the occlusal rims try in of the waxed dentures was done in the mouth to check tooth positions and occlusal relationships .
trial dentures were processed with heat cure acrylic resin [ figure 4 ] . during denture insertion , final necessary adjustments were done and the dentures were delivered to the patient [ figure 5 ] .
advice about speech , eating and daily maintenance of the denture was given to parents .
photograph showing the dentures fabricated with heat cured acrylic resin photograph showing patient wearing complete dentures after the final necessary adjustments after 24 hours of denture insertion , the patient was recalled for the inspection of mouth , to check whether any necessary corrections to the dentures should be made and for any traumatic ulcers .
the child was recalled after a week , parents were shown to examine the child for evidence of trauma or signs of tooth eruption .
after insertion of dentures , right and left maxillary permanent first molar [ figure 6 ] , left mandibular permanent first molar and lower incisor started emerging [ figure 7 ] .
necessary trimming of the dentures were done from the areas of erupting teeth , so that the erupting teeth can be accommodated in the modified dentures [ figure 8 ] .
photograph showing eruption of upper right and left permanent first molars photograph showing eruption of lower permanent left first molar and left central incisor photograph showing modified dentures to allow eruption of permanent teeth patient and parents were given instruction with regards to maintenance of proper oral hygiene .
after 24 hours of denture insertion , the patient was recalled for the inspection of mouth , to check whether any necessary corrections to the dentures should be made and for any traumatic ulcers .
the child was recalled after a week , parents were shown to examine the child for evidence of trauma or signs of tooth eruption .
the child was recalled every three months for check - up . around two years after insertion of dentures , right and left maxillary permanent first molar [ figure 6 ] , left mandibular permanent first molar and lower incisor started emerging [ figure 7 ] .
necessary trimming of the dentures were done from the areas of erupting teeth , so that the erupting teeth can be accommodated in the modified dentures [ figure 8 ] .
photograph showing eruption of upper right and left permanent first molars photograph showing eruption of lower permanent left first molar and left central incisor photograph showing modified dentures to allow eruption of permanent teeth patient and parents were given instruction with regards to maintenance of proper oral hygiene .
preschool children wearing complete denture are not very common , however , it is not uncommon to find preschool children with grossly carious primary teeth .
most of the parents are negligent towards restorations and maintenance of primary teeth , may be because of lack of knowledge about importance of primary teeth , reluctant to have prosthetic rehabilitation of their child at such an early age , economic constraints and lack of time to follow up with lengthy treatment procedure .
although the clinical procedure for fabrication of complete denture is similar to that in adults , cooperation of the child and also the parents will influence the treatment .
so , when a child presents with premature loss of teeth , it is important to determine the level of cooperation expected from both the child and parents .
clinician can concur that children adapt extremely well to dentures and functions is rarely a problem .
tray selection is carried out by explaining the procedure of impression making to the child .
operant conditioning is utilized in which at first visit the patient is given upper stock tray to take home and practice under the parents supervision .
silicon based impression material is used for making primary impression because of its pleasant taste and acceptable smell , and it is well tolerated by the children than alginate impression material .
impression making is first done for the lower jaw followed by the upper since gagging is frequently encountered in the upper arch . in case of younger children , jaw relation is difficult to record since the neuromuscular development completes only by 7 years of age .
however , there is need to improve the clinical performance of dentures in the child patient .
it may be beneficial to improve patient compliance by additional guidance , in the presence of a parent , about denture insertion , removal and overall care , together with accompanying written instructions .
complete dentures prove to be very useful to the preschool children suffering from induced anodontia ( due to rampant caries ) , and also , the dentures are simulated to resemble the deciduous dentition and thereby provide psychological satisfaction to the patient .
prosthetic management of children with premature loss of primary teeth should be followed up for many years .
periodic recall visits should be advised , as modification has to be made to the dentures with the changes in growth and eruption of permanent teeth . | a case of prosthetic rehabilitation with complete dentures for a three year nine months old child is presented .
dental prosthesis are frequently used to avoid psychologic , speech , or swallowing problems in preschooler children .
the case was followed up for a period of three years .
these prosthesis were modified as the child grew and as the permanent teeth erupted into the oral cavity the dentures were trimmed from the areas of erupting teeth to facilitate their eruption . | Introduction
Case Report
Follow up
Discussion
Conclusions |
a 68-year - old woman presented with decreased visual acuity ( va ) , and 20 hours earlier had noticed a visual field defect in the superior half of her right eye .
slit lamp biomicroscopy and fundus examination revealed intraretinal edema with inferior retinal whitening in addition to a visible hollerenhorst plaque .
fluorescein angiography ( fa ) revealed an arterial filling defect along the inferotemporal arcade , distal to the corresponding embolus with foveal ischemia ( fig .
the initial treatment involved anterior chamber paracentesis , use of an iop - lowering agent , and ocular massage .
a goldmann three mirror lens was used to focus an nd : yag laser onto the arterial embolus .
the laser energy level commenced at 0.5 mj and was increased to 1 mj ( about 12 pulses ) until the embolus was partially or completely shattered .
after shattering the embolus , a small amount of vitreous hemorrhage was noted inferior to the optic disc .
five days after treatment , fa showed no evidence of the emboli , and retinal arteriolar blood flow had been restored .
bcva improved to 20/80 . at two months after nye , bcva was 20/25 with no remaining vitreous hemorrhage . at three months after nye , bcva was 20/20 and fa showed that the branch retinal artery and all arterioles were patent with good flow . at ten months after nye ,
an 82-year - old woman presented with a four - day history of progressive decrease in va in her left eye .
her medical history was unremarkable . at presentation , bcva was 20/200 , which was lower than the reading of 20/32 taken six months earlier .
slit - lamp biomicroscopy revealed a large plaque in the associated branch retinal artery on the disc of the left eye .
fa showed an arterial filling defect along the superotemporal arcade distal to the corresponding embolus ( fig .
2 ) . a macular contact lens was used to deliver the nd : yag laser to the plaque .
pulsing commenced with one pulse at 0.5 mj , and the energy was increased to 1 mj , at which a total of eight pulses were applied to the plaque .
a small amount of vitreous hemorrhage over the arteriole was noted which resulted in cessation of the laser treatment despite the remaining embolus .
the following day , vitreous hemorrhage decreased slightly and laser treatment was recommenced ( 1 mjten pulses ) .
however , a partial embolus fragment remained in the associated arteriole . on the second day after nye ,
bcva had improved to 20/100 , and the vitreous hemorrhage remained present . on the third day after nye , fa showed restoration of the retinal arteriolar blood flow .
forty days after nye , bcva had improved to 20/32 . at four months after nye , fa showed no retinal neovascularizations , and bcva remained at 20/32 .
a 68-year - old woman presented with decreased visual acuity ( va ) , and 20 hours earlier had noticed a visual field defect in the superior half of her right eye .
slit lamp biomicroscopy and fundus examination revealed intraretinal edema with inferior retinal whitening in addition to a visible hollerenhorst plaque .
fluorescein angiography ( fa ) revealed an arterial filling defect along the inferotemporal arcade , distal to the corresponding embolus with foveal ischemia ( fig .
the initial treatment involved anterior chamber paracentesis , use of an iop - lowering agent , and ocular massage .
a goldmann three mirror lens was used to focus an nd : yag laser onto the arterial embolus .
the laser energy level commenced at 0.5 mj and was increased to 1 mj ( about 12 pulses ) until the embolus was partially or completely shattered .
after shattering the embolus , a small amount of vitreous hemorrhage was noted inferior to the optic disc .
five days after treatment , fa showed no evidence of the emboli , and retinal arteriolar blood flow had been restored .
bcva improved to 20/80 . at two months after nye , bcva was 20/25 with no remaining vitreous hemorrhage . at three months after nye , bcva was 20/20 and fa showed that the branch retinal artery and all arterioles were patent with good flow . at ten months after nye ,
an 82-year - old woman presented with a four - day history of progressive decrease in va in her left eye .
her medical history was unremarkable . at presentation , bcva was 20/200 , which was lower than the reading of 20/32 taken six months earlier .
slit - lamp biomicroscopy revealed a large plaque in the associated branch retinal artery on the disc of the left eye .
fa showed an arterial filling defect along the superotemporal arcade distal to the corresponding embolus ( fig .
2 ) . a macular contact lens was used to deliver the nd : yag laser to the plaque .
pulsing commenced with one pulse at 0.5 mj , and the energy was increased to 1 mj , at which a total of eight pulses were applied to the plaque .
a small amount of vitreous hemorrhage over the arteriole was noted which resulted in cessation of the laser treatment despite the remaining embolus .
the following day , vitreous hemorrhage decreased slightly and laser treatment was recommenced ( 1 mjten pulses ) .
the large embolus was ruptured , and recirculation around the macula was noted . however , a partial embolus fragment remained in the associated arteriole . on the second day after nye , bcva had improved to 20/100 , and the vitreous hemorrhage remained present . on the third day after nye , fa showed restoration of the retinal arteriolar blood flow .
forty days after nye , bcva had improved to 20/32 . at four months after nye , fa showed no retinal neovascularizations , and bcva remained at 20/32 .
recent reports showed immediate restoration of retinal blood flow after nye , in addition to rapid restoration of bcva .
however , complications such as vitreous hemorrhage and subretinal hemorrhage were observed.3,4 in the present cases , laser treatment resulted in dramatic improvement in bcva and restoration of retinal blood flow despite a small amount of vitreous hemorrhage .
mason et al.5 reported that visual prognosis after brao appeared to correlate with presenting va , and that eyes with an initial va of 20/40 or better usually remained at 20/40 or better .
individuals with poor va ( e.g. , 20/100 or worse ) generally did not show significant improvement .
they concluded that it may be misleading to indicate that 80% of eyes with brao improve to 20/40 or better ( as described in previous reports ) when final bcva is so closely related to presenting bcva . in conclusion
therefore , nye should be considered an effective treatment in patients with brao with visible emboli . | the purpose of this paper was to report a successful treatment of transluminal nd : yag laser embolysis ( nye ) for branch retinal artery occlusion ( brao ) with visible emboli .
two patients with acute , severe vision loss secondary to a branch retinal artery occlusion with visible emboli in one eye underwent nye .
a complete ocular examination was performed which included biomicroscopy of the posterior pole of the retina , intraocular pressure measurement , fundus color photographs , and fluorescein angiography ( fa ) .
after the nye , the two patients showed dramatic improvements in best - corrected visual acuity , as well as , immediate and dramatic restorations in flow past the obstructed arteriole in fa .
nye is a treatment modality to be considered in patients with brao who present acutely with severe vision loss and a visible embolus . | Case Reports
Case 1
Case 2
Discussion |
uterine fibroids are benign monoclonal smooth muscle tumours of the uterus , and are the most common pelvic tumour in women .
asymptomatic in at least 50 % of cases , fibroids are nonetheless an important cause of morbidity and a common reason for surgery .
a recent study estimated the annual cost of treating uterine fibroids in the united states at $ 34.4 billion dollars .
prevalence rates ranging from 5 to 77 % have been reported , reflecting differences in case definition and population studied .
fibroids are an indication for a large proportion of hysterectomies ; estimates based on pathological findings are likely to overestimate prevalence , and may preferentially identify women with specific symptoms , such as pain or bleeding . reported risk factors for uterine fibroids include black ethnicity , family history , parity , and obesity [ 1 , 2 , 911 ] . factors that increase exposure to oestrogen appear to increase the incidence of uterine fibroids .
fibroids are not seen before puberty , occur most commonly in women of reproductive age , and are commonly said to regress after the menopause . the reduction in size or resolution of fibroids in postmenopausal women [ 1012 ]
is thought to be due to the lower average levels of endogenous ovarian hormones after the menopause .
however , in postmenopausal women average oestrogen levels have been found to increase with increasing body mass index ( bmi ) .
use of exogenous hormones in the form of menopausal hormone therapy ( ht ) might also be expected to increase risk , although evidence for this has been relatively scarce .
the million women study provides a unique opportunity to examine the relationship between adiposity , ht use and the incidence of uterine fibroids in postmenopausal women .
this prospective cohort study collected detailed information at recruitment on participants menopausal status , use of ht , and height and weight .
there is virtually complete follow - up for cause - specific hospital admissions , incident cancers and death . in order to clarify the public health impact of fibroids in postmenopausal women
, we used a strict case definition , including only clinically relevant disease involving surgical detection and/or treatment .
we aimed to determine whether postmenopausal women who were overweight or obese were more likely to have surgically - confirmed fibroids than normal weight women .
we also investigated whether women who had ever used ht were at an increased risk of fibroids , and whether the use of ht modified the association with bmi .
incidence rate throughout the study , however surgically - confirmed fibroids in postmenopausal women represent a subset of true incidence ; cases in our analysis are those in which uterine fibroids , or a co - morbid condition , have resulted in a surgical diagnosis of fibroids .
the million women study is a large prospective cohort study which recruited 1.3 million women , most aged 5064 years , between 1996 and 2001 via the national health service ( nhs ) breast screening programme .
approximately one in two women in this age range in the recruitment areas agreed to participate in the study , or around one in four women in this age range in the entire united kingdom .
participants gave written informed consent for use of their questionnaire data for research , and for ongoing linkage to nationally held registry and health data .
information about personal characteristics including height and weight , reproductive history , medical history , family history , menopausal status , and ht use was collected at baseline .
postmenopausal women were defined as those who reported having undergone a natural menopause or having had a bilateral oophorectomy prior to recruitment .
body mass index ( kg / m ) was calculated from self - reported weight and height at recruitment .
this has been shown in million women study participants to correlate closely with values based on measured variables .
standard world health organization definitions were used to categorise women with a bmi of < 25 kg / m as normal , 2529.9 kg / m as overweight , and 30 kg / m or more as obese. ht use was self - reported by participants at baseline .
reliability of reporting has been checked on a subset of participants and found to be excellent when compared with general practice prescription records .
all study participants are flagged on the nhs central registers , so that cancer registrations and deaths are routinely notified to study investigators .
in addition , participants are linked using their name , date of birth , and nhs number ( a unique personal identifier on all nhs health records ) to nhs hospital admission databases , hospital episode statistics ( he s ) in england and the scottish morbidity records ( smr ) in scotland [ 18 , 19 ] .
the hospital records include day case and overnight stays to all nhs hospitals from april 1997 to march 31 , 2011 ( england ) ; and from january 1981 to december 31 , 2008 ( scotland ) . for each hospital admission ,
diagnoses are coded according to the international classification of diseases 10th revision ( icd-10 ) and procedures coded according to the office of population censuses classification of surgical operations and procedures ( opcs-4 ) .
the outcome of interest ( surgically - confirmed fibroids ) is defined as a first hospital admission ( including day - case admissions ) after recruitment into the study with a primary diagnosis of uterine fibroids ( icd-10 d25 ) together with a related surgical procedure during the admission , limited to one ( or more ) of the following : dilation of cervix uteri and curettage of uterus ( d&c , opcs-4 code q10 ) ; diagnostic endoscopic examination of uterus ( q18 ) ; abdominal hysterectomy ( q074 ) ; vaginal hysterectomy ( q089 ) ; endoscopic resection of lesion of uterus ( q171 ) ; open myomectomy ( q092 ) ; open excision of lesion of uterus ( q093 ) ; vaginal excision of lesion of uterus ( q161 ) ; or diagnostic laparoscopy ( t43 ) . women who reported a natural menopause or bilateral oophorectomy at recruitment were included in the main analysis .
exclusion criteria included self - reported hysterectomy at baseline , hospital diagnosis of fibroids or record of hysterectomy prior to recruitment .
pre- or peri - menopausal women , those with indeterminate menopausal status , and those who began using ht prior to menopause were also excluded , as were those with missing information on height , weight , ht use or recruitment date .
we conducted an additional analysis looking at rates of surgically - confirmed fibroids in women who reported being pre - menopausal or peri - menopausal at recruitment , in order to compare them with postmenopausal women of the same age ( 5054 ) .
each participant contributed person - years to the analysis from the date of recruitment until the first identified end - point : date of hospital admission with surgically - confirmed fibroids , hysterectomy , date of death , emigration , or the end of the follow - up period . for women recruited in england follow - up
was to 31 march 2011 , and for women in scotland to 31 december 2008 .
for the small proportion of women ( 5 % ) recruited in england before 1 april 1997 , person - years were calculated from this date .
earlier he s data , which was available in england from 1989 , does not carry the individual s nhs number thus we were only able to link to records from 1997 onwards .
multivariate cox regression models , with attained age as the underlying time variable , were used to estimate the relative risk of surgically - confirmed fibroids associated with bmi and ht use .
analyses were stratified for recruitment region ( 10 geographic regions corresponding to the areas covered by the cancer registries ) , and adjusted for deprivation using townsend deprivation index quintiles , smoking ( never , past , current ) , oral contraceptive use ( never / ever ) , parity ( 0/1/2/3 + ) , bmi ( < 25 , 2529 , 30 + ) , and ht use ( never vs. ever ) .
missing values were included in the analysis as a separate category for each adjustment variable .
each participant contributed person - years to the analysis from the date of recruitment until the first identified end - point : date of hospital admission with surgically - confirmed fibroids , hysterectomy , date of death , emigration , or the end of the follow - up period . for women recruited in england follow - up
was to 31 march 2011 , and for women in scotland to 31 december 2008 .
for the small proportion of women ( 5 % ) recruited in england before 1 april 1997 , person - years were calculated from this date .
earlier he s data , which was available in england from 1989 , does not carry the individual s nhs number thus we were only able to link to records from 1997 onwards .
multivariate cox regression models , with attained age as the underlying time variable , were used to estimate the relative risk of surgically - confirmed fibroids associated with bmi and ht use .
analyses were stratified for recruitment region ( 10 geographic regions corresponding to the areas covered by the cancer registries ) , and adjusted for deprivation using townsend deprivation index quintiles , smoking ( never , past , current ) , oral contraceptive use ( never / ever ) , parity ( 0/1/2/3 + ) , bmi ( < 25 , 2529 , 30 + ) , and ht use ( never vs. ever ) .
missing values were included in the analysis as a separate category for each adjustment variable .
610,604 participants in the million women study were postmenopausal , had not had a prior hysterectomy or recorded diagnosis of fibroids at recruitment , and provided information on their height , weight and ht use .
47 % were of normal weight , 36 % were overweight and 17 % were obese .
obese women were less likely to smoke , use ht , take regular vigorous exercise , or to come from the highest socioeconomic groups ( table 1).table 1participant characteristics and follow - up , by body mass index ( bmi)characteristicall womenbmi<25 kg / m
kg / m overweight30 kg / m obesenumber of women ( % ) 610,604 ( 100.0)287,208 ( 47.0)217,546 ( 35.6)105,850 ( 17.3)mean age at recruitment ( sd)57.8 ( 4.7)57.6 ( 4.8)58.0 ( 4.6)57.9 ( 4.5)mean age at menarche ( sd)13.1 ( 1.6)13.2 ( 1.6)13.0 ( 1.6)12.7 ( 1.6)past use of oral contraceptive % 52.2 ( 316,194)54.5 ( 155,394)50.9 ( 109,948)48.5 ( 50,852)mean number of full term pregnancies ( sd)2.1 ( 1.3)2.0 ( 1.2)2.2 ( 1.3)2.3 ( 1.4)nulliparous ( n % ) 12.3 ( 75,103)13.6 ( 39,107)11.0 ( 23,982)11.4 ( 12,014)current smoker ( n % ) 20.4 ( 117,234)22.8 ( 61,944)19.1 ( 39,012)16.5 ( 16,278)mean alcohol intake , units / week ( sd)3.9 ( 5.2)4.3 ( 5.4)3.8 ( 5.1)2.8 ( 4.6)ever user of ht ( n % ) 34.0 ( 207,634)36.3 ( 104,338)33.5 ( 72,889)28.7 ( 30,407)lowest quintile of socioeconomic status ( n % ) 26.3 ( 121,477)23.6 ( 49,064)26.5 ( 44,050)33.0 ( 28,363)vigorous physical exercise at least once a week ( n % ) 38.6 ( 227,161)44.1 ( 122,443)36.9 ( 77,301)27.0 ( 27,417)follow - upall women<25 kg / m normal25.029.9
kg / m obesewoman - years ( 1000s)6974330224841188mean length of follow - up ( sd)11.4 ( 2.5)11.5 ( 2.5)11.4 ( 2.5)11.2 ( 2.6)number of women with surgically - confirmed fibroids356115081273780 participant characteristics and follow - up , by body mass index ( bmi ) during 7 million person - years of follow - up , on average 11.4 years per woman , 3561 postmenopausal participants were admitted to hospital with a primary diagnosis of fibroids and a related surgical intervention .
overall , the 5-year incidence rate of surgically - confirmed fibroids in the cohort was 0.3 % , or around 1 in 300 participants .
this rate fell with age ( fig . 1 ) , from 0.50 admissions per 100 women ( 0.50 % , 95 % ci 0.450.0.55 ) at ages 5054 , to 0.11 ( 95 % ci 0.080.15 ) in women aged 7579 .
women who reported never using ht had 5-year incidence rates which increased from 0.1 % ( around 1 in 1000 ) in normal weight women to 0.3 % ( 1 in 300 ) in obese women , a three - fold increase ( fig . 2 ) .
higher rates were seen in all bmi groups in women who reported ever using ht , giving an overall rate of 0.4 % , ( or 1 in 250 ) over 5 years , with a smaller increase in rate with increasing adiposity compared with that seen in never users of ht.fig .
1five - year incidence rates per 100 women of surgically - confirmed fibroids in postmenopausal women aged 5079 , by 5-year age groupfig .
2five - year incidence rates per 100 women of surgically - confirmed fibroids by body mass index and menopausal hormone therapy use five - year incidence rates per 100 women of surgically - confirmed fibroids in postmenopausal women aged 5079 , by 5-year age group five - year incidence rates per 100 women of surgically - confirmed fibroids by body mass index and menopausal hormone therapy use at age 5054 , premenopausal women had a 5-year incidence rate of surgically - confirmed fibroids of 1.94 % ( 95 % ci 1.852.02 ) , which was about four times that seen in postmenopausal women of the same age ( 0.50 % , 95 % ci 0.450.55 ) . just over half ( n = 1810 , 51 % ) of the women with surgically - confirmed fibroids had an abdominal or vaginal hysterectomy during their admission .
a small number had another open procedure : myomectomy / excision of uterine lesion ( n = 30 , 1 % ) , and 7 % had a diagnostic laparoscopy ( n = 244 ) .
the remainder had less extensive procedures , most commonly hysteroscopy , dilatation and curettage , or vaginal or endoscopic resection of uterine lesion .
ever - use of ht increased the risk of surgically - confirmed uterine fibroids ( table 2 ) . among women who reported ever using menopausal hormone therapy ,
the relative risk was 2.33 ( 95 % ci 2.182.49 ) , compared with never users , adjusted for smoking , oral contraceptive use , parity and deprivation.table 2adjusted relative risks of surgically - confirmed fibroids by menopausal hormone therapy ( ht ) use ( ever vs. never)ht usenumber of womennumber with fibroidsadjusted rr ( 95 % ci)never402,97016071.00 ( reference group)ever207,63419542.33 ( 2.182.49 )
adjusted for smoking , oral contraceptive use , parity and deprivation , stratified by region adjusted relative risks of surgically - confirmed fibroids by menopausal hormone therapy ( ht ) use ( ever vs. never )
adjusted for smoking , oral contraceptive use , parity and deprivation , stratified by region body mass index also had a significant impact on risk ( table 3 ) . compared with women of normal weight , those who were overweight ( bmi 2529.9 ) had a rr of 1.15 ( 95 % ci 1.071.24 ) , and those who were obese ( bmi 30 + ) a rr of 1.46 ( 95 % ci 1.331.59 ) , adjusted for smoking , oral contraceptive use , parity and deprivation.table 3adjusted relative risks of surgically - confirmed fibroids by body mass index ( bmi)bminumber of womennumber with fibroidsadjusted rr ( 95 % ci)<25287,20815081.00 ( reference group)2529.9217,54612731.15 ( 1.071.24)30 + 105,8507801.46 ( 1.331.59 )
adjusted for smoking , oral contraceptive use , parity and deprivation , stratified by region adjusted relative risks of surgically - confirmed fibroids by body mass index ( bmi )
adjusted for smoking , oral contraceptive use , parity and deprivation , stratified by region while both ht use and bmi significantly increased risk of surgically - confirmed fibroids in postmenopausal women , when we looked at the interaction between the two risk factors , it was clear that bmi had a stronger effect in never - users of ht than in those who reported use of ht ( table 4 ) . in never users of ht ,
there was a doubling of risk associated with obesity , rr 2.00 ( 95 % ci 1.772.26 ) compared with that seen in normal weight women . in women who reported
ever having used ht , the absolute risks overall were much higher , although the increase in relative risk associated with rising bmi was smaller .
the additional increase in risk associated with adiposity was still present , but the magnitude of this effect was smaller in women who used ht.table 4adjusted relative risks of surgically - confirmed fibroids by hormone therapy use and body mass indexmenopausal hormone therapy usebminumber of womennumber with fibroidsadjusted rr ( 95 % ci)never<25182,8705761.00 ( reference group)2529.9144,6575721.30 ( 1.161.46)30 + 75,4434592.00 ( 1.772.26)ever<25103,3389322.78 ( 2.503.08)2529.972,8897013.05 ( 2.733.41)30 + 30,4073213.30 ( 2.863.79 )
adjusted for smoking , oral contraceptive use , parity and deprivation , stratified by region adjusted relative risks of surgically - confirmed fibroids by hormone therapy use and body mass index
adjusted for smoking , oral contraceptive use , parity and deprivation , stratified by region the fully adjusted relative risk of fibroids in ever users of ht of normal bmi was 2.78 ( 95 % ci 2.503.08 ) , which rose to 3.05 ( 95 % ci 2.733.41 ) in the overweight group , and further to 3.30 ( 95 % ci 2.863.79 ) in women who were obese ( table 4 ) .
we examined the relationship between adiposity , menopausal hormone therapy and the risk of surgically - confirmed uterine fibroids in postmenopausal women amongst participants in a large uk prospective cohort study .
use of ht doubled the risk of incident surgically - confirmed uterine fibroids in postmenopausal women at any bmi .
adiposity alone had a lesser , but still significant effect , with a 46 % increase in risk of fibroids in obese women irrespective of history of ht use .
when stratified by ht use , increasing adiposity had a much stronger effect in women who had never used ht , with a doubling of risk in the obese group .
the effect of bmi was also present in ever users of ht , but they had much higher risks at any bmi , and the additional effect of increased adiposity was smaller .
other groups have reported that uterine fibroid risk is increased in overweight and obese women , although published findings are inconsistent .
the majority of studies show an increasing risk with increasing bmi [ 11 , 2224 ] but some have reported a j - shaped association [ 2527 ] , no association [ 2830 ] , or even a decreased risk in obese women .
two other cohort studies , the nurses health study ii and the black women s health study used surgically - defined cases , and both found strong associations between bmi and the risk of fibroids , which our findings support .
there has been comparatively little published on the effect of ht on uterine fibroid risk .
several small studies with short - term follow - up have been published , and results have been contradictory . for example , yang et al
. followed 37 early postmenopausal ht users and 35 matched controls who did not receive ht .
fibroids were measured by transvaginal ultrasound at baseline and then annually for 3 years . in the 3rd year
there was a significant increase in the fibroid volume in the ht group , but not in the control group .
the women treated with ht were also significantly more likely to have an increase in fibroid volume of > 25 % , however numbers were small .
a review by parker which included yang s study , concluded that despite being hormone - sensitive tumours , for the majority of postmenopausal women with fibroids hormone therapy did not stimulate uterine or fibroid growth .
two previous epidemiological studies that have examined the association between fibroids and ht use both reported increased risks . in a retrospective case
progestagen ht was associated with an increased risk of fibroids , although the excess risk was largely confined to women who were not overweight ( with bmi < 24 kg / m ) . in the california teachers
study women taking ht had a higher risk of surgically treated fibroids than women who had never used ht .
an increased risk was seen both in women who used oestrogens alone ( rr = 2.03 , 95 % ci 1.173.52 ) , and in those using combined ht ( rr = 2.38 , 95 % ci 1.663.41 ) , similar to our overall findings .
early menarche and obesity increase the incidence of fibroids , whereas high parity appears protective .
fibroids are hormone - sensitive tumours , and so it is perhaps not surprising that use of ht doubles incidence rates in postmenopausal women .
adiposity is thought to affect the risk of fibroids in postmenopausal women because peripheral tissues , principally body fat , become the major source of circulating oestrogens ( when women are not taking ht ) .
rates fall with age , and in women 5054 , the only age group which includes substantial numbers of both premenopausal and postmenopausal women , menopausal status affects incidence .
at all ages , uterine fibroids occur more frequently in postmenopausal women who are obese , and those who use ht .
data in our study were collected prospectively , and we limited our analysis to women with a primary diagnosis of uterine fibroids during their hospital admission who also had a related operative procedure .
a limitation of our study is that we were not able to assess whether having a raised bmi or using ht increased the risk of developing new fibroids in the postmenopausal period , or whether these factors decrease the likelihood of regression or encourage growth of pre - existing fibroids .
over half of the postmenopausal women with surgically - confirmed fibroids in our cohort underwent a hysterectomy or other major abdominal surgical procedure associated with their disease .
approximately 50,000 hysterectomies are performed annually in the uk . at around 3282 per case ,
the 1810 hysterectomies in our study alone would have cost the nhs about 6 million .
the cost in terms of quality of life , lost earnings , and morbidity to the women may have been even greater .
surgically - confirmed fibroids continue to occur in postmenopausal women , especially those who are obese or using menopausal hormone therapy . | to examine the association between body mass index ( bmi ) , use of menopausal hormone therapy ( ht ) , and incidence of uterine fibroids in postmenopausal women , 610,604 postmenopausal women without prior hysterectomy or diagnosis of fibroids were followed as part of a large united kingdom prospective cohort study .
we used cox regression models to calculate adjusted relative risks ( rrs ) of surgically - confirmed fibroids ( defined as a hospital admission with uterine fibroids as a primary diagnosis with a related surgical procedure ) , in relation to bmi and use of ht . during an average of 11.4 years of follow - up
, 3561 women were admitted to hospital with surgically - confirmed fibroids .
five - year incidence rates decreased with age , from 0.50 % ( 1 in 200 women ) at age 5054 , to 0.11 % ( 1 in 1000 women ) at age 7579 .
the 5-year rate in postmenopausal women aged 5054 was about a quarter that seen in premenopausal women of the same age ( 1 in 200 vs. 1 in 50 ) .
compared with normal weight women , obese women had a rr of surgically - detected fibroids of 1.46 ( 95 % ci 1.331.59 ; p < 0.0001 ) .
ht use was associated with a rr of 2.33 ( 95 % ci 2.182.49 ; p <
0.0001 ) in ever versus never users .
when we analysed ht use and bmi together , obese vs. normal weight never users had a rr of 2.00 ( 95 % ci 1.772.26 ) : the highest risks were seen in women who were obese and had ever used ht , rr = 3.30 ( 95 % ci 2.883.79 ) .
uterine fibroids continue to occur in postmenopausal women ; obesity and hormone therapy use are important modifiable risk factors . | Introduction
Materials and methods
Statistical analysis
Results
Discussion |
during august and september 2015 , an outbreak of mers - cov infection occurred in hospitals in jordan ( 10 ) .
mers - cov positive throat swab or bronchiolar lavage samples were obtained from each patient ; of these , 13 samples were labeled jordan-1 - 2015 through jordan-13 - 2015 and shipped on dry ice to erasmus university medical center ( rotterdam , the netherlands ) for testing .
total rna was isolated from 140 l of each sample by use of a qiaamp viral rna mini kit ( qiagen , hilden , germany ) and quantified by taqman assay targeting the envelope gene , as described previously ( 11 ) .
full mers - cov genome sequencing was performed on the sample with the highest viral load , obtained on august 24 , 2015 , from a 60-year - old man who died of the disease 3 days later . at the end of july 2015
, this man had traveled from jeddah , saudi arabia , to jordan for his annual vacation . from the throat swab sample from this man ,
76,082 sequence reads of which 851 were specific for mers - cov were obtained by using 454 deep - sequencing ( 12 ) , revealing 85% of the mers - cov genome with coverage of 1178 reads at single - nucleotide positions .
missing sequences and low - coverage regions were obtained by conventional sanger sequencing , except for 6 nt at the 5 end .
phylogenetic analysis of the complete viral genome of this sample revealed that this virus , tentatively called jordan-1 - 2015 , clusters with viruses detected in humans in riyadh in 2015 ( figure 1 ) .
alignment of the full genomic sequences of this virus with the riyadh 2015 isolates showed that they were > 99.7% similar .
amino acids in the spike protein were the same in jordan-1 - 2015 and the consensus sequence of the riyadh 2015 viruses .
of note , jordan-1 - 2015 contained a 48-nt ( 16 aa ) in - frame deletion in open reading frame ( orf ) 4a that had not been detected in any other mers - cov ( figure 2 , panel a ) .
this orf is present only in lineage 2c betacoronaviruses and encodes protein 4a , which has recently been shown to inhibit type i interferon production , presumably by binding and masking double - stranded rna from detection by rna sensors such as rig-1 ( retinoic acid inducible gene 1)like helicases and the double - stranded rna - binding protein pact ( protein activator of the interferon - induced protein kinase ) ( 13,14 ) .
subsequent sequence analysis showed that all 13 viruses from patients sampled during this outbreak contained the deletion and that the flanking regions were identical , indicating that this deletion mutant was transmitted ( figure 2 , panel b ) .
phylogenetic analysis of middle east respiratory syndrome coronavirus ( mers - cov ) isolated from jordan ( jordan-1 - 2015 ; boldface ) compared with reference strains .
genome sequences of representative isolates were aligned by using clustalw , and a phylogenetic tree was constructed by using the phyml method in seaview 4 ( http://pbil.univ-lyon1.fr/software/seaview ) ; the tree was visualized by using figtree version 1.3.1 ( http://tree.bio.ed.ac.uk/software/figtree ) .
values at branches show the result of the approximate likelihood ratio ; values < 0.70 are not shown .
genomic characterization of middle east respiratory syndrome coronaviruses ( mers - covs ) from jordan .
a ) nucleotide differences between mers - cov strain jordan-1 - 2015 and the consensus sequence of the riyadh 2015 cluster .
nucleotide changes are indicated in green ( a ) , red ( t ) , blue ( c ) , and black ( g ) .
b ) protein sequence alignment of open reading frame ( orf ) 4a ( residues 1076 ) of all 13 mers - cov strains from jordan in 2015 compared with the consensus sequence of the riyadh 2015 cluster .
no nucleotide substitutions were observed in this region between mers - covs from jordan . predicted secondary structures are indicated ( 13 ) .
c ) protein sequence alignment of orf3 ( residues 2577 ) of all 13 mers - cov strains from jordan in 2015 compared with the consensus sequence of the riyadh 2015 cluster .
alignments were generated with the clustalw program ( http://pbil.univ-lyon1.fr/software/seaview ) and visualized by using jalview 2.9 ( http://www.jalview.org/ ) .
boxes in panels b and c indicate regions where amino acids have been deleted in the viruses from jordan . to further characterize the viruses in the clinical samples , we subjected all samples to titration on vero cells ( american type culture collection no .
we observed cytopathic changes in cells inoculated with samples jordan-1 - 2015 and jordan-10 - 2015 ( obtained on september 17 from a 29-year - old man , who survived the illness ) ; however , no cytopathic changes were observed in the cells inoculated with the other samples .
subsequently , we obtained complete genomes by deep sequencing of the second - passage viruses , with high coverage of reads at single - nucleotide positions .
no cell culture adaptive mutations in the genome sequence were observed for the virus isolated from clinical sample jordan-1 - 2015 .
pairwise comparison of jordan-1 - 2015 with jordan-10 - 2015 revealed that both viruses were highly similar ( 99.99% nt identity ) and confirmed that both carried the 48-nt deletion in orf4a .
however , in addition to this deletion , a 9-nt ( 3-aa ) in - frame deletion was observed in the genome of jordan-10 - 2015 .
this deletion was located in orf3 , which encodes a protein with an unknown function .
the presence of this deletion in the virus from clinical sample jordan-10 - 2015 was confirmed by sanger sequencing , excluding the possibility that it was an adaptation to cell culture .
next , sequencing of orf3 from all other clinical samples revealed that the orf3 deletion was present in a subgroup of clinical samples ( figure 2 , panel c ) .
these samples had been collected toward the end of the outbreak , suggesting that the orf3 deletion may be an adaptation resulting from sustained transmission between humans .
identical deletions in orf3 and orf4a in the same sample set from jordan were independently confirmed by erasmus university medical center and the centers for disease control and prevention ( atlanta , ga , usa ) .
despite the human - to - human transmission capacity of mers - cov and substantial sequencing efforts , major genomic changes associated with human adaptation have not been described ( 38 ) .
we observed a large - scale deletion in orf4a and a small deletion in orf3 in mers - covs that caused the outbreak in jordan .
one of the most striking genomic changes observed in sars - cov isolated from humans soon after its transmission from animals was the acquisition of a 29-nt deletion in orf8 , an accessory protein with an unresolved function ( 9 ) .
the sars - cov orf8 deletion disrupted the reading frame of the early - stage human isolates and created 2 new orfs , designated orf8a and orf8b .
although no clear roles have been designated for both orfs , viruses containing this deletion predominated later during the epidemic , showing that these viruses were able to spread efficiently from human to human .
the deletion we describe in orf4a of mers - cov does not result in the removal of an entire gene , because the reading frame is not disrupted , suggesting that this mutation is not a loss - of - function mutation . however , whether this orf4a deletion variant can still bind double - stranded rna needs to be assessed because the deleted region contains a predicted -sheet belonging to the classical double - stranded rna binding fold of this protein ( 13 ;
figure 2 , panel b ) .
the data from this study indicate that adaptive pressures possibly exerted by the human host may operate on the orf4a and orf3 regions .
alternatively , proteins encoded by these orfs could have functions specific for the camel host , causing them to be redundant in humans and enabling accumulation of mutations that do not affect viral fitness in humans .
future studies of the roles of these proteins are needed . the deletion located in the type
another small - scale deletion was detected in orf3 in a subset of samples collected later in the outbreak .
overall , the data suggest that mers - cov orf4a and orf3 can acquire deletions by selection or by chance .
whether these deletions affect the transmissibility or pathogenicity of this particular mers - cov strain needs to be addressed .
the finding that all viruses analyzed contained the orf4a deletion suggests that all patients were infected with the same virus .
although unlikely , some mers patients may have indirectly become infected through independent transmission of viruses in dromedaries carrying highly related viruses , some of which acquired deletions in their genome . therefore , a more detailed epidemiologic investigation of this outbreak is needed .
these data underscore the need for close monitoring of the molecular evolution of mers - cov . | we characterized middle east respiratory syndrome coronaviruses from a hospital outbreak in jordan in 2015 .
the viruses from jordan were highly similar to isolates from riyadh , saudi arabia , except for deletions in open reading frames 4a and 3 .
transmissibility and pathogenicity of this strain remains to be determined . | The Study
Conclusions |
a cross - sectional descriptive study was performed with two hundred patients who referred to a psychiatry center and underwent methadone maintenance therapy in the city of sari between 2006 and 2009 .
it should be noted that , only a small number of eligible patients were available in the clinic .
therefore , patients data were studied first and then completed by an interview ; this process was time consuming .
patients were selected through random sampling and data were obtained by a last year medical student through an interview and filling a questionnaire .
the number of samples was calculated using krejcie and morgan table and review of recent studies ( references ) .
two questionnaires were used in this study : the first was about demographic characteristics , a history of drug abuse such as opium , alcohol , etc .
, a history of drug usage which caused erectile dysfunction , type and amount of use of methadone , a history of mental disease and a history of high risk behavior ; the second questionnaires was about erectile problems .
in international index of erectile function ( iief ) questionnaire ( 33 , 34 ) , there are 15 questions that contain questions about orgasm , sexual desire , erectile function and satisfaction .
persons who had a history of erectile dysfunction and took drugs for erection such as diuretics , anti - cholinergic agents and anti - psychotics , and patients with chronic diseases like dm and esrd were excluded .
also , the patients who had a history of arthrosclerosis based cardio - vascular problems such as mi , cva and peripheral vessels diseases were excluded from the study , because these problems can cause erectile dysfunction . before the study , the nature and main purpose of the study
data were analyzed with descriptive statistics such as measures of variability and central tendency and also chi - squared ( ) test using spss ver.18.0 software .
persons who had a history of erectile dysfunction and took drugs for erection such as diuretics , anti - cholinergic agents and anti - psychotics , and patients with chronic diseases like dm and esrd were excluded .
also , the patients who had a history of arthrosclerosis based cardio - vascular problems such as mi , cva and peripheral vessels diseases were excluded from the study , because these problems can cause erectile dysfunction . before the study ,
data were analyzed with descriptive statistics such as measures of variability and central tendency and also chi - squared ( ) test using spss ver.18.0 software .
the study included two hundred addicted men with the mean age of 39.5 years ( sd = 11.304 ) . in terms of age ,
the most common age was 50 years ( 21 patients ) , 32 years ( 11 cases ) , 30 ( 9 cases ) and 34 years ( 9 cases ) , respectively .
most patients ( 142 patients ) lived in the urban and 58 ( 29% ) lived in the rural areas . in this study , most cases were married ( 156 patients ) . in terms of education level ,
86 persons ( 43% ) had an elementary school degree , 34 cases ( 17% ) have studied at high school , 44 ( 22% ) had finished high school and 36 ( 18% ) persons had higher education ( table 1 ) .
unfortunately , due to the inappropriate response of the patients to the prepared questionnaire , some data such as the time and amount of drug abuse were not available .
the review of the history of drug abuse showed that 155 patients used opium that allocated the most statistics .
of the total patients , almost 127 cases were smoker , 80 used crack , 62 used crystals and 46 patients consumed cannabis .
the main input substance of most patients was opium ( 103 cases ) and crack ( 65 cases ) , respectively ( figure 1 ) .
based on the findings of this study the following results were obtained in terms of risky behaviors of the subjects : 32 ( 16% ) patients had a history of drug injection ; among the total subjects , 38 ( 19% ) had suspicious sexual relations in the past year ; during the past year , 27(13.5% ) patients had a history of imprisonment for at least one time ; and 20(10% ) patients mentioned an experience of physical beatings and conflict . based on the results of the interview , 43(21.5% ) patients had a history of chronic disease ; of whom , 40(20% ) received medical treatment for their physical illness .
associated psychiatric disorders were determined from patients records and based on dsm - iv criterion for diagnosis : 67(33.5% ) patients had depression , 63(31.5% ) had anxiety , 29(14.5% ) had illusion , 68(34% ) had aggressive behavior , 14 ( 7% ) had self- injuring behavior , 24 ( 12% ) had a history of suicide and 19(9.5% ) were hospitalized for their psychiatric disorders .
laboratory results of hiv and hcv tests in patients record showed that only 2(1% ) persons were hiv positive and 1)0.5% ) was hcv positive . in this study
, we also reviewed the occupation of the patients and we found that 66(33% ) had full - time jobs , 67(33.5% ) were unemployed and 49(24.5% ) had part - time jobs .
also , 3 patients ( 1.5% ) were under education and 15(7.5% ) were retired .
an interesting point in the results of our interviews was the rate of substance abuse in the first degree relatives ; 41 patients mentioned this disturbing history in their family ( table 2 ) .
the patients who had erectile dysfunction were 36.81 years old in average , and the persons who did not have this problem were 40.34 years old in average .
the results showed that among the 156 patients who used opium , 37 became impotent . in the meantime , among the patients who used heroin ( 6 persons ) , crack ( 26 persons ) , neurjizak ( 2 persons ) , teramadol , codeine and dyphnoxilate ( 2 persons ) , alcohol ( 17 persons ) , canabis ( 17 persons ) , metamphetamine ( 5 persons ) , sedatives ( 2 persons ) , cocaine ( 1 person ) , hallucination agents ( 2 persons ) and cigarette and tobacco , 28 persons became impotent ( table 3 ) .
table 4 shows the distribution of different sexual problems based on iief which was filled by all the patients .
the results showed that 19% of the patients had severe problems in erectile function , 20.5% in intercourse satisfaction , 19.5% in orgasm , 19.5% in desire and 20.5% in overall satisfaction .
moreover , the moderate , moderate to mild , mild and no dysfunction cases were studied thoroughly and their data are demonstrated in table 5 .
the overall results showed that 27 persons ( 13.5% ) had severe erectile dysfunction , 26 ( 13% ) had moderate problems , 66 ( 33% ) had mild to moderate problems , 53 ( 26.5% ) had mild problems and 28 persons ( 14% ) had no erectile dysfunction ( table 5 ) .
the overall prevalence of erectile dysfunction in our study was pointed 30 and less which covers the severe and moderate cases ; the number of patients with erectile dysfunction was 53 ( 26.5% ) .
erectile dysfunction , as a public health problem , affects the quality life of the families .
the prevalence of erectile dysfunction in the general population is about 16.1% or more ( 8 , 9 ) , and in diabetic population and esrd is reported to be up to 90% .
psychotropic drugs show their adverse effect through central inhibitory neuroendocrine mechanisms and local neuromuscular junction , and they also affect the hormone system ( 10 , 11 ) .
the relationship between drugs and erectile dysfunction is not always direct , but depends on social , cultural and other peripheral factors .
therefore , this theorem can rationalize the differences between the prevalence of this dysfunction in different studies .
just a few studies have been done on the synergism effects of different drugs on erectile dysfunction ; this problem has led us to study all the drugs in this paper thoroughly . in this paper , the impact of each drug and prevalence of erectile dysfunction have been studied and the mechanism of this dysfunction has been reported
. the results of this study show that erectile dysfunction , as a side effect of mmt in drug abusers , can be of concern .
it should be noted that the direct relationship between methadone usage and erectile dysfunction was not evaluated in this study .
they conducted a study in pursuit of finding the etiology of hypogonadism caused by methadone .
they chose 103 addicted males and found that the amount of free testosterone in the males under mmt decreased . in our paper ,
erectile dysfunction due to each drug and their total prevalence in the society on the basis of iief were studied , but the mechanism of dysfunction was not . in a study done by brown et al .
( 13 , 14 ) , age was found to be a very important risk factor for erectile dysfunction with mmt .
the age range of the subjects in our study was 18 to 55 with the average of 39.5 ; of the total subjects in this study , 26.5% had erectile dysfunction , whereas in study of gianluca quaglino ( 15 ) the subjects were 18 to 47 with the average of 31 and 42% of them had this problem .
besides , in a study ( 2000 ) conducted on italian males aged 18 to 39 years of age , this number was 2% ( 16 ) .
the differences between the result of our study and that of others may be due to the existence of some factors like sample size , social condition and different human populations .
the mean age of the patients with ed were lower than those of non - infected patients and this difference could be due to sexual activity in the younger patients . in this study ,
used 60 patients in their study which definitely could not be representative of the majority of the population . in our paper , those drugs that caused erectile dysfunction were studied .
most of the patients had a history of opium misuse ; of whom , 37 patients ( 23.71% ) had erectile dysfunction . in another study performed by venkat chekuri et al .
long duration of opium addiction can block the gonadotropin releasing hormone and therefore decreases the testosterone and ultimately decreases sexual desire in males .
in contrast to other countries where opium derivations are used most frequently , in iran opium is used directly ; and unfortunately , the specialists and physicians do not pay enough attention to the side effects of mmt .
the prevalence of erectile dysfunction due to heroin addiction was 30% in our study which is close to other studies that have found it to be 39 to 48 percent .
nicotine rises in blood after smoking and increases the sympathetic tone in penis and interacts with relaxation of smooth muscles in corpus cavernous ( 21 , 22 ) .
wolf and shalman ( 23 ) concluded that changes in relaxing factors derived from endothelium , no , prostaglandin , prostacyclin and tromboxan are important in cigarette related erectile dysfunction . in their study , the prevalence of erectile dysfunction in cigarette smokers was about 22.7% ; in this study , it was 22.04% .
getting everyone to quit smoking should be seen as a goal to reduce erectile dysfunction .
survey on alcoholic males showed that amount , frequency and duration of alcohol use are related to erectile dysfunction and libido ( 24 ) . in our study , the prevalence of erectile dysfunction due to alcoholism was 27.41 , while ofarrell reported the minimum prevalence of alcoholism in these people to be 25% .
amphetamines increase the cns function by stimulating , releasing or inhibiting uptake of norepinephrine and dopamine .
long duration of using these drugs causes a condition called crystal dick , with strong libido , high energy and decrease in sexual blockage , but the person can not reach to full erection ( 26 , 27 ) .
bang - ping ( 20 ) and colleagues have reported the prevalence of erectile dysfunction to be 34.5% .
the effect of mdma ( ecstasy ) is due to releasing serotonin and inhibiting dopamine uptake .
zemishlany(28 ) reported that these substances have about 40% negative and 40% positive effect on erectile function .
cocaine causes erection malfunctions through hyperprolactinemia and negative feedback of hypothalamic dopamine receptors ( 29 , 30 ) .
mcduland ( 31 ) reported that 66% of patients who used cocaine for more than one year became impotent . in a very vast study done about canaboids ( marijuana , hashish )
it was reported that 70% of males who used these substances showed increase in sexual satisfaction ( 32 ) .
the impacts of long duration of these drugs are still ambiguous ( 24 ) . in our study , according to the ieff questionnaire , moderate and sever data were considered as erectile dysfunction . in terms of sexual desire , 29.5% of the patients had problems ; this number was lower than what was fund in the study of venkat chekuri et al .
( 38.4% ) ( 18 ) . also , in this study , 33.5% of the patients had an orgasm dysfunction that was roughly similar to the study of quaglio et al .
, 13.5% of the patients had severe and 13% had moderate dysfunction ; while these problems in hallinan r ( 4 ) study were 53% and 26% , relatively .
the frequency of erectile dysfunction in our study was 26.5% which was more than the result of brown .
statistical differences between the result of our study and that of others may due to the differences in the number of samples and the diagnostic tools .
there was no simultaneous access to all patients , so this limitation resulted in prolonged duration of the study .
the limitations of this study were as follow : some abused drugs had low range of usage in the patients ; therefore , it was not possible to make comparisons between groups .
a noticeable part of this survey was the large sample size and studying erectile dysfunction caused by each drug . however , one of the limitations of this study was sampling of people coming to only one of the mmt centers in sari .
the followings are highly recommended for the future studies : larger sample size , conducting studies in multiple centers in the province , and even in all parts of the country .
also , a very extensive cohort study should be conducted to investigate the erectile dysfunction and evaluate the hormones , neurotransmitters , some blood substances changes and morphologic and physiologic changes in testis and penis in persons using illegal drugs .
in this study , all the factors related to sexual function such as erectile function , sexual desire , orgasm , sexual and overall satisfaction have decreased considerably .
this study proved that erectile dysfunction and other sexual dysfunctions have a high prevalence in drug abuser males .
this issue has not drawn enough attention yet and is still unknown in the public health .
this problem has its own effects on patients health and worsens the prognosis of drug abuse .
therefore , they should be warned about the side effects and dysfunctions caused by the medications used at these centers .
moreover , this issue should be recognized as a protective factor to prevent addiction in the future . | objectiveerectile dysfunction affects quality of life and is a common dysfunction in drug abusers .
the aim of this study was to evaluate the frequency of erectile dysfunction in drug abusers on methadone maintenance therapy to reduce this drug side effect in the future studies.methodsthis cross - sectional study was conducted with two hundred addicted individuals on methadone maintenance therapy .
erectile dysfunction was surveyed using the international index of erectile function ( iief ) .
finally , all data were collected and analyzed by descriptive statistics such as measures of variability and central tendency and chi - squared ( 2 ) test using spss version18.0 software.resultsthe amount of the total frequency of erectile dysfunction was pointed 30 or less among the two hundred patients and included the moderate and severe cases . in this study ,
the number of patients with erectile dysfunction was fifty three ( 26.5%).conclusionthe frequency of erectile dysfunction in our study was approximately 1.5 times of prevalence of the public ( 16.1% ) . in this study , all the factors related to sexual function such as erectile function , libido , orgasm , and sexual pleasure showed a decline among drug abusers . | Material and Methods
Inclusion criteria
Exclusion criteria
Result
Discussion
Conclusion |
we reviewed final reports of nontyphoidal salmonella outbreaks investigated by cdc from 1984 to 2002 .
we excluded outbreaks that occurred in a healthcare setting or outside the united states , including on cruise ships . when antimicrobial susceptibility was not recorded in the final report , we searched cdc microbiology records for susceptibility test results on isolates collected as part of the outbreak .
outbreaks were only included if susceptibility data were available for > 1 isolate . because different laboratories performed susceptibility testing , the antimicrobial agents tested and the methods used varied between outbreaks .
we classified outbreaks as resistant when the outbreak strain was resistant to 1 antimicrobial agent ; other outbreaks were considered pansusceptible .
outbreaks caused by resistant strains were additionally classified as r - type ac / kssut when the outbreak strain was at least resistant to ampicillin , chloramphenicol or kanamycin , streptomycin , sulfamethoxazole , and tetracycline .
outbreaks were additionally classified as resistant to a clinically important agent when the outbreak strain was at least resistant to ampicillin , trimethoprim - sulfamethoxazole , aminoglycosides , fluoroquinolones , or a third - generation cephalosporin .
data from the final investigative reports were used for the analysis . when analyzing data according to outbreaks , we calculated the medians for percentage hospitalized and percentage who died and compared medians with the wilcoxon rank - sum test . when analyzing data according to ill persons , we pooled data from the reports , calculated proportions , and compared proportions with chi - square or , when appropriate , fisher exact test .
the denominators for percentage hospitalized and died varied depending on the number of persons in whom outcome data were ascertained .
data were analyzed by using sas v.9 ( sas institute , cary , nc , usa ) . from 1984 to 2002 , cdc investigated 48 community outbreaks of nontyphoidal salmonella strains in the united states .
of these , 47 ( 98% ) had a final report available for review ( table a1 ) .
we restricted our analyses to the 39 ( 83% ) outbreaks in which data about antimicrobial susceptibility were available .
the largest outbreak occurred in 1985 , in which culture - confirmed s. typhimurium infection associated with milk consumption developed in 16,659 persons ( 11 ) .
strains from 11 ( 28% ) outbreaks were resistant , and 28 ( 72% ) were pansusceptible .
the 11 outbreaks caused by resistant strains involved 18,698 persons . of these 11 outbreaks ,
7 ( 64% ) , involving 17,182 persons , had strains that were at least r - type ac / kssut , and 9 ( 82% ) , involving 17,919 persons , had strains that were resistant to a clinically important agent .
the hospitalization rates were higher for each type of outbreak caused by resistant salmonellae compared with outbreaks caused by susceptible salmonellae ( p<0.01 for each comparison ) ( table ) . to account for differences in the size of outbreaks
, we compared the median proportion of persons hospitalized and compared hospitalization rates after excluding a large salmonella outbreak that occurred in 1985 .
the median proportion hospitalized for each type of outbreak caused by resistant strains ( 26% ) was > 2.5 times higher than the median proportion hospitalized for outbreaks caused by pansusceptible strains ( 10% , p<0.05 for all resistance patterns ) .
the difference in hospitalization rates between outbreaks caused by resistant and susceptible strains was similar after we excluded the large 1985 outbreak of resistant s. typhimurium , in which the percentage hospitalized was 22% .
the results also remained similar after excluding s. enteritidis outbreaks , in which rates of hospitalization and isolate resistance were low . * ac / kssut , ampicillin , chloramphenicol , kanamycin , streptomycin , sulfamethoxazole , tetracycline .
mortality data were available for 24 outbreaks involving 21,927 persons . a greater proportion of persons died in resistant outbreaks than in pansusceptible outbreaks , but the difference was not significant ( 0.1% in outbreaks caused by resistant strains vs. 0.06% in outbreaks caused by pansusceptible strains , p = 0.57 ) ( table ) .
three ( 38% ) outbreaks were due to s. enteritidis and 2 ( 25% ) to s. typhimurium . in the 6 outbreaks for which hospitalization data were available , 70 ( 20% )
of 353 persons were hospitalized . in the 4 outbreaks for which mortality data were available , 7 ( 0.4% ) of 1,708 persons died .
outbreaks caused by antimicrobial - resistant , nontyphoidal salmonellae were associated with an increased rate of hospitalization compared with outbreaks caused by pansusceptible salmonellae .
persons who take antimicrobial drugs for reasons unrelated to gastroenteritis have an increased risk of developing antimicrobial - resistant salmonella infections ; such patients may be taking antimicrobial drugs because they have medical conditions that increase their risk for hospitalization ( 79 ) .
we doubt that this explains the differential hospitalization rate observed in our study , because the outbreaks we studied occurred in diverse community settings .
a second explanation for the higher hospitalization rate is that persons with resistant salmonella infections may fail empiric antimicrobial treatment , and their physicians subsequently hospitalize them for inpatient therapy .
a third explanation is that resistant salmonellae may be more virulent because of some unknown factor . in the united states ,
resistant salmonellae are more often associated with hospitalization and bloodstream infection compared to pansusceptible salmonellae ( 13 ) . in canada and denmark , studies have also found excess death rates associated with resistant salmonella infection ( 14,15 ) . in england and wales ,
a study found no association between resistance and bloodstream infection , but that study had substantial limitations , including the failure to use pansusceptible salmonellae as a referent group and the failure to adjust for confounders , such as age ( 16 ) . since the review of salmonella outbreaks published in 1984 , several changes have occurred in the epidemiology of antimicrobial - resistant salmonellae .
first , s. typhimurium dt104 has emerged as a cause of antimicrobial - resistant salmonella infections .
five outbreaks in this study were caused by s. typhimurium with r - type ac / kssut ; isolates with this resistance pattern are often dt104 ( 2,17 ) .
the hospitalization rate was higher in these 5 outbreaks ( median proportion hospitalized : 17% ) than in 3 outbreaks caused by pansusceptible s. typhimurium , which suggests that resistance or related factors , rather than just serotype , contribute to the differential rates of hospitalization .
second , a strain of s. newport resistant to at least 9 agents and with diminished susceptibility to ceftriaxone recently emerged in the united states ; 1 outbreak in this review was caused by this strain and had a hospitalization rate greater than that seen in most other resistant outbreaks ( 18 ) .
third , s. enteritidis has emerged as a frequent cause of foodborne outbreaks ( 19 ) .
s. enteritidis strains are infrequently resistant to antimicrobial agents , and in this review , s. enteritidis outbreaks were associated with low hospitalization rates . excluding s. enteritidis outbreaks did not change the results of our analysis . the previous analysis by holmberg et al .
found that the rate of death was significantly greater in outbreaks caused by resistant strains ( 4% ) compared with outbreaks caused by pansusceptible strains ( 0.2% ) ( 10 ) .
because this study covered a more recent period , improvements in medical care may explain the overall lower death rates .
studies with larger sample sizes are needed to determine whether antimicrobial resistance is associated with increased fatality rates .
state health departments investigate hundreds of salmonella outbreaks annually . occasionally , state health departments request assistance from cdc in investigating these outbreaks .
the reasons for such requests vary but may be related to size , severity , setting , or other features of the outbreak . while concern about a high hospitalization rate might encourage a state health department to request assistance from cdc in a salmonella outbreak , susceptibility results are usually not known initially and are unlikely by themselves to influence the request for assistance .
another limitation is that our study was based on final reports , not raw data . as a result
, we were unable to adjust for the different patient populations affected , including age , medical condition , and other factors that could affect hospitalization .
excluding outbreaks that occurred in healthcare settings may have reduced some of this bias . adjustment for serotype , which could also affect hospitalization rates , was also not possible because of the small sample size . despite these limitations
, we believe this study adds to the weight of evidence about the health effects of antimicrobial - resistant salmonellae .
the evidence now includes data from outbreaks and sporadic illness in the united states from 1970 to 2002 .
data from sporadic illness and 1 outbreak is also available from denmark ( 14,20 ) . across these diverse studies ,
such data about the adverse human health effects of resistant salmonellae should be incorporated into programs that promote appropriate antimicrobial use in humans and animals . | few studies have evaluated the health consequences of antimicrobial - resistant salmonella strains associated with outbreaks . among 32 outbreaks occurring in the united states from 1984 to 2002 ,
22% of 13,286 persons in 10 salmonella - resistant outbreaks were hospitalized , compared with 8% of 2,194 persons in 22 outbreaks caused by pansusceptible salmonella strains ( p<0.01 ) . | The Study
Conclusions |
primary cerebellar agenesis is rare as only eleven living cases have been reported till date to the best of our knowledge .
our case is unique in that the child presented with isolated cognitive impairment with no motor impairment .
mild to profound motor impairment , mental and behavioral abnormalities have been described in these patients .
this case is among the few cases which shows that it is possible to have normal motor function even in the absence of the cerebellum and that the cerebellum has an important role to play in nonmotor functions .
a 5-year - old male child was brought to the pediatric outpatient department on account of parental concern for impaired academic performance at school .
he was referred for magnetic resonance imaging ( mri ) study of the brain in view of learning disability and high degree of parental anxiety .
t1-weighted images in the axial and sagittal plane , t2-weighted images in the axial and coronal plane , and axial fluid - attenuated inversion recovery images were obtained .
images revealed a normal - sized posterior fossa which was empty and showed cerebrospinal fluid ( csf ) signal intensity on all sequences [ figures 1 and 2 ] .
the brainstem ( pons and medulla ) was hypoplastic as seen on sagittal t1-weighted images [ figure 3 ] .
small remnants of the cerebellum were seen anteriorly adjacent to the pons and likely represented residua of the anterior quadrangular lobes [ figures 1 and 2 ] .
axial t1-weighted magnetic resonance image shows an empty , normal sized posterior fossa ( large white arrow ) filled with hypointense cerebrospinal fluid .
remnants of the quadrangular lobes are seen anteriorly ( small white arrows ) axial t2-weighted magnetic resonance image shows an empty , normal sized posterior fossa ( large white arrow ) filled with hyperintense cerebrospinal fluid .
remnants of the quadrangular lobes are seen anteriorly ( small white arrows ) sagittal t1-weighted magnetic resonance image shows hypoplastic brainstem ( white arrow ) with empty posterior fossa ( black arrow )
sometimes , small remnants of the cerebellum may be present on account of which the term subtotal cerebellar agenesis has also been used in the literature .
the first case of primary cerebellar agenesis was described by combettes in 1831 . to the best of our knowledge
this may point toward neuronal plasticity which allows the supratentorial brain to compensate for cerebellar function .
a case of cerebellar agenesis has been reported to show improvement in motor function over the years , which lends support to the proposed theory that the remaining brain may compensate for cerebellar function over a period of time . moreover , small remnants of the palaeocerebellum may be present , which could compensate for the motor function .
a review of imaging in a case of cerebellar agenesis reported earlier in a man who died in 1951 without any motor impairment revealed a small residual cerebellum .
remnants of cerebellar tissue were also seen in a case of cerebellar agenesis in a 22-year - old male by velioglu et al .
our patient had remnants of the anterior quadrangular lobes which may account for the lack of motor manifestations .
recent studies have revealed the role of cerebellum in important nonmotor functions including attention , language , working memory , and learning , which explains cognitive and behavioral abnormalities in cerebellar agenesis .
the pons and medulla are hypoplastic due to the lack of afferent input from the cerebellum .
primary cerebellar agenesis needs to be differentiated from secondary degeneration of the cerebellum ( vanishing cerebellum ) in chiari ii malformation .
chiari ii is characterized by a small posterior fossa unlike the normal sized fossa in primary cerebellar agenesis .
the other differential diagnosis for primary cerebellar agenesis is dandy walker syndrome and its variants . in that case
, the posterior fossa is enlarged with superior displacement of the tentorium , straight sinus , and torcular herophili . | primary cerebellar agenesis is a rare entity . to the best of our knowledge
, eleven living cases have been reported till date .
most of these were associated with some degree of motor impairment .
we present a case of cerebellar agenesis in a child who presented with cognitive abnormalities leading to poor performance at school .
no motor impairment was seen . among the eleven cases reported earlier , only one case showed lack of motor impairment . | Introduction
Case Report
Discussion
Financial support and sponsorship
Conflicts of interest |
several external and internal factors influence an individual s ability to balance , including genetics , the state of vestibular apparatus , age , the area of support , center of mass positioning , emotional state , strength , coordination , flexibility , frequency of participation in motor activities , and training status .
independent of these factors , static and dynamic balance continues to be an indispensable motor skill because it is at the center of all human movement ( 30 ) .
not only is falling risk a result of poor balance , the ability to maintain balance is necessary to complete activities of daily living both safely and correctly ( 25 ) as well as to excel in sport - specific activities .
the definition of balance is most often related to the goals of the present investigation . because balance is required during movement as well as during stance , several laboratory measures have defined two major types of balance .
dynamic balance is the preservation of an upright body position throughout locomotion ( 30 ) , whereas static balance is the process of maintaining the center of mass vertically over the base of support with minimal movement ( 22 ) while maintaining specific poses for an extended period of time ( 30 ) .
our study focused exclusively on static balance since the literature has shown that collegiate females ( gymnasts and soccer players ) do not differ in terms of static and dynamic balance for single and double leg stance on stiff and compliant surfaces ( 8) . further , although the definition of a stable body position includes the ability to maintain and return to the proper positioning of body segments during the execution of a task or following a perturbation ( 30 ) , our study only included the components of static balance that demonstrated minimal movement during a specified pose .
center of pressure ( cop ) measurements have been shown to be a reproducible measure of static balance ( 17 ) .
the ability to minimize displacement of the cop while maintaining an upright stance during proper orientation or desired locomotion is controlled by the central nervous system .
further , because the central nervous system processes afferent input from visual , vestibular , and somatosensory systems the utilization of multiple senses provides for greater balance ability .
when one of the sensory inputs is reduced or eliminated other systems compensate for the loss .
of the three systems though , it is common for the somatosensory input to dominate the balancing task ( 12 ) .
dance training strengthens the accuracy of the somatosensory system and effectively shifts the vision - dominated , sensorimotor control of balance to an internal - based system of reference ( 12 , 19 ) .
therefore , the way dancers adjust in static and dynamic circumstances and self - correct body positioning is a function of strength , responsiveness of their proprioceptive system ( 18 ) , and advanced spatial skills ( 4 , 12 , 16 ) . due to enhanced balance abilities seen among dancers the purpose of this study was to determine if dancers demonstrate better single - leg static balance ability compared with non - dancers .
specifically , we hypothesized that dancers would show better static single - leg balance over the prescribed 30 second time interval more efficiently than non - dancers , would have lower excursion of the cop and would keep their ground reaction force ( grf ) balance more lateral and anterior indicating better balance over the four conditions .
the four conditions were static balance on the ( 1 ) dominant leg ( dl ) , ( 2 ) non - dominant leg ( ndl ) , ( 3 ) shod ( s ) and ( 4 ) barefoot ( bf ) .
two questionnaires and a consent form were completed by those who volunteered as participants . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) .
the pre - participation survey helped identify whether a participant had any known neurological conditions or symptoms that would interfere with the demonstration of static balance .
questions that were included in the survey that identified medications that could alter balance or cause dizziness as well as visual deficits that could interfere with balance .
because the survey was given immediately prior to the testing period , we further asked if adequate amounts of food had been consumed since this could potentially alter balance performance and negatively affect concentration and focus .
the lefs determined who was qualified to participate in the study since decreased functioning of the lower limb may affect balance .
the score for the survey was calculated using the equation below : the lower the score , the greater the disability
. we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college .
inclusion criteria for d included seven years or more of dance experience as well as participation on the collegiate level dance team .
individuals in the nd control group had less than one year of dance experience or no experience at all .
single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level .
their gaze had to remain fixed on an x that was taped two meters away at the individual s eye level .
cop was defined as the resulting position of the force vector for all vertical grfs measured by the force plate .
copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively .
total balance time represented the average time of the three trials that the subject maintained single - leg stance .
maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) .
the grf coordinate system for the force plate was positive ( + ) for anterior and negative ( ) for the posterior direction .
positive ( + ) was the sign for the medial direction and negative ( ) for the lateral direction .
maximum grf variables for the three planes were normalized by dividing the resultant force vectors by subject body weight ( grf / subject weight in newtons ) .
the dimensions of the force plate measured 600 mm by 1200 mm . in each corner , a piezoelectrical transducer measured the reaction forces occurring in the ap , ml , and vertical planes .
data from the transducer were filtered with a low - pass 12 hz filter using a fourth order zero - lag butterworth frequency and processed by an analogue - to - digital converter , thereby eliminating high - frequency noise .
these data were then transferred to the microcomputer and saved to the data collection software .
single - leg stance was demonstrated to each of the 14 participants immediately prior to testing .
participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe .
subject shoe familiarity provided for no learning effect of different and unfamiliar shoe types among subjects , thereby optimizing shod balance .
each subject performed single - leg stance with her supporting leg on the center of the force plate .
the force plate was on an elevated , flat , and stable surface to reduce the amount of vibrations from the ground and surroundings .
for each of the four conditions , participants balanced for 30 seconds . in order to obtain a valid measurement , each subject had three trials for each condition .
however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded .
the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg .
we controlled for the effect that external and environmental conditions could have on our measurements .
specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance .
also , the room was consistently well - lit and of the same temperature and humidity .
data analysis was conducted through ibm spss version 19 for windows ( chicago , il ) . prior to statistical analysis ,
because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions .
a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 .
the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl .
two questionnaires and a consent form were completed by those who volunteered as participants . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) .
the pre - participation survey helped identify whether a participant had any known neurological conditions or symptoms that would interfere with the demonstration of static balance .
questions that were included in the survey that identified medications that could alter balance or cause dizziness as well as visual deficits that could interfere with balance .
because the survey was given immediately prior to the testing period , we further asked if adequate amounts of food had been consumed since this could potentially alter balance performance and negatively affect concentration and focus .
the lefs determined who was qualified to participate in the study since decreased functioning of the lower limb may affect balance .
the score for the survey was calculated using the equation below : the lower the score , the greater the disability
. we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college .
inclusion criteria for d included seven years or more of dance experience as well as participation on the collegiate level dance team .
individuals in the nd control group had less than one year of dance experience or no experience at all .
single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level .
their gaze had to remain fixed on an x that was taped two meters away at the individual s eye level .
cop was defined as the resulting position of the force vector for all vertical grfs measured by the force plate .
copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively .
total balance time represented the average time of the three trials that the subject maintained single - leg stance .
maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) .
the grf coordinate system for the force plate was positive ( + ) for anterior and negative ( ) for the posterior direction .
positive ( + ) was the sign for the medial direction and negative ( ) for the lateral direction .
maximum grf variables for the three planes were normalized by dividing the resultant force vectors by subject body weight ( grf / subject weight in newtons ) .
the dimensions of the force plate measured 600 mm by 1200 mm . in each corner , a piezoelectrical transducer measured the reaction forces occurring in the ap , ml , and vertical planes .
data from the transducer were filtered with a low - pass 12 hz filter using a fourth order zero - lag butterworth frequency and processed by an analogue - to - digital converter , thereby eliminating high - frequency noise .
these data were then transferred to the microcomputer and saved to the data collection software .
single - leg stance was demonstrated to each of the 14 participants immediately prior to testing .
participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe .
subject shoe familiarity provided for no learning effect of different and unfamiliar shoe types among subjects , thereby optimizing shod balance .
each subject performed single - leg stance with her supporting leg on the center of the force plate .
the force plate was on an elevated , flat , and stable surface to reduce the amount of vibrations from the ground and surroundings .
for each of the four conditions , participants balanced for 30 seconds . in order to obtain a valid measurement , each subject had three trials for each condition .
however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded .
the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg .
we controlled for the effect that external and environmental conditions could have on our measurements . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance .
also , the room was consistently well - lit and of the same temperature and humidity .
data analysis was conducted through ibm spss version 19 for windows ( chicago , il ) . prior to statistical analysis ,
because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions .
a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 .
the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl .
in table 1 , the demographics of the seven dancers and seven controls are shown .
significant differences existed between groups for balance time ( d 30.00.0s ; nd 28.55.9s p0.03 ) . in 6 of the 84 trials for the non - dancer group , single - leg stance
statistically significant differences also existed for : ap grf in bf dl ( d 0.0090.057 n ; nd 0.1490.067 n p0.001 ) and ndl ( d 0.0690.062 n ;
nd 0.1880.105 n p0.001 ) ; ml grf in bf ndl ( d 0.0120.015 n ; nd 0.0130.025 n p0.001 ) ; ap grf in s dl ( d 0.0110.061 n ; nd 0.1620.041 n p0.001 ) ; ml grf in s dl ( d 0.0030.015 n ; nd 0.0180.006 n p0.001 ) ; ap grf in s ndl ( d 0.0800.041n ; nd 0.1680.097 n p0.001 ) ; and , ml grf in s ndl ( d 0.0080.012 n ; nd 0.0120.013 n p0.001 ) as shown in tables 2 and 3 . within group analyses for dancer and non - dancers can be found in tables 4 and 5 .
statistically significant differences existed among dancers for : ap grf in bf ( dl 0.0090.057 ; ndl 0.0690.062 p0.001 ) and s ( dl 0.0100.062 ; ndl 0.0800.041 p0.001 ) ; and , copap in bf ( dl 0.000010.00003 ; ndl 0.000040.00006 p0.001 ) and s ( dl 0.000020.00004 ; ndl 0.000050.00005 p0.002 ) .
statistically significant differences existed among non - dancers for : copml in bf ( dl 0.000020.00003 ; ndl 0.0000020.00003 p0.013 ) and s ( dl 0.000040.00002 ; ndl 0.0000020.00003 p0.001 ) .
for the dl , a significant difference was found in the ap directional grf between dancers and non - dancers .
for the ndl , significantly different grf was found in both the ap and ml directions .
specifically , non - dancers demonstrated greater posterior - directed grf compared with dancers as well as more grf directed medially compared to the dancer group , which exhibited lateral grf . between group analyses for the s condition can be found in table 3 .
for both the dl and ndl , significant grf differences were found in both the ap and ml directions .
these significant differences were the result of non - dancers demonstrating greater posterior grf compared with the dancers .
also , while dancers demonstrated a lateral grf in the f ml plane , a medially grf was seen among non - dancers .
more posterior grf in both bf and s conditions indicates a greater loss of balance in the non - dancer group since , when in a static stance . sensory awareness in movement
has received increased interest in sports and clinical literature as a feed - forward and feedback mechanism which may by extension contribute to static balance ( 6 ) .
the greater medial grf for non - dancers in the bf and s conditions may also indicate more global instability . further , for the dl , a statistical trend ( p=0.019 ) was observed for the cop ml .
non - dancers had a more medial cop , which we believe is indicative of greater perturbation , and therefore instability .
it is also important to note that in the shod ndl condition , cop ap distances were different from each other , but did not reach significance ( p = 0.053 ) . in this condition , dancers showed more posterior grf , which is difficult to explain . since strength and proprioception play an essential role in injury prevention ( 3 ) , current athletic shoe construction may hinder balance ability without shoes .
athletic shoes are often designed with features that enhance stability of the foot and body which is important for prevention and protection against injury ( 31 ) .
there is evidence however that the rigid constraints of a traditional athletic shoe contribute to a lack of muscle development and proprioception ( 23 ) . because dancers perform movements primarily without shoes , it is possible that poor balance in the shod condition among dancers may be due to loss of sensory information that is available only when barefoot . in the within group analyses
there were more statistical differences within the dancer group , indicating more variability in dl and ndl , as seen in tables 4 and 5 .
dancers were statistically different in the ap grf in both the bf and s conditions .
specifically , the results found that dancers show more posterior grf in the ndl , which would support the concept of greater instability on the non - dominant side and more stability on the dominant side .
the copap was also statistically different between dl and ndl in bf and s conditions among dancers .
both indicated more posterior deviation on the ndl , which supports the notion that the ndl shows greater instability when shod and barefoot .
lastly , the bf ndl ml grf showed a statistical trend ( p = 0.016 ) .
although dancers had more lateral grf on the non - dominant side , both measures support the position that a dancer s balance is directed more laterally . among the non - dancers
the only statistically significant differences were found in the copml for both bf and s conditions .
specifically , a more lateral deviation was observed in the copml for the ndl when barefoot .
this finding seems unusual considering dancers seem to direct their grf and cop more lateral to maintain balance , yet in the non - dancers , the bf condition shows more laterality for the ndl , which would indicate greater stability for this side . in the s condition , there was less medial deviation in the ndl than in the dl , which again indicates greater stability for the ndl than the dl
. the higher number of statistically significant differences between the dl and ndl within the dancer group may lead to the conclusion that a dancer s balance is not equally distributed between dl and ndl .
it is important to recognize , though , that the less variability in these measures for non - dancers may be because they have poor balance in both legs , and therefore , differences can not be demonstrated .
the results from two different studies demonstrate the impact that dance activity and associated movements have on enhancing static balance .
ricotti and ravaschio measured static balance three times over a course of six months in three homogenous groups of 9-year - old soccer athletes ( 29 ) . for single - leg stance , there were no significant improvements in the dominant limb s cop area values neither for the group involved in soccer activity only nor for the group involved in soccer and swimming activity over the 6-month period .
the group involved in the soccer and break dance activity , however , demonstrated a significant improvement in cop area values at month 0 and month 6 for the dominant limb .
further , for the non - dominant limb , the combination of soccer and break dance activity yielded a greater number of significant differences in cop area values between month 6 and month 2 as well as between month 4 and month 2 ( 29 ) .
stankovi m and radenkovi showed enhanced balance among 39 male and female school age children among those involved in a dance program ( 30 ) . in our study , it is evident that the seven or more years of dance experience increased balance ability when performing single - leg stance .
although there was not a defined pattern of cop measurements in the ml and ap directions between dancers and non - dancers throughout all four conditions , dancers consistently demonstrated less deviation in the ap and ml grf during bf and s conditions compared to non - dancers .
ambeganokar and colleagues also showed that dancers demonstrated significantly greater balance than non - dancers , especially statically ( 1 ) .
our results of a subject s ability to maintain single - leg static balance for a 30-second duration is closely related to the results shown by ambeganokar et al . in our study dancers were able to maintain single - leg stance on the dominant and non - dominant limb for the entire 30-second duration while non - dancers were unable to do so in 6 of the 84 trials .
many authors support the notion that balance plays an important role in sport - specific movements and injury reduction both of which are needed for dance success ( 15 , 22 ) . between dancers and soccer players , dancers performed significantly better on tasks measuring sway index during single - leg stance on stable and unstable surfaces ( 15 , 17 , 22 ) .
postural control , balance , coordination , proprioception and consistency in movement patterns are successful dance characteristics and are also linked to reduced risk of ankle injury , re - injury and acl tears ( 5 , 15 , 16 , 20 , 3234 ) . diminished or exaggerated
proprioceptive responses may be the direct result of musculoskeletal injury and surgical interventions ( 5 , 16 , 27 ) . a deficit in proprioception , independent of its extent , will compromise postural control , and therefore , offset balance and increase injury risk ( 11 , 13 , 26 ) .
failure to restore proprioception in the lower extremity following surgery and rehabilitation , even if the athlete regains complete strength , will increase the risk for re - injury because of the joint instability that is perceived by the individual ( 16 , 21 ) . further , peripheral and central alterations in function , including joint laxity and instability , misalignment , localized weakness , diminished muscle reaction time , and altered center motor programming ( 4 , 22 , 24 ) may result if proprioceptive deficits are left untrained . thus , extending proprioceptive training and balance protocols used in dance for treatment of lower - extremity injuries may decrease the risk of injury since proprioceptive training is believed to be beneficial in preventing injuries in the lower extremity , protecting against re - injury , and reducing recovery time ( 4 , 22 , 35 ) . in athletes with recurrent ankle sprains
, proprioceptive testing has shown that some with recurrent ankle sprains have lower ankle joint position sense compared with individuals without such injuries ( 7 , 28 ) . following knee and ankle injury in athletes ,
proprioceptive training improves postural stability ( 14 ) , flexibility ( 13 ) , joint position sense , and faster muscle reaction time ( 24 ) .
it is important to note , though , that the ability to reduce the risk of specific injuries in specific sports through proprioceptive balance training remains unclear due to the inclusions of strength and agility throughout the training programs ( 3 , 9 ) .
for example , ambegaonkar and colleagues sought to understand why female dancers have lower acl injury rates compared with physically active females ( 1 ) . prior to the study
, the authors believed that better balance by experienced dancers contributed to decreased musculoskeletal injury risk .
the authors concluded that the examination of balance between dancers and non - dancers does not provide a complete explanation regarding why physically active females experience higher acl injury rates compared with dancers .
acl injury risk is multifaceted , with balance and proprioception being important components , however joint structure , including the direction and magnitude of the destabilizing force , the rate at which loads are applied , and joint position all contribute to risk .
therefore , balance appears to be one of several components that contributes to acl injury .
static testing conditions that use a fixed base of support may not translate into the functionally dynamic movements that occur in sport .
thus , an athlete may not demonstrate a deficiency in balance ability during static conditions when in fact one exists throughout his movement patterns ( 15 ) .
selective destabilization of a joint through the incorporation of perturbations in balance training should also be included in the protocol for rehabilitation ( 10 ) .
the application of graded , controlled forces across injured joints is effective in activating higher neural centers that evoke postural synergies , reducing injury potential ( 2 , 5 ) , allowing the center of mass to remain over the base of support .. following this study , we hoped to better understand how dance impacted balance and whether or not these results could be applied to lower - extremity rehabilitation .
our study defined balance as the ability to maintain a specified pose for a duration of 30 seconds as well as the ability to demonstrate minimal movement throughout this time interval .
more specifically minimal movement was understood as a decrease in posterior and medial grf and deviation in the cop .
the duration that an individual maintained single - leg stance also highlighted static balance ability .
rehabilitation should provide balance training as a major component in both rehabilitation as well as pre - season injury prevention programs for their athletes . | the purpose of this study was to compare kinetic differences of static balance between female dancers ( d ) with at least seven years of dance experience and female non - dancers ( nd ) who were typical college students .
participants were tested in single - leg stance .
both the dominant leg ( dl ) and non - dominant leg ( ndl ) were tested with the participants shod ( s ) and barefoot ( bf ) .
kinetic variables ( vertical , medio - lateral [ ml ] , antero - posterior [ ap ] maximum ground reaction forces ( grf ) , and center of pressure ( cop ) ml and ap ) were measured by a bertec force platform at 1000 hz with participants s and bf .
each subject s stance was measured over 3 30-second intervals .
no significant differences ( p0.05 ) existed between groups for height , body mass , or age .
significant differences existed between groups for balance time , ap grf in both bf and s conditions for both dl and ndl , and ml grf in bf ndl and s dl and ndl conditions .
d and nd in bf and s conditions with dl and ndl static stance demonstrate different ap and ml grf when balancing over a 30-second time interval .
data may suggest that nd are more prone to lose their balance .
further investigation is warranted to understand whether individuals in the rehabilitative field and athletic populations can use dance therapy for injury prevention and rehabilitation . | INTRODUCTION
METHODS
Participants
Protocol
Statistical Analysis
RESULTS
DISCUSSION |
14 special attention has been paid to the cytotoxicity of benzalkonium chloride ( bak ) , included in solutions as a preservative.511 glaucoma surgery using mitomycin c leads to a reduction in the density of corneal endothelial cells ( cecs).12 by comparison , little is known about the toxicity of antiglaucoma eye drops to cecs.1316 the aim of the present study is to evaluate the toxicity of antiglaucoma medications on human ( h ) cecs using a cell culture system .
our investigation will contribute to better long - term patient care especially in the cases with decreased cec density and history of invasive intraocular surgeries , since glaucoma medication may continue throughout life .
the present study was approved by the institutional review board of showa university , fujigaoka rehabilitation hospital ( formerly the affiliation of the corresponding author ) .
we established primary cultures of hcecs derived from human eye bank specimens ( sightlife , seattle , washington , usa ) , as described previously.17 when sufficient cell numbers for bioassay were obtained , cells were harvested .
a 100-l aliquot of the culture , containing approximately 2 10 cells , was replated , and cells were cultured for 2 days before exposure to test solutions .
preservative - free 0.5% timolol maleate and preservative - free 1% dorzolamide were prepared for the present investigation only .
test solutions were used undiluted or diluted 2- , 10- , 100- , 1,000- , or 10,000-fold .
0.01% and 0.005% bak ( wako pure chemical industries , osaka , japan ) were also tested . after exposure to the test solutions for 10 , 30 , or 60 minutes , or 48 hours , cell viability was assayed using 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ( wst-1 ; dojindo laboratories , kumamoto , japan ) .
the wst-1 assay is a quantitative colorimetric assay that measures mitochondrial activity as an index of cell viability and proliferation .
it only detects living cells , and the signal generated is directly proportional to the number of live cells . at the completion of the assays ,
absorbance was read on a spectrophotometer ( benchmark microplate reader ; bio - rad , hercules , california , usa ) .
control wells were incubated with growth media without test solution . to calculate cell viability ,
the value of the signal from the treated culture well was expressed as a percentage of that of the control well .
experiments were repeated between 8 and 16 times , and results were expressed as the mean standard deviation .
the first established cell line was used for assays after 48 hours exposure , and the second one was for 10 , 30 , and 60 minutes exposure .
statistical analysis with student s t test was performed using microsoft excel ( tokyo , japan ) .
we expressed the results of the viability of cells after 10 , 30 , or 60 minutes exposure as a cell viability score ( cvs ) to enable easy comparison of the effects of different drugs .
this concept is similar to the minimum inhibitory concentration ( mic50 ) of a drug required to inhibit the growth of 50% of organisms .
cvss are calculated as follows : ( 1 ) the toxicological tests are performed in undiluted , 2-fold or 10-fold diluted solutions for 10 , 30 , or 60 minutes exposure .
( 2 ) the cell viability value in any solution is found to be < 80% in any conditions , and confirmed cell viability is 80% after adequate dilutions for every solution .
( 3 ) the cvs50 is determined for each solution in each condition as the number of experiments for a viability value 50% .
the cvs40/80 is calculated as follows : cvs40/80 = ( number of experiments for a viability value > 80% ) = ( number of experiments for viability value < 40% ) .
( 4 ) a total of cvs for multiple ( 2 or more concentrations or exposure times ) conditions is relevant for general evaluation .
although some cvs for a single condition seems suitable to determine the order of toxicity such as 2-fold dilution after 30 or 60 minutes exposure ( eg , figure 1 ) , note especially that these results are not always consistent with the result from a total of cvs or the general trend observed in graphic expression .
( 5 ) as for determination of the order of toxicity of the tested solutions , the significant difference in actual scores should be > 20% of a total number of experiments under the condition for cvs . consequently , the order of toxicity is expressed by
< and > for the difference 20% , , , and
a total of 9 experiments were conducted , and we defined significant difference as 2 or more scores .
when the orders determined by cvs50 and cvs40/80 are conflicting , a final decision is made by uniting both results or by considering cvs for individual concentration or exposure time to explore the trend .
the cvs system is subject to change according to tested solutions , cell lines , toxicological methods , and other factors .
comparisons of cell viability were also made between a drug with bak and the corresponding drug without bak .
hcec viabilities after exposure to 10-fold dilutions of test solutions for 48 hours were , eg , 48.5% for 0.5% timoptol ( banyu pharmaceutical , tokyo , japan ) , 80.9% for preservative - free 0.5% timolol maleate , 47.0% for 1% trusopt ( banyu pharmaceutical ) , 71.7% for preservative - free 1% dorzolamide , 55.5% for travatan ( alcon laboratories , fort worth , texas , usa ) , 88.5% for travatan z ( alcon laboratories ) , and 52.5% for xalatan ( pfizer , new york , ny , usa ) .
cell viability > 80% was seen after incubation with 100-fold dilutions of test solutions , with the exception of preservative - free 1% dorzolamide , which resulted in 72.0% viability .
we confirmed cell viability exceeded 80% in 1,000-fold or 10,000-fold dilution for every solution including 48 hours exposure . using cvs as an indicator of cytotoxicity , the order of cell viability after short exposure ( 10 , 30 , or 60 minutes ) to the solutions was travatan z preservative - free timolol maleate = preservative - free dorzolamide > 0.5% timoptol = 1% trusopt > travatan
xalatan ( table 1 ) . as for the cell viability after 48 hours exposure ,
the difference between the solutions with bak and those without bak was clear in 10-fold dilution ( figure 1 , far right panel ) .
however , cell viabilities were > 70% in all of the 100-fold diluted solutions and the difference was not clear .
we assessed the effect of antiglaucoma medications with and without bak statistically , which revealed that cell viability in the presence of bak was markedly lower , especially with higher concentrations and following longer exposure ( figure 1 ) .
cell viabilities following treatment with bak alone or antiglaucoma solutions containing comparable bak concentrations are shown in figure 2 .
cell viabilities were lower for 0.5% timoptol and 1% trusopt than for 0.005% bak ( p < 0.01 ) . in comparison ,
higher cell viabilities were seen with xalatan ( used at a 2-fold dilution , containing 0.01% bak ) and travatan ( used at a 2-fold dilution , containing 0.0075% bak ) than for 0.01% bak alone ( p < 0.01 , p < 0.01 ) .
bak is toxic for cecs , and several clinical cases have been reported2125 for accidental introduction of bak into the anterior chamber and for instillation of bak - containing solutions to the eyes with or without disrupted ocular surface barrier function .
the antiglaucoma medications containing bak were significantly more toxic to hcecs than solutions without bak .
our results are similar to those of other studies using ocular surface cells.111 considering the actual concentration of drugs ( 1,000- to 10,000-fold dilution in aqueous humor)26,27 to which hcecs are exposed after topical administration and the fact that toxicity decreased markedly after dilution in the present study , hcec loss due to antiglaucoma medication may only occur in rare cases .
previous studies of its side effects on ocular surface cells demonstrated that timolol maleate itself has acceptable toxicity and that bak and other ingredients in commercial timolol maleate eye drops , as well as interaction of each component , play a major role in their toxicity,9,28,29 which was confirmed by the present results for hcecs .
treatment with trusopt has been reported to result in corneal edema ; however , this side effect only appeared in patients with compromised corneas and a previous history of corneal pathology.30 previous studies and the results of the present study suggest that trusopt and preservative - free dorzolamide are not significantly toxic to cecs under usual ocular conditions.15,29,3133 travatan z was less toxic to hcecs than travatan containing the preservative bak .
travatan z uses sofzia , a unique ionic buffering preservative system containing borate , sorbitol , propylene glycol , and zinc.11,34 our assessment of the combined effect of antiglaucoma compounds and bak revealed that cell viability for timolol maleate ( timoptol ) and dorzolamide ( trusopt ) eye drops with bak was much lower than that for comparable concentration of bak , whereas latanoprost ( xalatan ) , and probably travoprost ( travatan ) did not exhibit lower cell viability to hcecs than bak alone ( figure 2 ) .
our results on hcecs could not confirm previous findings for a protective effect of latanoprost and travoprost against the toxicity of bak on conjunctival cells.35 owing to the limited number of cultured hcecs available , we were not able to examine the effect of bak at exactly the same concentration as that contained in travatan .
further investigations with additional methodologies are necessary to properly evaluate the cytotoxicity of antiglaucoma medications .
the comparison of drug toxicity with graphic expression of a series of measurements is often difficult .
using cvs reference values of 40% , 50% , or 80% enables numerical expression , leading to a better comparison of solution toxicity .
80% for cvs40/80 is a border between nontoxic and toxic , and 40% is a border between toxic and very toxic according to our experience of assay with numerous cells and reagents .
the cvs was a useful evaluation method for comparison of cell viability values obtained from multiple series of toxicity assays .
the concentration of tested solutions was very high compared with the actual concentration in aqueous humor after instillation , because the present study is a so - called accelerated experiment .
we were unable to construct a culture system to observe toxicity over a long time in the solution of very low concentration .
consequently , we set up experiments in solutions of high concentrations to detect significant toxicity after reasonable incubation .
the major difficulties in eye drop toxicity studies include setting exposure time and concentration , since both tear and aqueous humor are continuously exchanged in a living eye .
this is the reason why our setting covers many exposure times and dilutions to explore toxicity in various environments .
the short exposures may simulate accidental introduction of the drug to endothelial cells , and 48 hours exposure may simulate long - term or repeated instillations .
antiglaucoma eye drops produced hcec toxicity that appeared to depend on the presence of bak .
the cvs was useful in comparing the cytotoxicity of different drugs , and the order of cell viability in tested solutions determined by cvs was travatan z preservative - free timolol maleate = preservative - free dorzolamide > 0.5% timoptol = 1% trusopt > travatan xalatan . because dilution of the antiglaucoma solutions resulted in markedly lower hcec toxicity , hcec damage due to antiglaucoma medication | purposethe toxicity of antiglaucoma medications to ocular surface cells has been evaluated extensively ; however , the toxicity to corneal endothelial cells ( cecs ) remains elusive .
our aim is to evaluate the toxicity of antiglaucoma medications to cecs using an in vitro toxicity assay.methodsprimary cultures of human ( h ) cecs derived from eye bank specimens were established . following exposure of hcecs to test solutions for 10 , 30 , or 60 minutes , or 48 hours , we measured cell viability using a wst-1 assay .
test solutions were diluted in culture media and included 0.5% timoptol , preservative - free 0.5% timolol maleate , 1% trusopt , preservative - free 1% dorzolamide , travatan , travatan z , xalatan , and benzalkonium chloride ( bak ) . to assess cell viability , the value of the test culture well after treatment was expressed as a percentage of that of the control well .
toxicity of each solution was compared using the cell viability score ( cvs).resultsafter exposure to 10-fold dilutions of test solutions for 48 hours , hcec viabilities were 48.5% for 0.5% timoptol , 80.9% for preservative - free 0.5% timolol maleate , 47.0% for 1% trusopt , 71.7% for preservative - free 1% dorzolamide , 55.5% for travatan , 88.5% for travatan z , and 52.5% for xalatan .
exposure to test solutions diluted 100-fold or more resulted in hcec viabilities > 80% , with the exception of preservative - free 1% dorzolamide , which resulted in a viability of 72.0% at a dilution of 100-fold .
based on cvs , the order of cell viability was travatan z preservative - free timolol maleate = preservative - free dorzolamide > 0.5% timoptol = 1% trusopt > travatan xalatan .
assessment of the combined effect of drug and bak revealed that latanoprost reduced the toxicity of bak.conclusionantiglaucoma eye drops produced hcec toxicity that appeared to depend on the presence of bak . because dilution of the antiglaucoma solutions resulted in markedly lower hcec toxicity , hcec damage due to antiglaucoma medication
may occur only in rare cases .
the cvs was useful for comparison of the toxicity of the drugs . | Introduction
Methods
Results
Discussion
Conclusion |
this year in the united states approximately 24,000 women will be diagnosed with ovarian cancer , and there will be approximately 14,000 associated deaths , predominantly from epithelial ovarian cancer ( eoc ) .
worldwide , it is estimated that 204,449 patients with ovarian cancer will be diagnosed this year with an estimated 124,860 disease - related deaths .
the incidence of ovarian cancer has been steadily increasing over the past 10 years , with an overall lifetime risk of 1.8% . despite improvements in surgical techniques and new chemotherapeutic regimens ,
the overall survival for women with stage iii / iv eoc has remained relatively unchanged ( 15% ) over the past 40 years .
in contrast , women diagnosed with disease confined to the ovary ( stage i ) require less morbid surgical intervention , may not require adjuvant chemotherapy , have a significantly improved quality of life , and most importantly have an overall 5-year survival approximating 90% . unfortunately
it is thought that accurate diagnosis of eoc at an earlier stage may decrease overall disease - related mortality .
evaluation of adnexal masses can be performed with several imaging methods , including tvs , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and positron emission tomography ( pet ) .
ct can detect large adnexal masses but has lower sensitivity for small adnexal masses , especially in thin patients in whom adnexal lesions can be misinterpreted as other pelvic structures or loops of pelvic small bowel .
furthermore , the ability of ct to characterize adnexal lesions as benign or malignant is limited by low inherent tissue contrast , with the notable exception of ovarian dermoids that can be characterized based on the presence of macroscopic fat and/or calcification . mri offers higher spatial and contrast resolution than ct and can characterize a wider spectrum of adnexal lesions based on magnetic signal properties or enhancement behavior , but accuracy of mri may diminish for borderline ovarian tumors and small ovarian masses .
pet can identify aggressive adnexal lesions on the basis of increased fluorodeoxyglucose ( fdg ) uptake , but it suffers from low specificity for small lesions , noting that normal premenopausal ovaries will demonstrate increased metabolic activity at mid cycle , and a physiological corpus luteum can therefore mimic an aggressive malignancy . the accuracy of pet is also limited secondary to false negatives in borderline ovarian neoplasms .
tvs is widely available and offers high - resolution imaging without the use of ionizing radiation . for these reasons ,
tvs is the initial diagnostic modality of choice for the evaluation of most patients with a pelvic mass .
as previously mentioned , tvs has limited sensitivity and specificity for the definitive diagnosis of ovarian cancer because of overlapping morphologic features seen in benign and malignant lesions . recently , however , significant technologic advances have yielded vast improvements in the sonographic depiction of early - stage ovarian cancer , and these improvements have translated into improved sonographic discrimination of benign from malignant disease in preliminary studies .
combined evaluation of sonographic morphology and cds forms a set of basic simple rules for sonographic distinction of benign from malignant ovarian masses based on the data derived from a european multicenter study which included 1,223 adnexal tumors ( sensitivity 93% ; specificity 90% ) ( table 1 ) .
three - dimensional transvaginal sonography ( 3d tvs ) has improved the morphologic depiction of ovarian cancers beyond the capabilities of traditional tvs .
improvements in transvaginal color doppler sonography ( tv - cds ) have enhanced sonographic assessment of large tumor vascular networks , and contrast - enhanced transvaginal sonography ( ce - tvs ) now allows for interrogation of tumor microvascularity [ 79 ] .
this paper discusses these newer techniques , specifically ce - tvs , with emphasis on their advantages and areas for potential improvement .
conventional sonographic criteria for ovarian cancer diagnosis are based on morphological classification of ovarian masses .
ovarian malignancy is unlikely in simple cysts with smooth walls , but presence of a solid mass or solid projections ( papillary excrescences ) into the cyst cavity significantly increases the risk of malignancy .
hirai has described the morphologic features on tvs associated with stage i ovarian cancer in a lay - screening population in japan ( figures 1 , 2 , and 3 ) . in general , the stage ia ovarian cancers with normal ca-125 were small and had less solid components than stage ia cancers with elevated ca-125 .
papillary excrescences typically occur in areas of epithelial neoplasia and can be seen borderline rather than frankly malignant lesions . over the last 10 years , the diagnostic accuracy for conventional 2d tvs has been improving .
a 1997 study reported that gray - scale sonography identified malignant tumors with a sensitivity of 91% and a specificity of 84% , while a 2008 study found a sensitivity of 93% and specificity of 90% .
as the result of these studies , several morphological scoring systems have been developed for sonography , including features such as the presence of papillary projections or irregular and/or thick septae .
results of a meta - analysis provide evidence that sonographic techniques that combine gray - scale morphologic assessment with tumor vascularity mapping are significantly better in ovarian lesion characterization than doppler arterial resistance measurements , color doppler flow imaging , or gray - scale morphologic information alone .
the recent development of 3d - tvs improves the detection of morphologic abnormalities indicative of neoplastic ovarian masses .
in particular , small papillary excrescences or focal wall ( mural ) irregularities can be detected which are associated with epithelial malignant growth in ovarian masses .
the recent advent of matrix array transducers / probes may improve visualization of both internal and external wall ( capsular ) abnormalities , increase comfort for the patient , and increase reproducibility .
tv - cds provides depiction of the macrovascularity ( over 200 ) of tumors but does not delineate microscopic ( capillary ) tumor neovascularity . the vascular network in tumors
can be further interrogated using doppler techniques to indicate the impedance within vessels [ 7 , 8 ] .
combining morphologic assessment with tvs with color doppler features has allowed accurate assessments of whether a mass is benign or malignant by following
using color doppler techniques , the overall vascularity was classified as high , low , or intermediate , rather than determining vascular indices such as resistance or pulsatility . in a european multicentered study , it was shown that this paradigm resulted in 90% sensitivity and 92% sensitivity .
both micro- and macroscopically , tumor neovascularity is characterized as vessels that demonstrate irregular caliber and branching .
microvascular ( i.e. , capillary ) tumor neovascularity can be depicted using microbubble contrast ( figures 4 , 5 , and 6 ) . on dynamic ce - tvs malignant tumor neovascularity
usually demonstrates a higher peak of contrast enhancement and prolonged contrast washout when compared to benign tumors [ 1 , 9 , 14 , 15 ] ( figure 9 ) .
in our previous study , all malignant tumors and 50% of benign tumors showed detectable contrast enhancement ( image intensity > 10% above the baseline ) after microbubble injection .
when contrast enhancement dynamics were assessed , we found that malignant lesions had a similar time to peak ( tp ; 26.2 5.9 versus 29.8 13.4 seconds ; p = .4 ) , greater peak enhancement ( pe ; 21.3 4.7 versus 8.3 5.7 db ; p <
.001 ) , a longer half wash - out time ( ( /2)two ; 104.2 48.1 versus 32.2 18.9 seconds ; p < .001 ) , and a greater area under the curve ( auc ; 1807.2 588.3 versus 413.8 294.8 seconds ; p < .001 ) when compared with enhancing benign lesions ( figure 9 ) . auc greater than
787 seconds was the most accurate diagnostic criterion for ovarian cancer , with 100.0% sensitivity and 96.2% specificity .
additionally , pe greater than 17.2 db ( 90.0% sensitivity and 98.3% specificity ) and a ( 1/2)two of greater than 41.0 seconds ( 100.0% sensitivity and 92.3% specificity ) proved to be useful .
initial analysis of contrast - enhanced kinetic was done using time intensity curves for mean , standard deviation , and p value ( figures 9 and 11 ) and subsequently by receive operator characteristic curves for vascular index ( vi ) ; flow index ( fi ) ; vascular flow index ( vfi ) ( figure 12 ) .
the receiver operator characteristics of each parameter is shown in figure 13 with predetermined cutoff values as established with receive operation curves and compared to vi , fi , and vfi values .
these results show that contrast - enhanced nonlinear pulse inversion sonography is a more appropriate method for characterizing blood flow dynamics in ovarian tumors than by tv - cds and can provide an important tool to aid differential diagnoses between benign and malignant ovarian tumors . while the time - intensity curve has traditionally been calculated from mean signal intensities over a region of interest , parametric mapping of time - intensity curve variables on a pixel - by - pixel basis allows for more global visualization of tumor hemodynamics .
the use of this technique in ovarian cancer has been limited to selecting the pixel with greatest peak enhancement ( pe ) and using that pixel 's time - intensity curve for further analysis .
however , parametric maps of ce - tvs have recently been used with limited success for differentiation of benign and malignant focal liver lesions and breast lesions [ 17 , 18 ] .
preliminary results for using parametric mapping of ovarian tumors seem to indicate significant potential for improving diagnostic accuracy .
our preliminary results from a subset of 29 out of the 57 subjects analyzed in our previous region of interest ( roi ) study show potential for this technique to differentiate benign and malignant ovarian masses .
the methods of data acquisition are outlined in the previously described study . analysis with a quantification software prototype ( bracco suisse sa , geneva , switzerland ) utilized parametric maps of tp ( sec ) , pe ( db ) , and wash - in auc ( wiauc ; arbitrary units , a.u . ) .
the region of interest was kept constant in size between subjects and was corrected for motion .
the map color scales were adjusted such that abnormal hemodynamics were represented by red for pe > 24 a.u , tp < 11 s , and wiauc > 35 a.u .
( cutoffs chosen at optimum points on receiver operator characteristics curve ) , and the presence of any red color was used to differentiate benign and malignant tumors .
the preliminary results from the subanalysis of 18 benign and 11 histologically proven malignant ovarian masses showed greatest diagnostic accuracy for maps of pe ( sensitivity 100% , specificity 67% ) and wiauc ( sensitivity 73% , specificity 94% ) , while maps of tp were least accurate ( sensitivity 100% , specificity 17% ) .
final analysis of all 57 subjects is needed to determine the ultimate utility of these methods , but preliminary results are promising .
ce - tvs can significantly improve the diagnostic ability of transvaginal sonography alone to identify early microvascular changes that are known to be associated with early - stage ovarian cancer [ 1 , 9 , 14 , 15 , 20 ] .
currently , contrast agents play a pivotal role in the imaging modalities of ct and mri by increasing lesion conspicuity , accentuating morphologic features within a lesion , and defining time - resolved lesion enhancement patterns that serve as additional imaging parameters by which a lesion may be characterized .
indeed , contrast agents have received such widespread acceptance that a ct exam performed without intravenous contrast or an mri without contrast for many indications is now considered limited .
preclinical studies demonstrated that the intravenous contrast agents for sonography hold great promise in a multitude of potential clinical applications , especially in identifying aberrant vascular changes associated with malignancy [ 19 , 21 ] .
previous studies have addressed the use of ce - tvs for benign and malignant tumors by showing greater enhancement of malignant tumors on doppler imaging . according to the initial work reported by kupesic and kurjak
, the use of a contrast agent with 3d power doppler sonography showed very high diagnostic efficiency ( 95.6% ) that was superior to that of nonenhanced 3d power doppler sonography ( 86.7% ) .
however , simple documentation of tumor enhancement may not be sufficient because some benign tumors show detectable contrast enhancement .
only a few studies have been published that used kinetic parameters of the contrast agent to compare benign with malignant tumors in the power doppler mode .
demonstrated that after microbubble contrast agent injection , malignant and benign adnexal lesions behave differently in degree , onset , and duration of doppler us enhancement .
marret et al . reported that wash - out times and auc were significantly greater in ovarian malignancies than in other benign tumors ( p <
.001 ) , leading to sensitivity estimates between 96% and 100% and specificity estimates between 83 and 98% .
they concluded that doppler contrast - enhanced parameters had slightly higher sensitivity and slightly lower specificity when compared with transvaginal sonographic variables of the resistive index and serum ca-125 levels .
our preliminary clinical studies explored differences in enhancement parameters in benign versus malignant ovarian masses using a new method of ce - tvs termed pulse inversion nonlinear imaging .
this method produces more reliable estimates of tumor microvascular perfusion and provides more consistent results compared to doppler - based contrast - enhanced ultrasound .
our data suggest that , except for the tp , contrast enhancement parameters are significantly different in benign versus malignant ovarian masses .
the tp probably reflects intrinsic circulation depending on cardiac contraction , blood pressure , and overall vascular tone .
once blood circulates through the tumor , however , differences may reflect the unique branching patterns and vessel morphologic characteristics in the microvascularity of the tumors . as a general statement
the addition of a vascular sonographic contrast agent allows a more complete delineation of the vascular anatomy through enhancement of the signal strength from small vessels ( capillaries ) and provides an entirely new opportunity to time the transit of an injected bolus .
ce - tvs has higher sensitivity and specificity to differentiate between benign and malignant lesions than conventional tvs and for detecting occult stage i disease .
ce - tvs can detect tumor neovascularity ( figures 4 , 5 , 6 , 7 , and 8) .
tumor neovascularity is characterized by vessels with abnormal endothelial structure that are irregular in caliber and branching patterns . in order to recognize these features ,
these parameters may also be shown in a parametric map , which allows for stricter cutoff criterion than roi analysis , as peak values are visualized on a much finer scale than typical rois .
the traditional approach of calculating the time - intensity curve over rois chosen on morphology alone allows small areas of neovascularity to be missed , as they are averaged over larger heterogeneous areas .
the primary utility of parametric mapping of ovarian tumors may be guiding selection of rois to the area of greatest malignant potential . with improved imaging technology
specifically , this includes directed therapeutic measures after labeled microbubbles are used . in a murine model
, it is hoped that this paper may contribute to the development of new methods for diagnosing , enhancing therapy , and detecting tumor response for this dreaded gynecologic malignancy , possibly with the use of targeted microbubbles [ 2 , 2428 ] . | recently , there have been several major technical advances in the sonographic diagnosis of ovarian cancer in its early stages .
these include improved assessment of tumor morphology with transvaginal sonography ( tvs ) , and detection and characterization of tumor neovascularity with transvaginal color doppler sonography ( tv - cds ) and contrast - enhanced transvaginal sonography ( ce - tvs ) .
this paper will discuss and illustrate these improvements and describe how they enhance detection of early - stage ovarian cancer . our initial experience with parametric mapping of ce - tvs will also be mentioned . | 1. Introduction
2. Discussion |
the tissue dissolving ability of naocl depend on not only exposed time of the solution , concentration and its volume , but also its depend on the tissue surface area the effects of naocl solutions about tissue dissolution can be increased by :
raising the solution phincreasing the temperature of the naocl solutionsultrasonic activation of the solutionelongated working timecontinuous agitation of the irrigation solution .
raising the solution ph increasing the temperature of the naocl solutions ultrasonic activation of the solution elongated working time continuous agitation of the irrigation solution .
showed the dynamic balance of naocl as followed by the reaction : naocl + h2o naoh + hocl na + oh + h + ocl those chemical reactions between naocl and organic tissue were confirmed .
the dynamic balance of naocl resulted saponification reaction , amino acid neutralization reaction and chloramination reaction .
electrochemically activated solution ( eca ) are produced from water and low concentration solutions with salt .
the terms of eca is principally transferred liquids from the metastable membrane via to anode or cathode action using an elementary reactor . in vitro studies testing eca as an irrigant , suggested an alternative to naocl solution endodontic therapy , but eca
electrolysis is a method of using direct electric current by a potentiostat / galvanostat to drive an otherwise nonspontaneous chemical reaction . as a rule
, the activation liquid is performed in a diaphragm or nondiaphragm electrolysis device . during this activation , liquids
consequently , chemical structure of these liquids change and properties , as well as their microcluster structure , hydrogen concentration and oxidation reduction potential .
it is conceivable that electrically - activated sodium hypochlorite ( e - naocl ) could enhance its effect by electrochemistry .
however , there is no data available on the characteristics of electrolysis of naocl solutions relevant to the endodontist .
the purpose of this study was to compare the dissolution capacity achieved by solutions e - naocl on the dissolution of bovine muscle specimens at different concentrations and time periods .
stock solution of the 5% naocl ( naocl 5% , wizard , rehberkimya , istanbul , turkey ) was obtained from manufacturers .
1.25% and 2.5% solutions of the naocl were diluted from the stock solution using distilled tap water .
the solutions were covered amber glass bottles for protecting from co2 and stored at 4c and restored to room temperature before use .
bovine muscle tissue was used to test the dissolving capabilities of naocl at differing concentration , time and electrolysis activation .
the frozen bovine muscle tissues were cut into round pieces of 5 mm diameter and 2 mm thickness using sterilized tissue biopsy punch ( sterile dermal biopsy punch , kai industries ltd . , seki , japan ) .
( n = 154 ) , the tissue samples were preweighed using a digital scale ( presica 205a , dietikon , switzerland ) . after the weights were recorded ,
the samples were placed in 10 ml of the solution to be tested according to time periods . in the group of e - naocl
were conducted using autolab pgstat 302 potentiostat / galvanostat controlled by gpes 4.9 software ( ecochemie , the netherlands ) .
three electrodes were used for all computations ; a platinum electrode of 2 mm diameters as a working electrode , a pt electrode and an ag - agcl reference electrode [ figure 1 ] .
then , the electrodes were performed by triangular potential cycling 3v for 3 min or 5 min according to the groups .
schematic diagram of electrically activation of sodium hypochlorite ; ( a ) working electrode ( pyrolytic graphite electrode ) , ( b ) auxiliary electrode ( platinum sheet ) , ( c ) reference electrode ( ag / agcl ) all experiments were conducted at a room temperature and 10 ml liquids following experiment and control groups .
the samples pieces were then removed , dried , and then weighed again . the percentage of tissues weight loss were calculated .
the groups were as follows : group 1 : distilled water , 3 min ( n = 11 ) group 2 : distilled water , 5 min ( n = 11 ) group 3 : naocl 1.25% , 3 min ( n = 11 ) group 4 : naocl 1.25% , 5 min ( n = 11 ) group 5 : naocl 2.5% , 3 min ( n = 11 ) group 6 : naocl 2.5% , 5 min ( n = 11 ) group 7 : naocl 5% , 3 min ( n = 11 ) group 8 : naocl 5% , 5 min ( n = 11 ) group 9 : eca 1.25% , 3 min ( n = 11 ) group 10 : eca 1.25% , 5 min ( n = 11 ) group 11 : eca 2.5% , 3 min ( n = 11 ) group 12 : eca 2.5% , 5 min ( n = 11 ) group 13 : eca 5% , 3 min ( n = 11 ) group 14 : eca 5% , 5 min ( n = 11 ) .
the averages , standard deviation , minimum , maximum and median were calculated for each group .
resulting data were analyzed by statistically using the multi - way anova and tukey hsd tests .
stock solution of the 5% naocl ( naocl 5% , wizard , rehberkimya , istanbul , turkey ) was obtained from manufacturers .
1.25% and 2.5% solutions of the naocl were diluted from the stock solution using distilled tap water .
the solutions were covered amber glass bottles for protecting from co2 and stored at 4c and restored to room temperature before use .
bovine muscle tissue was used to test the dissolving capabilities of naocl at differing concentration , time and electrolysis activation .
the frozen bovine muscle tissues were cut into round pieces of 5 mm diameter and 2 mm thickness using sterilized tissue biopsy punch ( sterile dermal biopsy punch , kai industries ltd . , seki , japan ) .
( n = 154 ) , the tissue samples were preweighed using a digital scale ( presica 205a , dietikon , switzerland ) .
after the weights were recorded , the samples were placed in 10 ml of the solution to be tested according to time periods .
in the group of e - naocl were conducted using autolab pgstat 302 potentiostat / galvanostat controlled by gpes 4.9 software ( ecochemie , the netherlands ) .
three electrodes were used for all computations ; a platinum electrode of 2 mm diameters as a working electrode , a pt electrode and an ag - agcl reference electrode [ figure 1 ] .
then , the electrodes were performed by triangular potential cycling 3v for 3 min or 5 min according to the groups .
schematic diagram of electrically activation of sodium hypochlorite ; ( a ) working electrode ( pyrolytic graphite electrode ) , ( b ) auxiliary electrode ( platinum sheet ) , ( c ) reference electrode ( ag / agcl ) all experiments were conducted at a room temperature and 10 ml liquids following experiment and control groups .
the groups were as follows : group 1 : distilled water , 3 min ( n = 11 ) group 2 : distilled water , 5 min ( n = 11 ) group 3 : naocl 1.25% , 3 min ( n = 11 ) group 4 : naocl 1.25% , 5 min ( n = 11 ) group 5 : naocl 2.5% , 3 min ( n = 11 ) group 6 : naocl 2.5% , 5 min ( n = 11 ) group 7 : naocl 5% , 3 min ( n = 11 ) group 8 : naocl 5% , 5 min ( n = 11 ) group 9 : eca 1.25% , 3 min ( n = 11 ) group 10 : eca 1.25% , 5 min ( n = 11 ) group 11 : eca 2.5% , 3 min ( n = 11 ) group 12 : eca 2.5% , 5 min ( n = 11 ) group 13 : eca 5% , 3 min ( n = 11 ) group 14 : eca 5% , 5 min ( n = 11 ) .
the averages , standard deviation , minimum , maximum and median were calculated for each group .
resulting data were analyzed by statistically using the multi - way anova and tukey hsd tests .
tissue weight loss with different concentrations of naocl and e - naocl for two different time periods is shown in table 1 .
effect of electrically activation on tissue dissolution ( percentage of tissue weight change loss sd ) by the solutions compared with distilled
water tissue dissolution increased in concentration of naocl and e - naocl .
a significant weight loss was observed after exposure to 2.5% and 5% ( p < 0.001 ) of naocl compared with distilled water and 1.25% naocl .
the concentrations of 2.5% and 5% e - naocl solutions greatly increased the tissue dissolution to same concentrations of naocl groups ( p < 0.001 ) . when times were grouped for evaluation , it was shown that longer exposure times resulted in improved tissue dissolution in control and experimental groups ( p < 0.05 ) . comparing time periods within groups ;
tissue weight loss was significantly higher on the 2.5% and 5% e - naocl at 3 min than 2.5% and 5% naocl at 5 min ( p < 0.05 ) .
there were no significant differences between the 2.5% e - naocl and 5% naocl at 5 min ( p = 0.087 ) .
these results showed that the e - naocl was used , the less time or less concentrations that were required for solution contact to get similar tissue dissolution [ figure 2 ] .
bar chart depicting relative amounts ( percent of original weight ) of remaining bovine tissue , ( n = 11/group ) after 10 ml of sodium hypochlorite
this study focused on the effect of electrically activation of naocl at different concentration and time on the fresh bovine muscle tissue .
bovine pulp , muscle tissue of porcine , rabbit liver , connective tissue of rat , and oral mucosa of pig have been studied as a model to achieve dissolution capability of irrigants .
the main reason of using different type tissues except human dental pulp has been a possibility to obtained and the easier standardization of the surface area of each sample . the importance of many activation methods on the tissue dissolving capability of naocl has been reported , but there are no studies about the effect of electrically activation on naocl solution . the present study showed that the electrically activation of naocl raised its capacity of dissolving organic material .
for the same concentrations of naocl and e - naocl within the same time intervals , the percent of tissue loss was greater in e - naocl ( p < 0.001 ) .
therefore , this irrigation solution is supposed to use less concentration during the endodontic therapy preventing toxic effects .
previous studies have shown that if naocl as irrigation solutions is diluted , the tissue - dissolving ability decreases .
results from the present study showed that tissue weight loss after 5 min in 5% naocl was no significant difference to 2.5% e - naocl ( p > 0.05 ) .
it should be emphasized that lower concentrations of e - naocl have a similar effect to more concentrated naocl solutions on tissue .
tissue specimens immersed in 1.25% naocl , and distilled water increased in weight during the 5-min and 1.25% e - naocl at 3-min after exposure . low - concentration hypochlorite solutions and distilled water can probably cause hydration of the bovine tissue .
previous studies have also reported that a similar effect on the organic tissue by these concentrations and distilled water .
the e - naocl experiments conducted 3v potential and current did not exceed 10 ma .
it should be recorded that the present study was performed in an ideal in vitro study model to test tissue dissolving ability of e - naocl .
furthermore , it still unclear whether e - naocl solutions would be more or less effective than agitation and ultrasonic - activation methods of naocl at different temperatures . | objective : sodium hypochlorite ( naocl ) is a common antimicrobial and tissue - dissolving irrigant .
the aim of this in vitro study is to evaluate and compare dissolution capacities of sodium hypochlorite solutions after electrically activation ( e - naocl ) on bovine muscle specimens at various time periods and concentrations.materials and methods : three sodium hypochlorite solutions of 1.25% , 2.5% , and 5% were tested at 3-min .
and 5-min . with and without activation by electrically .
distilled water and naocl solutions without electrically activation were used as controls .
pieces of bovine muscle tissue ( 34 2 mg ) were placed in 10 ml of each solution at room temperature . in the group of e - naocl ,
electrically activation was performed through the potentiostat .
the tissue specimens were weighed before and after treatment , and the percentage of weight loss was calculated.results:weight loss of the tissue increased with the concentration of e - naocl and naocl .
higher concentration and electrically activation considerably enhanced the efficacy of sodium hypochlorite .
the effect of electrically activation on tissue dissolution was much greater than that of same concentrations in the groups of naocl ( p < 0.001 ) .
tissue weight loss was significantly higher in 2.5% and 5% e - naocl at 3 min .
than in 2.5% and 5% naocl at 5 min .
( p < 0.05 ) .
there were not any significant differences between the 2.5% e - naocl and 5% naocl at 5 min .
( p > 0.05).conclusion : electrically activation can improve the tissue - dissolving effectiveness of sodium hypochlorite . | INTRODUCTION
MATERIALS AND METHODS
Solutions
Tissue collection
Electrically activation of sodium hypochlorite
Data analyses
RESULTS
DISCUSSION
CONCLUSION |
patients typically present with solid food dysphagia and varied degrees of liquid dysphagia , in particular cold liquids , occasional regurgitations of undigested food , and heartburn .
clinical symptoms may also suggest respiratory complications ( nocturnal cough and aspirations ) [ 13 ] .
ninety percent of patients with achalasia have typical radiographic finding ( the so - called bird beak ) created by failure of the lower esophageal sphincter to relax . in the early phase of the disease , endoscopic and radiologic findings can be completely normal .
finally , the diagnosis is confirmed or ruled out by manometry of the upper digestive tract .
absence of effective peristalsis of the esophageal body and failure of the lower esophageal sphincter ( les ) to completely relax upon swallowing are the most characteristic .
the cause of the disease remains far from clear , but it is assumed to arise from a relative absence of inhibitory ganglion cells in auerbach s plexuses of the esophageal wall .
treatment is aimed at reducing les pressure and is still limited to mechanical or pharmacological disruption of its fibers . at present ,
both procedures have similar hazard of esophageal perforation , but multiple dilations may increase perforation ratio .
several authors reported divergence between patients subjective symptomatic improvement and objective result of the treatment . the most sensitive objective examination for les
disruption is esophageal manometry ; however , the examination is not sufficient in long - term follow - up ( evaluation of esophageal mucosa ) .
apart from revealing an enlarging esophageal diameter indirectly suggesting difficulty in its clearance , abnormalities of its wall , and a deteriorated antireflux barrier of les may also suggest coexisting upper digestive tract disorders such as impaired gastric empting .
the study cohort comprised of 71 patients who were treated for achalasia in the clinic of gastrointestinal and general surgery of the university of medicine in wroclaw between 2000 and 2007 .
achalasia was diagnosed by barium swallow study and upper digestive tract endoscopy in all the patients ; most patients ( 85% ) included in the study had esophageal manometry .
manometry was neglected only in patients older than 70 years , and/or with clinical record of the disease duration longer than 36 months , who presented typical bird beak on barium study , and who did not agree to the procedure , but presented no findings on endoscopy .
clinical evaluation of the patients was adapted from the subjective dysphagia score developed by eckardt et al . .
severe dysphagia was diagnosed if the patient experienced difficulty in swallowing during every meal and reported weight loss greater than 20% body weight .
five patients out of endoscopic group were classified as worse candidates for myotomy , mainly due to their age and co - morbidity .
we accepted other patients preferences : they could choose between myotomy and dilation after discussion with an experienced surgeon .
session of balloon dilation begun from size 30 or 35 mm , and duration of the dilatation was 2.53 minutes .
if the dilatation was unsuccessful ( radiogram a day after dilatation with soluble contrast after patient s subjective complaints ) , the second session was attempted on the 3 or 4 day with use of higher or the same diameter balloon and the same duration of the session .
patients who were treated endoscopically had to use ppi for 2 weeks after the procedure because we believe that a potential mucosal tear after mechanical disruption of the les heals quicker in a non - acidic environment .
surgery was considered after 3 trials of balloon dilatation within a period shorter than 12 months and the narrowest esophageal diameter on esophagogram smaller than 0.4 cm .
myotomy was performed 67 cm along the esophagus and 23 cm on to the gastric wall .
the anterior wall of the fundus was fixed to the diaphragmatic hiatus and the left and right walls of the esophagus .
the procedure aimed to reconstruct the abdominal portion of the esophagus and its antireflux barrier .
if a perforation occurred , it was sutured in 2 layers with 5.0 absorbable sutures , and myotomy was continued and completed with anterior fundoplication .
it was double - layer sutured , myotomy was performed laterally to the perforation , and the operation was finished with anterior fundoplication and drainage of the hiatal area .
all patients , regardless of the type of procedure , underwent a postoperative barium swallow study 6 to 8 days postsurgery or within 6 weeks after balloon dilation .
patients were also questioned , using the same eckardt s 4-grade scale , after they resumed oral feeding , to evaluate degree of dysphagia : very good result of the operation if a patient experienced no difficulty in swallowing of both solid and liquid food or experienced the sensation rarely in a month , good result when patient had minor difficulty with solid food or problems appeared sporadically ( no more than twice a week ) , and
all patients were also asked if they would choose to undergo the procedure again under similar postoperative outcomes .
patients were questioned 8 days after surgery or 1 day after balloon dilation , and between 6 months and 2 years after the procedure .
the most important parameters evaluated in control barium swallow were esophageal empting and the narrowest dimension along the lower esophageal sphincter .
if the narrowest dimension of the esophagus after myotomy or balloon dilation was not smaller than 0.4 cm and the esophagus cleared within 5 min , the procedure was considered successful .
barium study was performed in all the patients shortly after the procedure and in 83.1% of them in long - term follow - up ( between 1 and 2 years after the operation ) .
patients who had balloon dilation had a barium study a day after or within 6 weeks since the procedure .
we use the chi - square and fisher tests to show whether sex , age , disease duration , procedure type , or their combination influenced outcomes .
out of 71 patients included in the mid - term follow - up cohort , 12 patients were excluded : 1 patient ( 89 years old ) dilation was unable to complete the interview after balloon due to dementia .
one patient who underwent balloon dilation and 10 patients who underwent surgery did not appear for the control examination .
the study cohort consisted of 2 groups : the dilation group ( 21 patients ages 2382 years , mean age 55.6 years ) and the myotomy group ( 38 patients aged 2174 years , mean age 49.7 ) .
nine ( 15.3% ) patients required both procedures , in 2 ( 9.5% ) patients unsuccessful dilatation was followed by the operation , and in 6 ( 15.8% ) patients poor result of surgical intervention resulted in endoscopy with balloon dilation within 2 years after the surgery .
one ( 4.8% ) patient had esophageal perforation on the first trial of balloon dilation and underwent urgent surgical repair consisting of suturing the perforation and lateral myotomy completed with fundoplication .
all 6 ( 15.8% ) patients who had endoscopic dilation after surgery indicated they if they could have chosen again , they would have preferred endoscopy and balloon dilation instead of surgery .
the vast majority of patients treated with endoscopic dilations did not change their opinion and did not want to have surgery at 2-year follow - up .
although 8 ( 38.1% ) patients treated with balloon dilation 2 years after the first dilation scored the result as poor , only 2 of them agreed to surgical myotomy .
one patient , who had perforation after endoscopic intervention , decided he would not have chosen the dilation again and scored the outcome of surgery as good .
eleven ( 52.4% ) patients were satisfied with the dilation result and evaluated it as very good ( 9.5% ) or good ( 42.9% ) .
surgical complications consisted of 2 ( 5.2% ) perforations that did not interfere with the result of the surgery .
the tear was located in the anterior wall of the esophagus in both cases and was double - layer sutured ; myotomy was performed laterally to the perforation and was completed with anterior fundoplication .
patients who had perforation reported good and very good surgical outcome and the result of their esophageal radiogram showed good esophageal passage , with lower esophageal sphincter diameter between 0.6 and 0.8 cm 2 years after the surgery . at 2-year follow - up , 19 ( 50.0% ) patients in the surgical group scored the result of their surgery as very good , 4 ( 10.5% ) patients scored the result as poor , and their scoring of dysphagia corresponded well with the narrowest esophageal diameter ( 0.20.4 cm ) .
figure 1 presents mean values of eckardt s score at baseline and after 24-month follow - up for both groups . in
both groups mean time of dysphagia reoccurrence was similar , but frequency of reintervention was higher in the dilation group ( table 2 ) .
outcomes in the patients treated with myotomy were statistically significantly better than in the patients treated with dilation shortly after the procedure and during the 2-year follow - up ( figure 2 ) .
there were not statistically significant differences in sex , age , or disease duration between the groups .
few studies have compared short- or long - term results of treatment between balloon dilations and surgical myotomy supported by partial fundoplication in an adequate number of patients .
results of the first procedure are thought to last for a shorter period of time , but both procedures achieve similar control of dysphagia in short - term follow - up [ 68,1015 ] . according to the literature ,
perforation after dilation is a devastating complication ; usually it requires an urgent operation to save the patient s life , but perforation during surgery does not affect postoperative outcome .
we start dilation with a 30-mm balloon and prefer myotomy after the perforation instead of conservative treatment .
according to the literature and our own experience , myotomy decreases the risk of further interventions . as shown in figure 2 , dilation improved symptoms in 61.9% of patients evaluated 6 month after the procedure .
the success rate was highest at 12 months and declined after 24 months ( 81.0% and 52.4% , respectively ) .
myotomy had a greater percentage of patients with clinical improvement ( 94.7% , 92.1% , and 89.5% , p<0.05 ) . at 24-month follow - up , similarly to meta - analysis data , there were more treatment failures in the dilation group than in the myotomy group : 15.6% vs. 47.6% , respectively .
there are 2 types of failure after surgical myotomy for achalasia : early and late .
early failure is a consequence of incomplete myotomy , especially along the stomach wall or scarred myotomy , and is easily verified by difficulty in food intake and on esophagograms ( length and minimal width of the stenosis , time of esophageal empting ) .
reflux esophagitis , with all its consequences , as a result of deteriorating fundoplication occurs as a long - term failure .
similar times of dysphagia recurrence after myotomy and balloon dilation in our data support the thesis .
the observation time in our study might not have been sufficient to judge the outcome of balloon dilation .
successful single dilation may overcome the effect of incomplete or scarred myotomy but still end in treatment failure in long - term follow - up [ 1619 ] .
long - term follow - up is required to evaluate the influence of reflux on dysphagia control and overall success rate in patients treated by balloon dilations .
our experience and data from the literature show that significant proportion of patients do not consent to follow - up with use of invasive methods such as manometry , especially if the result of the procedure is satisfactory in their opinion . for this reason , we prefer less invasive techniques such as barium study and use of the same survey , which patients have to complete before and after the procedure . during the study ,
barium examination was evaluated or supervised by experienced radiologists who looked for simple criteria : the minimal and maximal esophageal width and length , and minimal width of the stenosis ( area of les ) during the first 14 minutes after the patient swallowed 150200 ml of low - density barium suspension . according to the survey ,
patients were questioned about details concerning difficulty with food intake and frequency of their occurrence .
exactly the same survey was completed by the patient pre- and post - operatively as an objective way to categorize symptom severity .
we also asked all patients if they would repeat the treatment considering all the postoperative circumstances .
the success or failure of the procedure from the patient s perspective is strongly determined by symptom relief ; however , attention should also be paid to mean esophageal diameter . in our opinion ,
barium study verifies the narrowest dimension and mean diameter of the esophagus , and is useful in early detection of treatment failures and in prognosing the risk of treatment failure in long - term follow - up .
increasing mean diameter of the esophageal body is an indicator of further deterioration of esophageal function that may lead to megaesophagus .
data from the literature suggests that some patients with subjective improvement after dilation may not present objective evidence of esophageal empting . in this regard , a barium esophagogram is a very useful and quite comfortable examination that may help identify patients with subjective improvement and with no or little difference in esophageal empting .
limitations of this study are its lack of manometry after the procedure , small sample size , and only short - term follow - up . according to some authors ,
the percentage of satisfied patients who underwent myotomy with successful dysphagia score after the first 24 months remains on a similar level in long - term follow - up in the majority of patients .
the attitude towards incomplete or scarred myotomy has evolved : both might be treated with dilation , but re - myotomy should be reserved for non - responders .
an important question is what kind of the procedure is the most appropriate for a particular individual .
the surgeon should consider the patient s general condition , age , personal expectations , attitude towards surgery , and the desire to avoid subsequent interventions .
subsequent interventions are common in patients treated for achalasia , although the risk of further intervention is relatively higher for patients treated with pneumatic dilations [ 6,2022 ] .
an algorithm updated from the american college of gastroenterology practice guidelines 1999 for the treatment of achalasia indicates that the therapeutic schedule depends on age and sex , but our results do not support this .
treatment results did not differ statistically by sex or age in both groups during 2-year follow - up .
stronger les muscle characteristic for male sex and younger age may need more than 1 session with wider balloon dimension in 1 treatment trial .
pastor et al . found increased tension in some cases of previously dilated cardia , but it did not increase perforation rate or influence results of the surgery .
the perforations in our material occurred in primarily operated patients and did not affect the outcomes ( good and very good result of the surgery ) .
the study has confirmed that in certain circumstances both procedures are of similar effectiveness and the decision of which option to use must be carefully discussed with the patient .
the average time for presentation of recurrent dysphagia was 10 months for both groups , although percentage of patients who developed dysphagia was higher in the dilation group ( figure 2 , table 2 ) .
our observation , similar to data from the literature , confirms the thesis that incomplete myotomy leads to dysphagia within 2 years after the surgery [ 19,20,2325 ] .
endoscopic dilation may achieve long dysphagia control , even in patients who did not benefit after the first session and who had to undergo a second session .
patients who do not respond to dilation therapy and present with rapid dysphagia recurrences should be offered surgery .
the observation was also made by snyder et al , who found worse outcomes of myotomy in patients who had undergone multiple endoscopic dilations pretreatment . in this context ,
barium esophagogram is a good diagnostic tool and might be used in follow - up , especially when there is no consistency in the predictive value of manometry in that aspect .
suggested that high les pressure after first dilation does not discriminate the patient from patients with low les pressure , although the group of patients with high les pressure shows the tendency towards the need for more than 1 dilation session to achieve clinical remission .
ever myotomy or balloon dilation might be useful in dysphagia control , but better results are obtained after myotomy . | backgroundthe optimal therapeutic schedule in patients with achalasia is still under discussion .
the aim of this study was to review our institution s experience with myotomy and dilation in patients with achalasia.material/methodsclinical data were available for 59 patients who had ever had myotomy ( n=38 ) , dilation ( n=21 ) , or both procedures ( n=8 ) between 2000 and 2007 .
patients were followed prospectively with objective ( a barium esophagogram ) and subjective ( a simple survey that scored dysphagia and overall patient satisfaction with the procedure ) diagnostic tools . in the group of patients after pneumatic dilatations ,
frequency of interventions was higher ( 1 , 2 ) than in the myotomy group ( 0 , 2 ) at 2-year follow - up .
patients after myotomy with recurrence of dysphagia were treated with dilations.resultsmean time of dysphagia occurrence was similar in both groups ( 10 months ) .
the statistically significant differences in treatment outcomes in both groups were in favor of myotomy during 2-year follow-up.conclusionsthe data indicates that both methods of treatment might be useful in dysphagia control , but better results are obtained after myotomy .
repeat interventions are more frequent after endoscopic dilation .
one method of treatment does not exclude the other .
a short period of symptom relief after myotomy may suggest the myotomy was incomplete . | Background
Material and Methods
Results
Discussion
Conclusions |
the design of translating research into action for diabetes ( triad ) has been previously described ( 12 ) .
the primary objective of triad was to determine how structural and organizational characteristics of health systems and health care provider groups influence the processes and outcomes of diabetes care .
six translational research centers collaborated with 10 health plans and 68 provider groups that served 180,000 diabetes patients .
health plans from california , hawaii , indiana , michigan , new jersey , new york , pennsylvania , and texas participated and included a racially and ethnically diverse membership .
the institutional review boards at all translational research centers reviewed and approved the triad study protocol , and all participants provided informed consent .
the triad study population was a random sample of adult enrollees with diabetes from participating health plans .
patients were eligible to participate if they were aged 18 years , community dwelling , spoke english or spanish , not pregnant , continuously enrolled in the health plan for at least 18 months , had diabetes for 1 year , and used services during their health plan enrollment .
in addition , eligibility required at least two outpatient visits or one inpatient stay with a diagnostic code for diabetes ( icd-9 250.xx ) , laboratory tests or values suggesting diabetes ( at least two hba1c tests ordered or a diagnostic hba1c or fasting blood glucose level ) , or a prescription for medications for diabetes ( e.g. , insulin or an oral antidiabetic agent ) . at the time of the survey
, patients who met these criteria were included only if they confirmed that they had diabetes and received most of their diabetes care through the participating triad health plan .
type 2 diabetes was defined as diabetes that was not currently treated with insulin or that was diagnosed after age 30 years , with or without current insulin treatment .
patient surveys were completed between july 2000 and october 2001 . of the 13,086 individuals who were contacted and eligible , 11,927 ( 91% ) completed the survey .
survey variables included age , sex , race / ethnicity , education , income , bmi , smoking , time since diagnosis of diabetes , treatment for diabetes , and hrqol as measured by the eq-5d . of the patients completing a survey ,
the participants whose records were reviewed were similar to the overall study population ( 13 ) .
centrally trained reviewers used standardized data collection forms to abstract each patient s records of medical treatment and prescribed medications during the 12 months before the survey date ; of these , 5% were abstracted by two abstractors . inter - rater reliability ( )
ranged from 0.86 to 0.94 for the main quality measures derived from the medical record data .
variables that were obtained from the record review were diabetes - related micro- and macrovascular complications and comorbid conditions .
we chose the eq-5d over other health utility measures , such as the short - form health survey-6d , because of its simplicity , sufficient responsiveness to changes in health states , relatively wide range of utility scores generated by the instrument , and availability of preference weight for our study population ( 11,14 ) .
briefly , the eq-5d consists of two parts : five questions relating to distinct dimensions of a person s functional capacity ( mobility , self - care , usual activity , pain / discomfort , and anxiety / depression ) and a visual analog scale .
there are three responses for each domain of the questionnaire . because the baseline survey was conducted primarily by phone , the visual analog scale of the eq-5d was not implemented .
the responses from the eq-5d were combined with preference weights derived from a sample of the u.s .
we constructed categories within each type of complication based on clinically defined disease states and data availability and ordered the individual disease categories based on disease severity .
the specific disease categories for each of the five diabetes - related complications include diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , stroke , and cardiovascular disease ( table 2 ) . within diabetic retinopathy ,
individuals with laser treatment were considered to have proliferative diabetic retinopathy . within cardiovascular disease , the category of other coronary heart diseases
was defined as having indicators of coronary heart disease or coronary artery disease or undergoing procedures to treat coronary heart disease such as coronary angioplasty and coronary bypass .
peripheral vascular disease was categorized as having or not having the disease .
a person had peripheral vascular disease if he or she had a record of peripheral vascular disease or had undergone procedures used to treat the disease such as peripheral vascular angioplasty or bypass .
means and standard errors are reported for continuous variables ( except age ) and proportions for categoric variables .
tests for differences between categories in eq-5d scores by patient level variables were tested using a test .
the eq-5d utility scores were modeled by a multivariate linear regression model adjusting for demographic and clinical characteristics and for disease states .
missing values for independent variables were imputed five times using a multiple imputation method ( 15 ) .
when estimates of adjacent response categories for any diabetes - related complication in the model were not significantly different from each other or were inconsistent with the order of increasing severity , the two response categories were combined and the model was rerun .
only those variables that had statistically significant estimated coefficients were kept in the final model .
the estimated coefficients of the indicator variables represent the penalty or deficit from optimal health associated with each variable .
subtracting penalty functions from the health utility scores for the healthy reference group created an additive model from which we derived the eq-5d utility score for any combination of treatments , complications , and comorbidities .
after combining some of the categoric variables , the reference group was defined as a nonobese man with type 2 diabetes who was not asian or hispanic , not using insulin treatment , and who had an annual household income of more than $ 40,000 with no diabetes - related complications , risk factors for cardiovascular disease , or comorbidities .
all statistical analyses were performed using sas 9.2 software ( sas institute inc . , cary , nc ) .
patient surveys were completed between july 2000 and october 2001 . of the 13,086 individuals who were contacted and eligible , 11,927 ( 91% ) completed the survey .
survey variables included age , sex , race / ethnicity , education , income , bmi , smoking , time since diagnosis of diabetes , treatment for diabetes , and hrqol as measured by the eq-5d . of the patients completing a survey ,
the participants whose records were reviewed were similar to the overall study population ( 13 ) .
centrally trained reviewers used standardized data collection forms to abstract each patient s records of medical treatment and prescribed medications during the 12 months before the survey date ; of these , 5% were abstracted by two abstractors . inter - rater reliability ( )
ranged from 0.86 to 0.94 for the main quality measures derived from the medical record data .
variables that were obtained from the record review were diabetes - related micro- and macrovascular complications and comorbid conditions .
we used the eq-5d to derive health utility scores . the validity and reliability of the eq-5d
we chose the eq-5d over other health utility measures , such as the short - form health survey-6d , because of its simplicity , sufficient responsiveness to changes in health states , relatively wide range of utility scores generated by the instrument , and availability of preference weight for our study population ( 11,14 ) .
briefly , the eq-5d consists of two parts : five questions relating to distinct dimensions of a person s functional capacity ( mobility , self - care , usual activity , pain / discomfort , and anxiety / depression ) and a visual analog scale .
there are three responses for each domain of the questionnaire . because the baseline survey was conducted primarily by phone , the visual analog scale of the eq-5d was not implemented .
the responses from the eq-5d were combined with preference weights derived from a sample of the u.s .
we constructed categories within each type of complication based on clinically defined disease states and data availability and ordered the individual disease categories based on disease severity .
the specific disease categories for each of the five diabetes - related complications include diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , stroke , and cardiovascular disease ( table 2 ) . within diabetic retinopathy ,
individuals with laser treatment were considered to have proliferative diabetic retinopathy . within cardiovascular disease , the category of other coronary heart diseases
was defined as having indicators of coronary heart disease or coronary artery disease or undergoing procedures to treat coronary heart disease such as coronary angioplasty and coronary bypass .
peripheral vascular disease was categorized as having or not having the disease .
a person had peripheral vascular disease if he or she had a record of peripheral vascular disease or had undergone procedures used to treat the disease such as peripheral vascular angioplasty or bypass .
means and standard errors are reported for continuous variables ( except age ) and proportions for categoric variables .
tests for differences between categories in eq-5d scores by patient level variables were tested using a test .
the eq-5d utility scores were modeled by a multivariate linear regression model adjusting for demographic and clinical characteristics and for disease states .
missing values for independent variables were imputed five times using a multiple imputation method ( 15 ) .
the initial model was developed using all variables . when estimates of adjacent response categories for any diabetes - related complication in the model were not significantly different from each other or were inconsistent with the order of increasing severity , the two response categories were combined and the model was rerun .
only those variables that had statistically significant estimated coefficients were kept in the final model .
the estimated coefficients of the indicator variables represent the penalty or deficit from optimal health associated with each variable . subtracting penalty functions from the health utility scores for the healthy reference group created an additive model from which we derived the eq-5d utility score for any combination of treatments , complications , and comorbidities . after combining some of the categoric variables , the reference group was defined as a nonobese man with type 2 diabetes who was not asian or hispanic , not using insulin treatment , and who had an annual household income of more than $ 40,000 with no diabetes - related complications , risk factors for cardiovascular disease , or comorbidities .
all statistical analyses were performed using sas 9.2 software ( sas institute inc . , cary , nc ) .
, they reflect the characteristics of the population with type 2 diabetes in the u.s .
patients with type 2 diabetes tend to be older , to be more racially diverse , to have higher bmis , and to have a higher proportion of individuals with low income and education levels compared with the general u.s .
the average hba1c level in the triad population with type 2 diabetes ( 8.0% ) was similar to that for the u.s .
general population with type 2 diabetes during the same period ( 7.5% ) ( 17 ) .
ore than 90% of patients were treated with oral antidiabetic agents , insulin monotherapy , or a combination of the two .
the mean charlson index , a weighted measure that incorporates 19 diseases to predict 10-year mortality risk according to severity of comorbidities , was 2.3 . for reference ,
the predicted 10-year mortality risk is 48% for individuals with a charlson index score of 2.0 ( 18 ) .
table 2 presents the eq-5d scores by demographic and clinical characteristics of the study population .
the eq-5d scores differed across subgroups by age , sex , race , level of education , and household income .
the scores also differed by time since diagnosis of diabetes , bmi , smoking status , and diabetes treatment . in general , patients with diabetes - related complications or comorbidities had lower health utility scores than those without . among those with the lowest eq-5d scores were patients who required dialysis , who had hemiplegia , or who had experienced the amputation of both feet .
unadjusted eq-5d health utility score by patient characteristics the penalty functions associated with each characteristic are presented in table 3 .
the intercept value of 0.920 was the mean health utility score in the reference group .
being a woman , being obese , smoking , using insulin , or having an annual household income of less than $ 40,000 were associated with a lower health utility score .
the most severe microvascular complications , painful neuropathy and amputations , were associated with a decrement of more than 0.10 in the health utility score .
complications associated with a reduction of more than 0.03 in the health utility score were hemiplegia , nonpainful diabetic neuropathy , cerebral vascular accident , congestive heart failure , and dialysis .
other complications , such as other coronary heart disease , transient ischemic attack , and peripheral vascular disease , were associated with relatively small reductions in eq-5d scores ( < 0.03 ) .
nonproliferative and proliferative diabetic retinopathy did not independently reduce health utility scores and were dropped from the final model . similarly , age , education , time since diagnosis of diabetes , and hypertension were not associated with significant independent reductions in health utility scores and were excluded from the final model . estimated coefficients for the multiple regression model
we derived health utility scores for adults with diabetes , its complications , and its comorbidities by using data collected in a large multicenter , prospective observational study of managed care patients from seven u.s . regions .
as one might expect , diabetes - related complications were associated with lower utility score , and the magnitude of the impact varied by the complication . in increasing order of deficit , the complications were peripheral vascular disease , other heart diseases , transient ischemic attack , cerebral vascular accident , nonpainful diabetic neuropathy , congestive heart failure , dialysis , hemiplegia , painful neuropathy , and amputation .
the variations in the effect of different diabetes complications on hrqol were as much as 10-fold .
however , it is not clear whether the lower health utility scores associated with insulin treatment were because insulin treatment did not improve functional capacity or because insulin treatment served as a surrogate for greater diabetes severity .
those treated with insulin have been diagnosed with diabetes longer and have experienced more diabetes - related complications than those who are treated with diet or oral antidiabetic agents alone . in our study ,
insulin treatment was still associated with a lower utility score after adjusting duration of diabetes and diabetes - related complications , implying insulin treatment lowered health utility scores independently .
studies that have assessed insulin use as part of overall quality - of - life have shown a decrease in quality - of - life as treatment moved from diet only to oral agents to insulin ( 19 ) .
however , studies comparing quality - of - life with specific treatment regimens and ongoing support strategies generally reported no decline , or even an improvement , in quality - of - life with insulin use ( 19 ) . on the one hand ,
using insulin could lower quality - of - life directly because of inconvenience , adverse effects of insulin injections , and hypoglycemia , or indirectly by stigmatizing a patient as being unable to control his or her diabetes adequately ( 20 ) . on the other hand , insulin use could also improve quality - of - life by leading to better glycemic control , which is positively associated with a high level of perceived quality - of - life ( 21 ) .
obesity , especially morbid obesity ( bmi 35 kg / m ) , was associated with a large reduction in health utility scores .
( 20 ) found a negative relationship between excess body weight and hrqol among type 2 diabetic patients , even after adjusting for their demographic characteristics , diabetes duration , diabetes treatment mode , and presence of cardiovascular diseases and other comorbid conditions .
obesity impairs physical functioning , decreases energy levels , increases health distress , and decreases self - rated health .
our model found a higher health utility score for asians and hispanics and a lower score for women and low - income individuals .
these differences in health utility scores by race , sex , or income level are consistent with findings from previous studies ( 21 ) .
however , these results do not imply that a diabetes intervention is more cost - effective for asians or hispanics compared with other races , for men compared with women , or for those of lower income compared with those of middle income , because no interactions were found among any of the three variables for type of diabetes treatment or diabetes - related complications ( data not shown ) .
coffey et al . ( 2 ) estimated health utility scores associated with different diabetic treatments , complications , and comorbidities among individuals with type 2 diabetes .
their study used a self - administered version of the quality of well being index to survey 1,257 people with type 2 diabetes who were patients at endocrinology , diabetes , and ophthalmology clinics at the university of michigan health system .
the study estimated the mean health utility score for a diet - controlled nonobese diabetic man without any complications , comorbidities , or cardiovascular risk factors to be 0.687 .
the closest comparable utility value in our study was the mean utility value for our reference group , 0.920 .
the difference in these values is likely attributable to the hrqol instruments used in the two studies .
health utility scores based on the eq-5d tend to be higher than those from other hrqol instruments .
in fact , about a quarter of our subjects had a utility value of 1.00 .
eq-5d can not distinguish small differences in physical or mental health status in relatively healthy populations .
this ceiling effect of eq-5d has been documented in other studies ( 3,4 ) . the estimated range and level of penalty scores associated with individual complications between the coffey et al .
they estimated that the penalty score , excluding blindness ( we did not collect information on blindness ) , varied from 0.011 to 0.105 , whereas our estimates varied from 0.012 to 0.108 .
clarke et al . ( 4 ) estimated health utility scores in 3,192 type 2 diabetic subjects who participated in the united kingdom prospective diabetes study ( ukpds ) using the eq-5d .
they estimated that a man with type 2 diabetes and no complications would have a utility score of 0.8500.962 , depending on the specific regression model .
their estimated range of penalty scores for individual complications was much larger ( 0.0550.280 ) .
several factors could have contributed to the different results : first , subjects in the ukpds represented a relatively healthy and homogenous newly diagnosed type 2 cohort .
our population included type 2 diabetic patients with a wide range of treatments , complications , comorbidities , and duration of diagnosed diabetes .
second , in calculating the utility score , we used the preference weights from the u.s .
finally , because of data availability , the variables included in the regression models were different .
having fewer variables in their regression model may have added weight to the variables they included .
( 22 ) assessed health utility scores using the eq-5d in a sample of 1,136 dutch type 2 diabetic subjects who participated in the code-2 study .
older age , obesity , woman , insulin therapy , and presence of complications were associated with lower health utility scores .
their relatively small study sample may have limited their ability to assess the effect of each individual complication on hrqol .
bagust and beale ( 3 ) also used data collected in the code-2 study to estimate utility scores but combined the dutch data used by redekop et al .
because of the much larger sample size of 4,641 , this study was able to estimate eq-5d utility scores associated with specific diabetes complications . except for coronary heart disease ,
our estimated penalty scores associated with individual complications were lower than those in the bagust and beale regression model ( 3 ) .
for example , the estimated penalty score for amputations was 0.108 in our study and 0.272 in the bagust and beale study .
the estimated penalty score for coronary heart disease was 0.042 in our study and 0.028 in the bagust and beale study .
the use of dissimilar preference weights in deriving the eq-5d score and dissimilar demographic variables and definitions of complications may have contributed to differences in estimated health utility scores .
the limited comparability of the estimated health utility scores between our study and the studies cited above might imply that penalty scores reported by different studies should be interpreted in the broad context of differences in study populations and quality - of - life instruments used and of the completeness and types of variables used in the regression models .
( 14 ) estimated the health utility scores associated with diabetes and its complications for patients enrolled in a clinical trial in australia .
the health utility score for those who had no any diabetes - related complication was 0.88 , a bit lower than the score in our study .
reduction in health utility scores by complications in decreasing order resulted in the following : stroke and/or transient ischemic attack , peripheral revascularization and/or amputation , unstable angina , myocardial infarction , coronary artery bypass graft , and eye disease .
differences in study population and definition of complications may have contributed to the different utility scores between the two studies . some limitations of our study should be noted .
health unity scores associated with diabetes and its complication can change due to improvements in physical , mental , and social functions of diabetes patients over time .
our study focused on assessing the relative decrement utility associated with each diabetes - related complication and its progression rather than the absolute level of health utility scores for those complications .
little evidence exists on the change in the relative function levels associated with different complications in the last 10 years .
thus , our estimates are very likely still applicable to the current diabetes population . because our income level was measured in 2001 u.s .
dollars , the income variable should be adjusted using an inflation calculator ( 23 ) if income is expressed in other years .
patients with no health insurance tend to have a lower quality - of - life than those with health insurance ( 24 ) . however , it is less clear that disutility associated with diabetes treatment mode and diabetes - related complications differ between the two groups .
in addition , our study population may not represent the entire managed care population with diabetes , and individuals in managed care might not be representative of the entire diabetic population ; for example , poorer , sicker , or older individuals with type 2 diabetes may have been less likely to participate in the triad survey .
finally , our study was cross - sectional , and variation in responses could occur if hrqol was measured at multiple points in time .
cost - utility analysis , the type of economic evaluation most often used to assess interventions used for the prevention and control of type 2 diabetes , requires valid and accurate health utility estimates by patient characteristics , treatment modalities , and the different clinical states of each diabetes - related complication .
we derived a set of such health utility estimates using data collected in a large multicenter diabetes study using u.s .- based preference weights .
our empirically derived health utility scores will allow researchers to calculate qalys for studies involving individuals living in the u.s . with type 2 diabetes and representing a wide variety of demographic characteristics , treatments , complications , and comorbidities .
the health utility scores provided should facilitate studies of the health burden of diabetes and the cost - utility analysis of alternative strategies for the prevention and treatment of diabetes in the u.s . | objectiveto estimate the health utility scores associated with type 2 diabetes , its treatments , complications , and comorbidities.research design and methodswe analyzed health - related quality - of - life data , collected at baseline during translating research into action for diabetes , a multicenter , prospective , observational study of diabetes care in managed care , for 7,327 individuals with type 2 diabetes .
we measured quality - of - life using the euroqol ( eq)-5d , a standardized instrument for which 1.00 indicates perfect health .
we used multivariable regression to estimate the independent impact of demographic characteristics , diabetes treatments , complications , and comorbidities on health - related quality-of-life.resultsthe mean eq-5d derived health utility score for those individuals with diabetes was 0.80 .
the modeled utility score for a nonobese , non insulin - treated , non - asian , non - hispanic man with type 2 diabetes , with an annual household income of more than $ 40,000 , and with no diabetes complications , risk factors for cardiovascular disease , or comorbidities , was 0.92 .
being a woman , being obese , smoking , and having a lower household income were associated with lower utility scores . arranging complications from least to most severe according to
the reduction in health utility scores resulted in the following order : peripheral vascular disease , other heart diseases , transient ischemic attack , cerebral vascular accident , nonpainful diabetic neuropathy , congestive heart failure , dialysis , hemiplegia , painful neuropathy , and amputation.conclusionsmajor diabetes complications and comorbidities are associated with decreased health - related quality - of - life .
utility estimates from our study can be used to assess the impact of diabetes on quality - of - life and conduct cost - utility analyses . | RESEARCH DESIGN AND METHODS
Data sources
Health utility measure
Diabetes-related complications
Statistical analysis
RESULTS
CONCLUSIONS |
it mimics a number of other clinical conditions such as ganglion , de quervan s disease , carpal tunnel syndrome , granulomatous gout , neurofibroma , and haemangioma .
we present case of 58-year - old male patient with swelling over thenar region of left hand where the initial fine needle aspiration cytology ( fnac ) report suggested diagnosis of ganglion / synovial cyst , but the patient did not improve with conservative treatment .
fnac was repeated which showed granulomatous inflammation consistent with the clinical diagnosis of tuberculosis and antitubercular therapy for 9 months was given .
swelling disappeared and normal range of movements was attained in adjacent joints at final follow - up of 12 months from initiation of antitubercular treatment ( att ) .
the literature on the management of tubercular tenosynovitis has mostly incorporated surgical method as one of the first - line treatments .
we report an unusual case , where the tenosynovitis of wrist completely responded within 12 months of att without any need of surgical treatment .
tuberculosis ( tb ) can affect any organs / systems of the human body and have variable presentation in each system . the musculoskeletal system is involved in approximately 10% of all extrapulmonary involvements .
tb tenosynovitis of the wrist and hand is an unusual condition [ 2 , 3 ] . the major dilemma in diagnosing these uncommon presentations is the presence of the non - specific clinical signs which can be misleading as they mimic a number of other clinical conditions such as ganglion , de quervain s disease , carpal tunnel syndrome , and granulomatous gout [ 4 , 5 , 6 ] .
we report an unusual case of tubercular tenosynovitis of the left hand in a 58-year - old male who had been treated as ganglion .
the purpose of this report is to highlight the importance of radiological and histopathological investigations in these suspicious cases .
further , there is only handful of literature available which highlights the management of tubercular tenosynovitis with anti - tubercular therapy only without operative treatment .
we present a case of 58-year - old male patient who presented to the outpatient department of the author s tertiary level institute with swelling over thenar region of the left hand .
he was a fully independent , nonsmoker , right handed , asthmatic patient on inhalational corticosteroids since 5 years .
he visited a local orthopedician who advised him to get fine needle aspiration cytology ( fnac ) of the swelling .
fnac report suggested the diagnosis of ganglion / synovial cyst and conservative treatment was started , but the swelling did not decrease in size , rather gradually increased in size and became painful with other signs of inflammation .
when the patient presented to the author s institute , there was a single , soft , fluctuant swelling of 5 cm 4 cm over left thenar region with ill - defined margins , erythematic overlying skin .
there was diffuse edema over the volar surface of the left wrist and distal forearm associated with erythema and tenderness .
there was no history of fever , trauma , joint pain , rashes , prolonged cough , decreased appetite , weight loss as well as no known or traceable history of contact with tb .
clinical photograph showing- ( left ) pretreatment lesion - single , soft , fluctuant swelling over left thenar region with ill - defined margins , erythematic and discolored overlying skin .
it revealed a large peripherally enhancing cystic lesion containing multiple loose bodies involving thenar eminence of left palm .
tenosynovitis of flexor carpi radialis was also seen , and a small cystic lesion ( 0.9 cm 0.5 cm 0.4 cm ) was seen superficial to flexor retinaculum laterally .
underlying carpal bones revealed focal area of altered signal intensity , with no evidence of joint effusion ( fig .
fnac was repeated which showed granulomatous inflammation with caseation , consistent with the clinical diagnosis of tb ( fig .
3 ) and the sample was sent for culture . the patient was started on antitubercular therapy with four first - line drugs ( isoniazid , rifampicin , pyrazinamide and ethambutol ) for 4 months followed by biotherapy ( isoniazid , rifampicin ) for 5 months .
complete blood count revealed elevated white blood cell count of 14.8 10/l with a high neutrophil differential count of 90% but low lymphocyte differential count of 5% .
erythrocyte sedimentation rate was also elevated ( 50 mm / h ) . magnetic resonance imaging wrist and hand showing a large peripherally enhancing cystic lesion containing multiple loose bodies involving the flexor tendons of forearm and thenar eminence of left palm .
the patient was compliant to the treatment and did not have any major adverse drug effects .
swelling disappeared and normal range of movements was attained in adjacent joints at final follow - up of 12 months from initiation of antitubercular treatment ( fig .
1 ) . supervised physiotherapy for fingers and wrist was initiated especially in the initial treatment phase to prevent adhesions and contractures and improve functional results .
mycobacterium tb infection is mostly seen in patients with impaired cell - mediated immunity , which may be disseminated readily resulting in high mortality rates .
the known predisposing factors include age > 60 years , malnutrition , alcohol abuse , low socioeconomic status , history of or exposure to tb , immunosuppression from any cause , and intake of corticosteroids .
the two theories regarding the pathogenesis of tb tenosynovitis direct inoculation and hematogenous dissemination from a primary focus .
infection appears to start in the synovium and then gradually spread to the tendons and even the bones .
tb tenosynovitis selectively targets the wrist and volar aspect of the hand , where it accounts for 5% of cases of osteoarticular tb .
the main setback remains the difficulty in diagnosing the disease because of non - specific clinical signs that point to a number of other possibilities such as other mycobacterial infections , pyogenic and fungal infections , brucellosis , foreign body tenosynovitis , sarcoidosis , rheumatoid arthritis ( ra ) , gouty arthritis , de quervain s disease , and ganglion [ 3 , 4 , 6 ] .
tb tenosynovitis may appear in different forms as a result of the long duration of the disease , the varying resistance of the individual , and the varying virulence of the organism .
patients with tubercular tenosynovitis usually present with an insidious , slow - growing , sausage - like mass along the inflamed tendon with minimal or no pain ; sometimes may present with discharging sinus and carpal tunnel syndrome .
this explains the presentation of our case , who initially presented to outpatient department with slow growing , insidious , painless swelling and later ( after being treated as ganglion only ) developed pain , increased size of swelling , and other signs of inflammation .
further , plain radiograph of the left hand showed only a large soft tissue mass on the thenar aspect of the hand with no bony involvement .
mri discovered a large peripherally enhancing cystic lesion containing multiple loose bodies with focal area of altered signals in underlying carpal bones ( fig .
finally , repeat fnac was performed which showed granulomatous inflammation consistent with tb ( fig .
mri may show thickening of the synovial membrane , increased vascularity and fluid within the tendon sheath , reactive inflammation and swelling around the tendon .
in contrast to acute suppurative tenosynovitis , where synovial sheath fluid is the predominant feature , relatively little synovial sheath fluid is characteristic of tb pathology [ 3 , 7 ] .
multiple rice bodies are composed of amorphous material surrounded by collagen and fibrin with unknown etiology .
although this is a rare occurrence related to ra , rice bodies can also be seen in systemic lupus erythematosus , seronegative arthritis , infectious arthritis ( tb , atypical mycobacterial infection ) , and nonspecific arthritis .
the literature on the management of tubercular tenosynovitis has mostly incorporated surgical method as one of the first - line treatment .
surgical debridement is controversial [ 9 , 10 ] tuli states that surgical debridement should be performed in patients who do not respond to four or five months of chemotherapy .
furthermore , surgery may cause adhesions especially in palmar side of hand so antitubercular therapy should be tried first .
only a few studies are available which managed the disease with antitubercular therapy ( att ) alone without the primary surgical procedure .
we report an unusual case , where the tenosynovitis of wrist completely responded within 12 months of att without any need of surgical treatment ( fig .
although rare , orthopedicians must consider tb tenosynovitis of wrist and hand as a differential , where patient presents even with an innocent mass lesion at the wrist .
the patient should undergo a thorough clinical , radiological and histopathological examination , to diagnose the tubercular tenosynovitis .
early diagnosis and instigation of att with or without surgical procedure gives good functional results .
tubercular tenosynovitis of hand is a rare presentation which can mimic many other clinical conditions due to which it can be easily misdiagnosed and mistreated .
once diagnosed , it can be treated with antitubercular chemotherapy without the need of surgical treatment . | introduction : tuberculosis tenosynovitis of the wrist and hand is an unusual condition .
it mimics a number of other clinical conditions such as ganglion , de quervan s disease , carpal tunnel syndrome , granulomatous gout , neurofibroma , and haemangioma.case report : we present case of 58-year - old male patient with swelling over thenar region of left hand where the initial fine needle aspiration cytology ( fnac ) report suggested diagnosis of ganglion / synovial cyst , but the patient did not improve with conservative treatment .
fnac was repeated which showed granulomatous inflammation consistent with the clinical diagnosis of tuberculosis and antitubercular therapy for 9 months was given .
swelling disappeared and normal range of movements was attained in adjacent joints at final follow - up of 12 months from initiation of antitubercular treatment ( att).conclusion : the literature on the management of tubercular tenosynovitis has mostly incorporated surgical method as one of the first - line treatments .
we report an unusual case , where the tenosynovitis of wrist completely responded within 12 months of att without any need of surgical treatment . | Introduction:
Case Report:
Conclusion:
Introduction
Case Report
Discussion
Conclusion |
palatal rugae ( transverse palatine folds or palatine rugae ) are anatomical structures present in the roof of the human mouth . among humans , they are recognized as asymmetrical , irregular mucosal ridges arranged in a transverse direction on either side of the median palatine raphe in the anterior third of the palate behind the incisive papillae [ 1 , 2 ] .
palatal rugae patterns are formed toward the third month of intrauterine life and initially occupy much of the length of the palatal shelves at the time of their elevation .
later , their pattern becomes less regular , with the anterior ones being more evident and compressed while the posterior ones disappear , resulting in their confinement to the anterior part of the secondary palate from the newborn stage onwards .
the regulation of the collagen fibers orientation in rugal morphogenesis has been attributed to the influence of certain genes .
following palatal rugae formation , their length continues to change during palatine growth , but their position remains the same throughout life .
therefore , it has been indicated that they gain their typical orientation pattern at birth and acquire their final shape in adolescence .
thereafter , they remain stable throughout life . however , this stability of the palatal rugae patterns is a matter of considerable debate in the literature ; some reports indicate that the rugae are closely related to teeth , following their movement after extraction of other teeth , resulting in a change in the original direction of the rugae ; in contrast , others indicate the retention of their stability [ 7 , 8 ] . the more posterior rugae were suggested as being more stable ; however , it was also noted that the first rugae are the most stable .
furthermore , pattern changes were observed following extreme finger sucking during infancy and orthodontic treatment due to persistent pressure [ 2 , 10 ] .
palatal rugae shapes were proven to resist diseases , chemical aggression , and postmortem intrusions ; additionally , they are well protected by the lips , a buccal pad of fat , and the teeth .
it was found that they express less pronounced changes compared to the general condition of the victim of 3rd degree facial burns . despite the similarity in rugae patterns between twins
therefore , the palatal rugae can be considered an ideal parameter for forensic identifications in terms of its uniqueness and postmortem resistance as well as stability following completion of growth [ 7 , 13 ] .
moreover , they can be especially useful in cases where fingerprints can not be attained , for example , when bodies are burnt or severely decomposed .
it is noteworthy that a comparison of rugae patterns between different ethnicities and within the subset of a single population showed diverse presentation patterns [ 2 , 1416 ] .
palatal rugae morphological studies and systemic trends in precise sex determination are lacking among sudanese arabs .
sudan is a country with extensive indigenous diversity , and sudanese arabs form the largest ethnic group of contemporary sudan and have a genetic mixture of arabs and africans [ 17 , 18 ] .
hence , the present study aimed to determine the prevalence of the different biometric characteristics of the palatal rugae in the sudanese arab population , to explore the existence of asymmetry in these sexes , and to determine its effectiveness in identifying sexes using logistic regression .
the age group involved in this study was between 18 and 23 years of age ( the mean age of males was 21.76 2.53 , and the mean age of females was 21.33 2.16 ) .
they were sudanese arabs with at least three generations on the mother 's side and the father 's side .
subjects with severe malocclusion , a history of palatal pathology , trauma or surgery to the palate , orthodontic treatment , active lesions , or any sign of allergy to impression material were excluded from the study .
the study received approval from the ethical committee of the faculty of medicine , university of khartoum , khartoum , sudan . after obtaining the informed consent , a maxillary impression of the subject
the casts were immediately poured with improved type iv dental stone ( zhermack , italy ) to obtain greater strength and accuracy .
all instructions by manufacturers were followed , including the water / powder ratio , vacuum mixing , and the use of a vibrator .
rugae patterns were delineated using a sharp 0.1 hb graphite pencil under adequate light and magnification utilizing a magnifying hand lens .
all the rugae patterns were assessed and measurements were conducted by a single examiner ( ah ) using a digital sliding caliper and protractor .
the rugae classification was recorded according to lysell and thomas and kotze [ 19 , 20 ] .
the palatine rugae lengths were measured on each side using a digital sliding caliper to an accuracy of 0.01 mm . based on their length ,
the rugae were divided into three categories : primary rugae with lengths of more than 5 mm , secondary rugae with lengths from 3 to 5 mm , and fragmentary rugae with lengths between 2 and 3 mm .
the shapes of the rugae ( figure 1 ) were classified into five major types : straight , curved , circular , wavy , and angular . on the other hand ,
the branching type had branches that extended 1 mm or more from the origin in a lateral direction .
the direction of each primary ruga was classified according to the angle between the line joining its origin and termination with a line perpendicular to the median raphae .
backward - directed rugae were associated with negative angles , and perpendicular rugae were associated with no angle .
the direction of the branched rugae was determined at their termination in the midpoint between the ends of the two branches . prior to recording the findings
, rugae observations were repeated twice for 20 casts to assess intraobserver error in interpretations .
association between length and directions and sex was assessed using the mann - whitney test .
bilateral differences were assessed using wilcoxon signed - rank tests . logistic regression analysis ( lra )
was used to assess the possibility of sex prediction utilizing a discrete variable ( shape ) and continuous variables ( dimensions and orientation ) using statistical package for the social sciences ( spss ) , version 14 computer software ( spss , inc . ,
the prevalence and percentages of different rugae lengths in both sexes are described in table 1 .
males tend to have a larger number of rugae than females ( 665 and 621 rugae , resp . ) . besides , the mean number of primary ( 505 ) and secondary ( 129 ) rugae in males was greater than that of females ( 486 and 103 , resp . ) , whereas the fragmented rugae were comparable in both groups ( 31 to 32 ) ; however , there were no significant differences between sexes in the means .
primary rugae were predominant in both sexes compared to secondary and fragmented rugae . when the shapes were considered ( table 2 ) , in males curved rugae are the most prevalent form , followed by wavy rugae and then by straight rugae .
in contrast , in females , wavy rugae are the most prevalent , followed by the curved rugae and then straight rugae .
however , none of the forms showed any significant differences between the sexes ( p > 0.05 ) .
the least common form in both sexes was the crosslink . also , a few nonspecific rugae were observed .
the females tend to have more wavy shapes compared to the males ( 31.24% to 27.67% ) , whereas the males tend to have more curved and straight forms .
when we assessed the direction of the palatal rugae in both sexes ( table 3 ) , the most common direction found in both sexes was forward , followed by backward and then perpendicular ones .
males tend to have more forward - directed rugae and fewer backward - directed rugae compared to females , but there are no statistically significant differences , except with forward - directed rugae ( p = 0.037 ) .
the bilateral differences in rugae lengths , shapes , and directions in both sexes were assessed and are summarized in tables 4 , 5 , and 6 .
there was no significant asymmetry in all rugae lengths in both sexes , though the primary rugae were more on the left side in both sexes and secondary rugae tend to be more prevalent on the right side .
the only significant differences in shapes between sides were found in the converging form in males ( p < 0.039 ) and circular one in females ( p < 0.013 ) .
males showed significant asymmetry in rugae directions for the three shapes , whereas in females , the forward- and backward - directed rugae are significantly asymmetrical .
lra was done for the combination of all shapes to assess the possibility of sex prediction .
a predictive value of 58% was obtained in assigning the sex correctly ( table 8) . when all continuous parameters ( dimension and orientation ) were used ( tables 9 and 10 ) , none showed any significance , but the perpendicular orientation was excluded in the developed equation .
however , sex prediction increased slightly to 60% with male prediction , better than with females ( 62% and 58% , resp . ) .
palatal rugae are a focus of interest due to their utilization in anatomy , anthropology , and genetics . in addition , they are important in biological profiling of individuals and can be an alternative method of identification when the application of conventional techniques , such as fingerprinting , dental recording , and dna analysis , is not possible or limited [ 21 , 22 ] .
moreover , palatine rugae have multiple uses in dental practice in terms of influencing prosthodontic treatment planes for edentulous patients , the diagnosis of submucosal clefts in the palate , and acting as reference points for measuring teeth migration during orthodontic therapy [ 1 , 23 , 24 ] .
various methods have been used to study palatal rugae patterns such as intraoral inspection , digital photography , stereoscopy , and stereophotogrammetry . in the present study ,
dental casts were used due to their simple analysis , easy manipulation , reliability , and the possibility of future comparative review , when needed .
the age of the studied participants was selected due to the lack of an agreement on rugae stability with aging .
while lysell indicated that the total number of rugae decreases after the age of 23 years , yamazaki found that the mean ruga count changes moderately in adolescence but increases markedly from the age of 35 to 40 years , as discussed by hauser et al . .
it has also been suggested that the characteristic pattern of palatal rugae remains stable from development up to seven days after death [ 11 , 25 ] . according to thomas and
kotze there is no universally acceptable classification and the importance of successful identification exceeds the importance of the adopted classification ; therefore , each examiner can use his own classification for comparative studies . in the current study , the method of rugae classification used [ 7 , 19 ]
was adopted as it is more practical than other methods , such as those followed by hauser et al . and dawasaz and dinkar .
it should be emphasized that no significant intraobserver errors were observed in this study ( p > 0.05 ) .
the dimensions of the palatal rugae , which are continuous , are affected by growth and tooth extraction , whereas the discrete variable , shape , remains stable . both were used and assessed by appropriate statistical methods .
lra can be used for continuous and discrete variables without the assumptions of a normal distribution , linearity , or equity of variance within each group .
earlier authors indicated that using of nonparametric discriminant procedure is more appropriate [ 13 , 27 ] than discriminant analyses determining ethnicity and sex .
the results of the present study showed that males have a larger number of rugae and more primary and secondary rugae than females . on the other hand , proportionally , the females showed more primary than secondary rugae compared to their male counterparts .
the mean of the total number of rugae was 13.30 in males and 12.42 in females , which is greater than what was reported in jordanians ( 8.8 in males and 8.5 in females ) .
in addition , the mean of the total number of primary rugae was found to be 10.10 in males and 9.72 in females , which is similar to australian aborigines ( 10 for males and 9.8 for females ) but higher than in rwandans ( 7.6 for males and 7.5 for females ) , saudis ( 7.3 for males and 7.2 for females ) , indians from maharashtra ( 7.30 for males and 7.25 for females ) , caucasian australians ( 8.6 each ) , indians ( 9.7 for males and 9.2 for females ) , and nepalese ( 8.9 for males and 8.5 for females ) .
however , some authors have indicated clearly that environmental factors play a minimal role in affecting the formation of rugae and that the main determinant factor in the formation is the genetic background [ 2 , 33 ] .
these highlighted differences can also be attributed to differences in palatal width ; it was indicated that differences in the mean number of rugae between populations are a reflection of greater ridge development , both qualitatively and quantitatively associated with the presence of broader palates and that males show more primary and secondary rugae than their female counterparts which can also be attributed to the fact that males have larger palates than females , allowing them to have more lengthy rugae .
the statistical analysis used in this study did not reveal any significant differences between sexes in terms of the mean total number of all rugae dimensions , and this is in line with findings among jordanians , aboriginals , and some indians [ 34 , 35 ] but contradicts the findings in western indians . in the present study
the most common rugae shapes are wavy and curved forms , accounting for more than 55% of rugae in both sexes . moreover ,
while the wavy shape was more common in females , the curved shape was slightly more common in males .
these findings are in accordance with the findings in egyptian and saudi children , saudi adults , and indians [ 32 , 33 , 38 ] , where wavy and curved rugae were the predominant forms , followed by the straight one .
furthermore , they are consistent with the findings in australian aborigines and caucasians populations , where wavy and curved rugae were found to form the majority . in contrast , in jordanians , the most prevalent shape was wavy , followed by diverging , while in rwandans and nepalese , the most prevalent shape was wavy , followed by straight and then curved ones . in our study , all the shapes were present .
in contrast to saudi children , who lack the crosslink shape , sudanese arabs have crosslink shapes similar to egyptians children . nonetheless , it was the least recorded shape in both sexes .
this variability in shapes between populations indicates the need for population - specific morphological studies to clarify the systemic trends among different populations .
also , the presence of some nonspecific rugae shapes may indicate the need for describing new rugae patterns in a larger sample .
the findings of the present study indicate that there is no sexual dimorphism in rugae shapes ( p > 0.05 ) , which concurs with the findings for jordanians and rwandans and egyptian and saudi children but contradicts the earlier findings in saudi adults , where converging and circular patterns showed sexual dimorphism .
this may suggest that either the degree of sexual differences in rugae patterns differs between populations or the differences expressed in some studies are individual - related variations .
the orientation of palatal rugae showed that the most prevalent direction is forward followed by backward and then perpendicular ones .
forward rugae are more common in males , and backward and perpendicular rugae are more common in females .
this finding is consistent with the results obtained among jordanians , but the reverse was reported among rwandans , where females have more forward - directed rugae .
our results showed that there is a significant sexual difference in forward - directed rugae consistent with jordanians . on assessing bilateral differences
, the present study showed that the left side tends to have more primary rugae in both sexes .
this observation is similar to the findings in australian aborigines , swedes , rwandans , mysoreans , and japanese .
the right side was found to have more secondary rugae than the left side in both sexes .
this observation gives credence to the assumption that there is an indirect relation between the primary and secondary rugae .
the findings of this study showed that there are no significant differences between the right and left sides in the means of the palatal rugae dimensions .
most of the shapes showed no significant bilateral differences , except for the converging shape in males ( p = 0.039 ) and circular shape in females ( p = 0.013 ) .
the orientations expressed the maximum degree of asymmetry in all directions in both sexes , except with the perpendicular direction in females .
forward - directed rugae were more on the left side in both sexes , whereas perpendicular and backward - directed rugae were more common on the right side in both sexes .
this asymmetry in directions contradicts the assumption that the development of rugae is a coordinated process that occurs throughout the palate , indicating that differential growth rates exist between sides among sexes .
when the ability of palatal rugae to assign sexes was assessed using logistic regression analysis , the results obtained indicated that the shapes of the rugae are a weak discriminator between sexes in sudanese arabs ( 58% for both sexes ) .
in contrast , the dimensions and directions yielded better prediction among males ( 62% ) , but female prediction did not improve .
previous studies among indians allocated sex with a 99.2% success rate using shapes in 120 subjects using logistic regression , a 73.08% success rate using shapes and dimensions in 100 coastal andhra indian population , and a 78% success rate using dimensions only .
when all continuous parameters were used , the assigning of sex among males improved slightly .
our findings revealed low success rate among sudanese arabs compared to the two previous mentioned studies ; this may indicate ethnic variability in the expression of sexual dimorphism using palatal shapes as most of the previous studies among indians indicated a lack of sexual dimorphism in palatal rugae , for example , that of puducherry .
previous studies among sudanese arabs using limbs [ 40 , 41 ] and crania found moderate to high degrees of sexual dimorphism .
the findings of the current study including palatal rugae dimensions , shapes , and directions did not show this discriminatory ability , which indicates that the palatal rugae can not be used as an effective tool in assigning of sex .
lastly , in the present study we used oral casts to assess morphological patterns which are two - dimensional ; however , it is worthwhile assessing palatal rugae among sudanese using other techniques , for example , stereoscopy and stereophotogrammetry to explore both rugae in three dimensions and the position of each ruga .
the evaluation of casts was conducted by a single investigator as complex shapes may generate interobserver variation in identification .
nevertheless , in spite of these shortcomings , this study has explored the utility of rugae as a biological profiling tool among the studied populations .
it has also established a baseline data for any larger - scale study among the sudanese that can be used for future comparative purposes among sudanese or other populations with genetic mixtures between arabs and africans .
the palatal rugae dimensions , shapes , and directions were analyzed in a sudanese population .
the most predominant rugae were primary , and the most prevalent shapes found were the wavy , curved , and straight forms .
there is a lack of any sexual dimorphism in the rugae dimensions and shapes , and the existence of sexual dimorphism was confirmed only in forward - directed rugae .
the findings obtained showed that the use of palatal rugae to assign sex among sudanese arabs is not recommended .
the possible differences in palatal rugae dimensions , shapes , and directions in different regions and even among different ethnicities with the same population necessitate further studies involving larger samples . | palatal rugae patterns have unique characteristics and have been proposed as an alternative method to establish identity when other means , such as fingerprints and dental records , are not attainable .
this study was conducted to determine the morphological characteristics of palatine rugae and to assess the existence of side asymmetry in them in sudanese arabs .
it also assesses the possibility of determining sex using logistic regression .
one hundred dental casts for 50 males and 50 females aged between 18 and 23 were studied for palatal rugae dimensions , shapes , and orientations , as well as sexual dimorphism and side symmetry .
the most predominant rugae were primary , and the most prevalent shapes in both sexes were wavy , curved , and straight forms .
the predominant orientation was forward .
side asymmetry existed more in the orientations than in the shapes , but no side asymmetry was recorded in the dimensions .
there was no significant sexual dimorphism in the rugae dimensions , shapes , and orientations , except for forward - directed rugae ( p < 0.037 ) . a predictive value of 60% was obtained in assigning sex using dimensions and orientations and of 58% using shapes alone . therefore , the palatal rugae are not recommended for assigning sex effectively among sudanese arabs unless it is the only means available . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
considering their poor prognosis and resistance to chemotherapy and radiotherapy , brain tumours in general , and high - grade astrogliomas in particular , are among the most devastating types of human cancer . at the cell population level ,
brain malignancies are typically heterogeneous lesions composed of cells with diverse morphologies that express a variety of neural lineage markers .
prognosis and response to treatment differ significantly not only among histologically different brain tumours , but also among those that appear similar in terms of morphology and patterns of phenotypic markers .
this notorious heterogeneity of brain tumours likely facilitates development of treatment - resistant clones , a process that is further fuelled by the ability of astroglioma cells to shift between quiescent and proliferating states .
given such unusual plasticity and the frightening rate of treatment failure , it is now commonly believed that treatment outcome and hence patients ' survival can only improve if the biology of the brain cancer cell populations is better understood .
classification of adult brain tumours is based on the world health organization ( who ) classification of nervous system tumours .
the who grading of astrocytomas establishes a malignancy scale based on histologic features of the tumour .
the histological grades are as follows : who grade i includes lesions with low proliferative potential , a frequently discrete nature , and the possibility of cure following surgical resection alone . who grade ii lesions are generally infiltrating and display low mitotic activity but recur .
some lower - grade lesions tend to progress to higher grades of malignancy . who grade iii includes lesions with histological evidence of malignancy , generally in the form of mitotic activity , clearly expressed infiltrative capabilities , and anaplasia . who grade iv lesions are mitotically active , necrosis - prone and commonly associated with a rapid pre - operative and post - operative evolution of disease
the most malignant types of astrogliomas are anaplastic astrocytoma ( grade iii astrocytoma ) and glioblastoma multiforme ( gbm , grade iv astrocytoma ) . in this article
, we commonly use the general term astrogliomas for simplicity , yet our discussion mainly concerns the grade iii and particularly grade iv tumours .
one of the key features of astrogliomas , affecting their biological and clinical behaviour , is dense vascularization . since the introduction of the concept of an
angiogenic switch as a pivotal component of tumour growth and metastasis by folkman et al .
, multiple therapies targeting the molecular regulators of this mechanism have been tested with variable clinical efficacy . despite such mixed initial results , anti - angiogenic therapy is regarded as a promising treatment strategy , and various pre - clinical experimental approaches targeting neovascularization turned out to be effective in vivo . for example
, one study showed that systemic therapy with a mono- clonal antibody against vascular endothelial growth factor receptor 2 ( vegfr2 ) inhibited tumour growth in mice by some 80% .
the ligand that operates through vegfrs to promote tumour angiogenesis is vegf , a versatile regulatory cytokine that is aberrantly up - regulated in a wide range of tumour types and acts as a potent modulator of tumour growth and metastasis in numerous pre - clinical tumour models . particularly relevant to this review ,
several pieces of evidence indicate prognostic significance of vegf in high - grade astrogliomas [ 68 ] . in this article
, we highlight the emerging role(s ) of vegf signalling in astroglioma biology , including the possible links with dna damage checkpoints in response to radiation treatment .
furthermore , we postulate and discuss a hypothetical function of autocrine vegf signalling in regulation of the so - called brain tumour - derived cancer stem - like cells ( btcsc ) and their intriguing resistance to therapy .
unlike the early - stage embryonic brain harbouring large numbers of neural stem and progenitor cells , the adult brain is mainly composed of highly differentiated and specialized cell types , including neurons , glia ( oligodendrocytes , astrocytes , microglia , ependymal cells ) , vascular endothelium and meningeal cells . over the past century
, the brain has traditionally been viewed as static with respect to its very limited turnover and regenerative capacity .
however , it has recently become clear that neurogenesis continues into adult life in restricted germinal zones [ 1013 ] .
a small percentage of quiescent cells present in the adult hippocampal dentate gyrus ( sub - granular zone , sgz ) , sub - ventricular zone of the lateral ventricles ( svz ) and olfactory bulbs are undifferentiated and multipotent neural stem cells ( nsc ) capable of self - renewal [ 1415 ] .
these stem cells divide to generate rapidly cycling transitamplifying cells with a limited proliferation and differentiation potential .
the transitamplifying cells give rise to restricted progenitor cells undergoing terminal differentiation . in the 1970s , an idea of specific anatomical locations termed
niches. in the adult mammalian brain , we are just beginning to identify the cellular and molecular features that characterize the neurogenic
niches in the svz and sgz , and the mechanisms by which the full range of adult nsc development is regulated .
nscs are grown in vitro either as neurospheres in suspension or as an adherent monolayer .
some of the cells within the neurospheres proliferate as multipotent , self - renewing nscs upon stimulation with either epidermal growth factor ( egf ) or basic fibroblast growth factor ( bfgf ) .
the nscs residing in the adult human brain are attractive candidates for isolation , in vitro expansion and autologous transplantation to replace neurons lost to neurodegenerative diseases , stroke and traumatic brain injury .
recently , a small population of cancer stem - like cells ( cscs ) has been identified in adult and paediatric brain tumours , as well as in established cell lines [ 2024 ] .
these cells express a number of nsc markers including cd133 , a cell surface protein which is extensively exploited for enrichment of stem - like cells using fluorescence activated cell sorting ( facs ) and/or magnetic bead separation , thereby providing cells to be examined in diverse applications [ 2526 ] . the human cd133 antigen , also known as ac133 , was originally identified as a marker of haematopoietic stem cells , however its function has remained unclear .
a characteristic feature of the cd133 protein is its rapid down - regulation during cell differentiation [ 2829 ] , which makes it a cell surface marker uniquely suited for the identification and isolation of stem - like and progenitor cells . besides expressing the cd133 antigen
, cscs express mrnas for several additional recognized nsc markers , including bmi-1 , sox2 and musashi-1 .
inspired by identification of cancer stem cells from leukaemia and breast cancer [ 2931 ] , btcscs were reportedly successfully cultured according to the following criteria : ( i ) expression of nsc markers cd133 and nestin ; ( ii ) generation of spheres morphologically indistinguishable from neurospheres ; ( iii ) self - renewal and proliferation and ( iv ) production of differentiated progeny in vitro or recapitulation of the parental tumour mass growth when implanted into immunodeficient animals [ 2022 , 24 , 3233 ] . as few as 100 cd133-positive human cells were sufficient to form brain tumours as xenografts in nod - scid mice .
importantly , the cd133-positive btcscs also exhibit high resistance to current chemotherapy and radiotherapy , in contrast to their cd133-negative counterparts [ 3334 ] , an intriguing difference discussed in more detail later in this article . among the unresolved questions of btcscs '
biology is also whether , or to what extent , do the cd133-positive btcscs require interaction with the cd133-negative bulk sub - population of cells .
another largely open issue concerns the emerging role of the local microenvironment in supporting the maintenance of these candidate cscs within tumour mass . as discussed below , identification of cd133-positive btcscs and attempts to understand their biology
provide powerful new tools and approaches to better understand tumourigenesis in the cns , a research area likely to become crucial in developing novel therapies based on btcscs as a target .
angiogenesis is a highly regulated process essential not only in early embryogenesis but also during tissue growth and repair , female reproductive cycle and diverse pathologies , such as inflammation or tumour development and progression [ 3536 ] .
localized breakdown of extracellular matrix ( ecm ) precedes proliferation , migration and tissue infiltration of endothelial cells . in time
these cells re - model back into capillary structures , and a new ecm is deposited .
vegf expression strongly correlates with tumour aggressiveness , metastatic potential , a short time to relapse and , consequently , it commonly indicates poor prognosis in patients with cancer [ 6 , 3843 ] .
recent work identified the btcscs to be a key source of angiogenic factors in brain cancer , suggesting that anti - angiogenic therapy targeting these stem - like tumour cell sub - populations might improve the therapy outcome .
understanding angiogenesis and its relation to tumour growth and resistance to therapy is of considerable interest , particularly because diffusely infiltrating astrogliomas are mostly refractory to current surgical and adjuvant treatments .
arrows indicate very week diffuse vegf staining in the cytoplasm of astroglial elements of a low grade astroglioma who grade ii ( a ) , in contrast to strong cytoplasmic positivity in astroglial elements in a high - grade astroglioma specimen who grade iv ( b ) .
both tumour grades exhibit certain level of cytoplasmic positivity in endothelial elements ( arrowheads ) ( original magnification 200x ) .
neovascularization in brain tumours correlates markedly with their enhanced aggressiveness , degree of malignancy and poor clinical prognosis and inversely with the post - operative survival time of patients [ 4951 ] .
newly formed tumour blood vessels possess an ineffective blood brain barrier that contributes to the pathogenesis of tumour - associated oedema .
the characteristic vascularity of astrogliomas has lead to a hypothesis that the formation of new blood vessels is crucial to tumour growth .
astroglioma cells have angiogenic activities , in that they promote capillary morphogenesis and endothelial proliferation in vitro .
as mentioned above , the pathologic features that distinguish gbm from lower grade astrogliomas are the presence of necrosis with pseudopalisades and a distinct form of angiogenesis , microvascular hyper - plasia [ 5456 ] .
these palisades are in part caused by vessel regression and increased tumour cell proliferation [ 56 , 58 ] .
analysis of the shapes and sizes of pseudopalisades suggests that these structures evolve and enlarge overtime , giving rise to a gradually expanding coagulative necrosis .
such features inspired formulation of a vaso - occlusive and prothrombotic model of pseudopalisade formation in gbm .
this emerging concept postulates hypoxic tumour cells migrating away from the central hypoxia that arises after a vascular insult .
hypoxia resulting from such conditions is then thought to induce new blood vessels that supply the tumour with necessary metabolites .
vegf is highly expressed in pseudopalisading tumour cells adjacent to necrotic zones and hyperplastic vessels in astroglioma .
this phenomenon reflects an elevated transcriptional activity of hypoxia - inducible factors 1 and 2 ( hif-1 and -2 ) [ 6264 ] .
secretion of vegf , in turn , causes endothelial cell proliferation and angiogenesis followed by microvascular hyperplasia and formation of glomeruloid bodies in gbm and other tumours [ 6566 ] .
overexpression of vegf by tumour cells frequently occurs not only in response to hypoxia [ 62 , 67 ] , but also upon loss of function of certain tumour suppressor genes [ 6869 ] and oncogene activation . under hypoxic conditions , transcriptional regulation of vegf
is dominated by hif-1 that , together with its target genes , plays a key role in astroglioma - induced angiogenesis [ 7172 ] .
chronic oncogenic stimuli , such as activated ras and pi3k pathways appear to enhance hif-1 expression and likely contribute to gbm progression [ 60 , 73 ] .
the protooncogene ets-1 and the transcription factor stat3 are also capable of vegf induction and/or activation .
ets-1 activates genes for vegf receptors , matrix metalloprotease proteins ( mmps ) and urokinase plasminogen activator ( upa ) , all features that can promote angiogenesis and tumour progression .
in addition , several cytokines and growth factors involved in astroglioma pathogenesis , including tgf- , egf , pdgf - b , bfgf , also up - regulate vegf .
genetic alterations common in astrogliomas , such as mutational activation of the epidermal growth factor receptor ( egfr ) and loss - of - function mutations of the pten tumour suppressor , lead to enhanced vegf expression and increased angiogenic activity [ 7576 ] .
astrogliomas frequently overexpress egfr , and its truncated mutant isoform egfrviii has been implicated in relapse and poor prognosis .
enhanced egfr signalling can up - regulate vegf production in brain cancer , while blockage of egfr inhibits secretion of vegf and other angiogenic factors .
overall , this accumulating evidence supports the notion that vegf plays a central role in the molecular pathogenesis of astrogliomas and strongly affects the biological behaviour of these tumours .
the importance of microvasculature and proper local microenvironment ( niche ) including a plethora of regulatory cytokines and growth factors for the maintenance of stemness of the normal nscs is known .
related critical issues for astrogliomas have been whether the btcscs also depend on their niche and , if yes , whether the mutual interdependencies of the niche microenvironment and the stem cells may differ between normal nscs and btcscs .
if such differences exist , the specific features of the btcsc - niche interplay might offer potential targets for novel therapeutic interventions .
there is little doubt that the nature of the niche microenvironment may represent an important factor in the behaviour of normal nscs and cscs in terms of their stem cell self - renewal and cell - fate decisions .
the microenvironment composed of the ecm , stem and progenitor cells , as well as mature , differentiated cells secreting a range of growth factors seems to be important in such processes
. balanced microenvironmental levels of various mitogens , including bfgf , egf and sonic hedgehog ( ssh ) , support the propagation of adult nsc in culture and appear to perform similar functions in vivo[8083 ] .
histological and ex vivo cell culture studies of mouse tissues suggest that nsc lie within a vascular niche in which endothelial cells regulate stem cell self - renewal [ 8487 ] .
growth factors , such as vegf , bfgf , pdgf and egf represent the mitogenic and trophic factors that regulate neurogenesis , while exerting direct neurotrophic and neuroprotective activities .
bfgf together with egf are crucial growth factors necessary for nsc , as well as for csc proliferation and maintenance of their self - renewal properties in vitro[22 , 8990 ] .
for example , vegf stimulates neurogenesis both in mouse brain cultures in vitro and in neuroproliferative regions ( sub - granular zone and sub - ventricular zone ) of the non - ischaemic mouse brain in vivo .
naturally coupled to astrocytes through astrocytic end - feet , endothelial cells are also important components of the niche structure and closely co - operate with astrocytes to regulate adult neurogenesis [ 8485 ] .
btcscs also interact with endothelial cells that secrete factors supporting the maintenance of these cells in stem - like cell state .
while current evidence supports the idea of vegf as a regulator of neuronal cell fate and a key ingredient of such stem - cell niche in both , normal brain and astroglioma scenarios , one possibly important difference may reflect the plausible role of the vegf as an autocrine factor in astrogliomas , as opposed to only paracrine role under the normal niche conditions .
tumours known to overexpress vegf have strong angiogenic potential which , in turn , could explain the documented importance of vegf in astroglioma progression . since vegf and
both vegfrs are co - expressed in tumour cells in the majority of nascent primary gbm lesions ( fig .
2 ) , an autocrine role of the vegf in gbm might significantly contribute to tumour growth and invasivity [ 9395 ] .
on the other hand , in contrast to vegfs well - established paracrine effects on endothelial cells , less is known about such potential autocrine role of vegf in glioblastoma . while some authors reported that exogenous vegf not only stimulates endothelial cells , but also directly enhances astroglioma cell motility , invasion and pro - liferation ( fig .
known receptor types are indicated and identified ligands / ligand isoforms are listed directly in the figure above .
cell membrane ( gc membrane ) receptors of the vegf tyrosine kinase receptor family consist of seven ig - like domains , transmembrane region and an intracellular tyrosine kinase binding domain interrupted by a kinase - insert sequence .
the aberrant sflt-1 receptor form lacking one ig - like domain as a result of vegfr1 truncation is also shown .
the neuropilin receptor acts as a co - receptor for vegfr2 , enhancing binding and biological activity of vegf165 .
ligand binding through electrostatic interactions with specific sequences of sulphation within the hs chains leads to receptor dimerization and tyrosine domain phosphorylation , thereby activating the corresponding signalling pathway .
( a ) vegf secreted by tumour endothelial cells ( orange ) induces angiogenesis in an autocrine manner , while vegf secreted by astroglial tumour cells ( pink ) stimulates tumour angiogenesis in a paracrine manner . as reports on other biological effects of the autocrine vegf function in astroglial tumour cells
( b ) hypothetical distinct effects of vegf on the cd133-positive ( vegf secreting and radioresistant ) versus cd133-negative ( radiosensitive ) astroglial cell population .
ir - induced vegf secretion by the cd133-positive astroglial cells ( cancer stem - like cells ) could enhance tumour angiogenesis and protection of endothelial cells against apoptosis .
furthermore , ir - induced vegf could modulate proliferation of the cd133-positive cells , thereby regulating their sensitivity to ir .
ir - induced vegf , massively secreted by the cd133-positive astroglial cell sub - population , might also help to protect these cells from apoptosis and increase their migration in an autocrine manner . despite our effort to understand the stem cell niche is in its infancy , particularly with regard to the btcsc niche and its potential aberrant features , recent studies in this area are encouraging and support a crucial role of a perivascular niche for brain tumour stem - like cells [ 92 , 98 ] .
the emerging significance of this concept for development of new astroglioma treatment strategies is discussed in the ultimate section of this article .
given the severity of the health problem posed by astrogliomas to the society , embarrassingly little is known about the biological basis and molecular mechanisms that allow brain tumours to survive treatment and recur . apart from surgery and some newly tested targeted therapies , the major modalities presently used in the clinic to treat astrogliomas are dna damaging treatments by ionizing radiation ( ir ) and chemotherapy . in this section ,
we briefly highlight how the cancer stem cell concept and recent advances in understanding the cellular machinery that responds to dna damage are beginning to converge to shed more light on the notoriously difficult issue of the treatment resistance .
although chemotherapeutics , particularly dna alkylating drugs , such as temozolomide are currently widely used in combination with traditional radiotherapy to treat astrogliomas , the molecular basis of sensitivity versus resistance to such drugs is relatively well understood [ 99100 ] , and it will not be further discussed here . rather , we focus our discussion on ir and cellular responses to the most toxic ir - induced lesions , the dna double strand breaks ( dsbs ) . in response to dsb - causing insults such as ir ,
human cells activate their dna damage response machinery , a sophisticated network of signalling and effector pathways that co - ordinate cell cycle check - points with dna repair and cell death mechanisms [ 101102 ] .
relevant for our discussion on cancer cells , dsbs can be generated not only through external genotoxic insults , but also from events within the cell itself , for example , due to metabolic reactive oxygen species , or errors during dna replication .
the latter insult , often referred to as replication stress , is commonly caused by various activated oncogenes and loss of some tumour suppressors , leading to constitutively activated dna damage checkpoint signalling in tumours [ 102104 ] .
such constitutive activation of the dna damage checkpoints is particularly apparent in borderline pre - malignant and early malignant lesions , resulting in enhanced apoptosis or induction of senescence as an inducible biological barrier against tumour progression [ 102107 ] . despite the existence of such physiological barrier response
has been demonstrated for multiple types of human solid tumours , particularly carcinomas and melanomas , it is largely absent in testicular germ cell tumours , and its relevance for astrogliomas remains to be explored .
what is important for our discussion about sensitivity versus resistance to dna - damaging therapies is the fact that during their progression , many malignant tumours , at least partially , disable the activated checkpoint barrier through mutations or epigenetic changes in relevant genes , such as p53 [ 102 , 109 ] . alternatively
, malignant cells might progress in the face of constitutive dna damage by enhancing the efficiency of their dna repair pathways , in either case altering the overall sensitivity towards potential subsequent therapy by dna damaging modalities , such as ir . at the molecular level , the key element of the cellular dsb response is activation of the apical signalling kinase atm ( and also the atr and dna - pk kinases ) , which rapidly phosphorylate a wide range of substrates including the effector kinases chk2 and chk1 . chk2 and chk1
become activated upon their phosphorylation by atm and atr , respectively , and further propagate the dna damage alert signal to diverse effectors such as the tumour suppressor p53 , or other checkpoint and dna repair proteins .
the severity of the dna damage , the effectiveness of the activated cell cycle checkpoint and dna repair mechanisms , as well as other parameters including the protective signals from the microenvironment , then jointly affect the final outcome and cell fate of the irradiated cell . in cancer cells
, such a cell - fate decision may be widely variable due to the heterogeneity of tumour cell populations , and it may also reflect alterations in the dna damage machinery acquired during cancer progression ( see above ) .
recent work on human glioblastomas now indicates that the cd133-positive stem - like cells may have an enhanced checkpoint response to radiation , and this may contribute to their selective survival and radioresistance .
after irradiation , the cd133- positive fraction of human gbm cultures and xenografts was enriched up to fivefold compared with cd133-negative cells .
this was not attributable to induction of cd133 expression in cd133-negative tumour cells , but to lower rates of apoptosis among the cd133-positive subset .
analysis of the checkpoint machinery pointed to an augmented activation of the chk2 and chk1 kinases , faster repair of the ir - induced dsbs , and overall better survival and ability to recur among the cd133-enriched btcscs .
furthermore , the authors used a chemical inhibitor to block the activities of the checkpoint kinases chk1 and chk2 after ir , and this treatment radiosensitized the cd133-positive subset more than the cd133- negative astroglioma cells from the same tumour .
collectively , these intriguing results identify a possible mechanism that contributes to radioresistance of btcscs and thereby of the tumour , and suggest that targeting the dna damage checkpoints may be worth considering as an option in the gbm radiotherapy scenario . whether this mechanism may be in any way linked with the effects of vegf or other cytokines in the btcsc niche , and how to exploit these new insights into therapy resistance for
in response to ir , vegf secretion by gbm cell lines was highly increased in a radiation dose - dependent manner [ 94 , 111 ] . among all the cell lines tested ,
the u87 mg is highly radioresistant and expresses the highest ir - induced vegf levels , a correlation that inspired a hypothesis about a potential involvement of ir - enhanced vegf secretion in radioresistance .
based on observations of decreased gbm cell proliferation in response to exogenous vegf , it was proposed that expression of vegf would at the same time induce growth of new blood vessels ensuring a better supply of oxygen and nutrients and reduce gbm cell proliferation resulting in decreased sensitivity to ir .
since proliferating cells are more sensitive to irradiation than quiescent cells and since vegf protects tumour blood vessels from irradiation- mediated toxicity , this suggests a possible mechanism through which gbm cells can escape the consequences of radiation treatment .
after irradiation , the vegf gene promoter becomes stimulated via multiple mitogenactivated protein kinase ( mapk ) dependent pathways in both cultured normal human astrocytes and gbm cell lines .
since hif-1 was not overexpressed under such conditions , hypoxia does n't seem to be involved in this mechanism . given the radioresistance of btcscs discussed in the previous section ,
could these data jointly indicate a connection between radiation - induced vegf secretion , increased angiogenesis and selective survival of the cd133- positive btcscs ? could ir - induced vegf secretion by btcscs selectively regulate their own migration and/or proliferation in an autocrine manner while protecting them from ir - induced apoptosis ( fig .
3b ) , possibly through some so - far unidentified link with dna damage checkpoint signalling or repair ? despite speculative at present , this idea seems indirectly supported by recent analysis of the so - called bystander effects of radiation .
this study showed that conditioned media from irradiated human glioblastoma cells contained factors including cytokines , whose membrane - mediated signalling to non - irradiated cells resulted in activation of the checkpoint kinase atr and cellular dna damage response without direct exposure of such bystander cells to radiation .
these results imply that secreted cytokines may be capable of inducing dna damage checkpoints , and that such bystander response differs in gbm cells compared with normal astrocytes , suggesting that these effects might be exploited through therapeutic targeting .
the arguments discussed so far point to critical roles of vegf and btcsc niche , as well as the status of the dna damage response machinery , both of which show unique features in the treatment - resistant astroglioma stem - like cells .
these accumulating results identify both vegf - mediated and dna damage signalling cascades as promising targets for treatment of astrogliomas , a notion that is supported also by recent successful attempts to experimentally target either vegf signalling alone [ 4 , 115 ] , or combine such vascular niche - targeting treatment with dna - damaging chemotherapy ( see below ) .
formation of multiple vascular btcsc niches , each possibly capable of giving rise to a recurrent tumour , may strongly facilitate tumour growth and invasion [ 116118 ] .
if btcsc are true tumour - initiating cells , then drugs selectively killing these cells could prove highly effective treatments for astrogliomas .
encouraging are recent data providing proof of principle that selective targeting of cscs is possible , at least in some types of malignancies [ 34 , 44 , 119 ] .
there is no doubt that additional factors of astroglioma biology may hinder attempts to successfully introduce combined vegf- and dna damage checkpoint - targeting treatment strategies .
for example , infiltration of tumour cells into surrounding brain contributes to the treatment - refractory nature of malignant astrogliomas .
moreover , the blood brain barrier represents a significant obstacle , preventing the delivery of large - molecular - weight polar compounds to brain tumour cells .
in addition , brain tissue highly sensitive to cytotoxic treatments , and our own data show that inhibition of checkpoint kinases such as chk1 causes endogenous dna damage in proliferating human cells , thereby raising concerns as to the suitability of this approach for therapy . with the exception of the modest activity associated with temozolomid
, there is no standard chemotherapy available for patients with high - grade astrogliomas , and resistance to chemotherapy is common . on the optimistic side bevacizumab ( avastin ) ,
a recombinant , humanized monoclonal antibody targeting vegf , has been recently approved for use in colorectal carcinoma - based on significant survival benefit observed following its addition to fluorouracil - based chemotherapy .
preliminary results from single - arm phase ii study of bevacizumab with irinotecan ( cpt-11 ) , currently underway at the preston robert tisch brain tumor center at the duke university medical center for patients with recurrent malignant astroglioma , indicate that the most effective therapy of gbm identified to date could be a combined treatment by bevacizumab with a dna - damaging drug such as cpt-11 .
thus , despite possible numerous obstacles that must not be underestimated , we believe that the available evidence justifies attempts to identify a clinically feasible , effective combination of therapeutic approaches that would allow complementary targeting of the vegf / niche , and the dna damage check - point aspects of malignant astrogliomas , with special emphasis on targeting the unique features of the candidate astroglioma stem cells ( fig .
4 ) . in general terms , such a combined strategy may include available or future drugs or antibodies to inhibit vegf and/or its receptor - mediated signalling , along with standard dna damaging treatment modalities , such as ir and alkylating drugs , complemented by selective inhibitors of checkpoint signalling or dna repair to counteract the mechanisms underlying astroglioma resistance to such treatments . in any case , given the close relationship between tumour microenvironment , vascular architecture and tumour response to therapy , it seems logical to investigate the potential role of vegf not only as an angiogenic factor stimulating formation of pathological vascular niche , but also as a potential regulator of btcsc cellfate . perhaps in concert with acquired defects within the apoptotic machinery and/or selective regulation of dna damage signalling / repair , vegf and
vascular niche might help btcscs escape from the toxic effects of chemotherapy and radiotherapy by protecting them from dna damage - induced apoptosis [ 126129 ] .
better mechanistic understanding of these biological processes appears to be more plausible based on the recent advances in the field , and such research will hopefully help to improve the presently dismal prognosis of astroglioma patients .
potential therapeutic approaches targeting pathological vascular niche and dna damage checkpoint as unique features of btcscs .
accumulating evidence suggests that the cd133-positive , candidate btcscs , harbour specific features that could be exploited in future combinatorial therapies of malignant astrogliomas .
such potential treatment combination might include classical dna - damaging modalities : ionizing radiation ( ir ) , chemotherapy ( ct ) or both ( therapy 1 ) , a concomitant blockade of dna damage checkpoint signalling and/or dna repair ( therapy 2 ) , and inhibition of the cytokine - mediated paracrine / autocrine circuitry ( asterisk ) such as the vegf effects ( therapy 3 ) . | abstractmalignant astrogliomas are among the most aggressive , highly vascular and infiltrating tumours bearing a dismal prognosis , mainly due to their resistance to current radiation treatment and chemotherapy .
efforts to identify and target the mechanisms that underlie astroglioma resistance have recently focused on candidate cancer stem cells , their biological properties , interplay with their local microenvironment or niche and their role in tumour progression and recurrence .
both paracrine and autocrine regulation of astroglioma cell behaviour by locally produced cytokines such as the vascular endothelial growth factor ( vegf ) are emerging as key factors that determine astroglioma cell fate . here , we review these recent rapid advances in astroglioma research , with emphasis on the significance of vegf in astroglioma stem - like cell biology .
furthermore , we highlight the unique dna damage checkpoint properties of the cd133-marker - positive astroglioma stem - like cells , discuss their potential involvement in astroglioma radioresistance and consider the implications of this new knowledge for designing combinatorial , more efficient therapeutic strategies . | Introduction
Adult brain a dynamic structure with active stem cells?
Cancer stem-like cells in brain tumours
Angiogenesis in astrogliomas
VEGF and the stem cell niche
Resistance to DNA-damaging therapy in astrogliomas
VEGF and DNA damage response: implications for astroglioma therapy |
dry socket is a common complication following a tooth extraction , with a peak incidence in the 4045 year - old age group .
it has an incidence of 1%4% for all routine dental extractions and is more frequent in female patients [ 1 , 2 ] .
it is a self - limiting disease that often will take 510 days to disappear , even without treatment .
the treatment of this disease has commonly been divided into two groups : the nondressing and dressing interventions .
the use of dressing interventions is controversial because no scientific studies have been carried out that specifically to investigate the incidence of potential side effects and tissue damage arising from the placement of them .
these dressings according to their active principle can be classified into antimicrobial dressings , soothing dressings , or dressings with local anesthetics .
one of the most common dressings reported in the literature is zinc oxide and eugenol , often mixed into a semisolid consistency [ 1 , 5 ] .
local complications have been described after the placement of intra - alveolar dressings ; some of them are neuritis , foreign body reactions [ 7 , 8 ] , and myospherulosis [ 9 , 10 ] .
this paper presents a case of a late complication related to a dry socket dressing that mimicked a trigeminal neuralgia during 3 years and caused a chronic osteomyelitis with foreign body reaction .
a 45-year - old female was referred to the oral and maxillofacial department of zacamil 's national hospital ; the chief complaint was a right trigeminal neuralgia that could not be managed by conservative treatment .
four days after the extraction her dentist diagnosed a dry socket , which was treated with an intra - alveolar dressing consisting of zinc - oxide eugenol paste ; this medication was placed directly into the alveolus without any other transport vehicle .
the patient experienced relief of pain and never went back with her dentist so paste remained inside the alveolus ( this information was obtained directly from the medical files of the dentist that treated the patient ) .
she visited different dentists to find the cure to her pain , and during a period of two years third molar , second molar , first premolar , and second premolar of the right maxillary side were extracted .
hemifacial pain persisted , and the patient was referred to the neurologist who confused by the symptoms treated the patient as a trigeminal neuralgia .
carbamazepine was prescribed for about a year without pain relief ; after this the neurologist sent the patient to our department .
the chief complaint was an intermittent right hemifacial pain , which was described as an ache with periods of intense shooting pain .
a visual analog scale was used to measure it during the intermittent periods finding a severe pain .
physical examination revealed no trigger zones and clinical absence of teeth 1 , 2 , 3 , 4 , and 5 .
a right maxillary foreign body was found in the position of a nonhealed alveolar bone of the maxillary first molar ; the image was in close proximity to maxillary sinus ( figures 1 and 2 ) . due to the signs and symptoms of the patient ,
the foreign body was removed and curettage of the affected area was done ; the findings during surgery were : a nonhealed alveolus , granulation tissue , free bone fragments , and a white solid foreign body that was in direct contact with maxillary sinus ( figure 3 ) .
all tissues were sent to the pathologist who reported a well - vascularized fibrous connective tissue , chronic inflammatory infiltrate , multinucleated giant cells and necrotic bone surrounded by bacterial forms .
the rest of the postoperative care was managed in a conventional manner without any further complications .
postoperative x - rays showed adequate healing and complete removal of the foreign body ( figures 4 and 5 ) .
visual analog scale was used during several months revealing no pain after foreign body was removed .
patient has been followed up for six months without any facial pain during this time .
the number of secondary complications to the placement of dressings in the treatment of an established dry socket is ignored ; most of the complications previously reported , myospherulosis , neuritis , and foreign body reaction , are related to intra - alveolar medication as a preventive methods and not as a treatment . bright et al .
described myospherulosis related to tetracycline in a petrolatum base , used as a preventive measure to avoid dry socket .
now is known that petroleum - based carriers interfere with wound healing by action of lipids on extravasated erythrocytes , producing myospherulosis . because of this , nowadays the usage of petroleum - based carriers has been discouraged .
zuniga and leist in 1995 reported a topical tetracycline induced neuritis six months after routine removal of an unerupted mandibular third molar .
moore and brekke in 1990 reported a foreign - body giant cell reaction related to placement of tetracycline - treated polylactic acid .
mainous in 1974 reported foreign body reaction after zinc oxide - eugenol packing in localized osteitis . bloomer in 2000 did an investigation of the prevention of alveolar osteitis by immediate placement of medicated packing but they used the medication for one week only and then they removed it , so they did not report complications in a long - term evaluation .
oil of cloves is eugenol in its unrefined form , and it has been used for centuries as a toothache remedy .
chisholm described its mixture with zinc oxide in 1873 to form a plastic mass for therapeutic uses .
this mixture of eugenol with zinc oxide relies on a setting reaction between them which produces zinc eugenolato .
eugenolato is not stable in the presence of water , and readily undergoes hydrolysis with the release of free eugenol .
the type and extent of oral tissues reactions to eugenol vary but eugenol is generally cytotoxic at high concentrations and has an adverse effect on fibroblasts and osteoblast - like cells .
this effect is dose related and will potentially affect all patients [ 5 , 12 ] .
kozam noted that eugenol at certain concentrations can extinguish impulse transmission of a nerve within 3 hours . also transient paresthesias have been reported after the use of eugenol as an endodontic medication [ 13 , 14 ] .
other treatment using a packing has been reported for the effective relief of alveolar osteitis pain which includes using iodoform gauze ( nu gauze , johnson & johnson wound management ) coated with a mixture of three to five drops of obtundant , eugenol , with or without other ingredients , and packed into the anesthetized socket .
this treatment should not be used if the patient is allergic to iodine . in our case ,
the intra - alveolar zinc - oxide eugenol medication , caused bone necrosis , foreign body reaction , delayed alveolar healing , and hemifacial pain that was confused with a trigeminal neuralgia .
symptoms of the patient confused dentists and neurologist , misleading them to the wrong diagnosis of a trigeminal neuralgia .
this case reveals the need to do more long - term scientific investigations about the usage of intra - alveolar dressings as treatment for dry socket and not as prevention of it , in order to determine the safety of them and their potential side effects to our patients in long - term studies . it also reveals the importance of a thorough clinical and radiographic evaluation in patients with a suspected diagnosis of trigeminal neuralgia to discard local jaws affections that could confuse or mislead to a wrong diagnosis and treatment . finally , providing patients with written postoperative instructions stating what was placed in the socket , how long it should stay in the socket , and when or if it should be removed , should not be overlooked by treating physician . | dry socket is often treated in dentistry with intra - alveolar dressings ; the use of them remains controversial and has been related to some side effects such as neuritis , foreign body reactions , and myospherulosis .
we present a case of an intra - alveolar dressing ( zinc - oxide eugenol paste ) that mimicked a trigeminal neuralgia for 3 years and caused a right maxillary chronic osteomyelitis and foreign body reaction in a zone corresponding to the alveolus of the maxillary first molar .
this long - term complication was successfully managed by complete removal of the foreign body and curettage of the affected area . | 1. Introduction
2. Case Report
3. Discussion |
percutaneous coronary intervention ( pci ) has become the first - line treatment for patients suffering from obstructive coronary artery disease ( cad ) .
drug - eluting stents ( des ) have significantly reduced the risk of restenosis and the need for repeat revascularization when compared to bare metal stent ( bms).1 in the ravel and sirius trials,2,3 when compared to bms , des improved restenosis rates and late lumen loss , and decreased target lesion revascularization ( tlr ) from 16.6% to 4.1% ( p<0.01 ) .
although first - generation des ( 1st gen des ) were introduced to disrupt neointimal growth by the use of antiproliferative drugs , this benefit was acquired at the expense of a substantial delay in vascular healing and the clinical consequences of late and very late stent thrombosis ( lst / vlst).4 second - generation des ( 2nd gen des ) were developed with newer alloys , biocompatible polymers , thinner struts , and different drugs kinetics , resulting in a reduction of lst / vlst .
small vessel cad accounts for up to 30% of all pci5 and remains an independent predictor of angiographic restenosis and tlr , even after the introduction of des.6 the cobalt chromium everolimus - eluting stent ( cocr - ees ) ( xience v ; abbott vascular , santa clara , california , usa ) has been reported as one of the most frequently used 2nd gen des with better event - free survival rates in small vessels.7 this review article discusses the preclinical , clinical , and pathological performance of cocr - ees in small vessel cad .
the antiproliferative drug used is everolimus , a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant .
it induces cell cycle arrest in the g1 phase by inhibiting the mammalian target of rapamycin , a serine / threonine protein kinase that regulates cell growth , proliferation , motility , protein synthesis , and transcription , among others.8,9 the polymer is a thin ( 7.8 m ) bio - inert , non - erodible , and ultra - pure fluorinated copolymer ( poly - n - butyl methacrylate [ pbma ] and poly - vinylidene fluoride and hexafluoropropylene [ pvdf - hfp ] ) that provides both elasticity and stability .
pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp , which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus , respectively , and no top - coat layer is applied .
the polymer composition provides mechanical integrity after stent deployment , followed by the controlled release of everolimus at a total dose of 100 g / cm , delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability , and at the same time , increasing its radiopacity , radial strength , and fracture resistance.12 the xience v and xience nano share the same platform design , delivery system , drug , and coating materials .
the differentiating features of the xience nano pertain to a balloon diameter of 2.25 mm with a nominal inner stent diameter of 2.25 mm , as compared to xience v , which is available at diameters of 2.5 mm , 2.75 mm , 3.0 mm , 3.5 mm , and 4.0 mm .
there are several stent - related factors that have been associated with lst / vlst , such as strut thickness , polymer characteristics , coating integrity , and drug dose , among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard , des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model .
two main factors were reported to determine acute thrombogenicity : 1 ) strut thickness ; and 2 ) polymer coating .
thrombogenicity within the various bms designs correlated with strut thickness ; stents with thicker struts were 49% more thrombogenic than stents with thinner struts ( 0.880.38 for struts < 100 m versus 1.440.65 for struts > 100 m ; p=0.036 ) .
after 3 days of implantation in porcine coronary arteries , stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts ( 0.210.041 mm versus 0.130.019 mm ; p=0.004 ) ; also , neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts ( 1.560.40 versus 0.830.41 ; p=0.016 ) .
the authors also evaluated overlapping stents , which were more thrombogenic than single length - matched controls , more so for thicker than thinner struts stents ( 2.320.96 and 3.250.11 versus 1.000.17 ; p<0.001 ) .
moreover , overlapping thinner strut des ( 0.510.019 ) were less thrombogenic than overlapping bms ( p<0.001 ) and even the single bms controls ( p<0.001).17 in this landmark study , kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms ( 0.760.02 versus 1.000.15 ; p<0.002 ) , and clot mass was also significantly lower for des when compared with bms ( 0.670.35 versus 1.030.54 ; p=0.011 ) .
hypersensitivity reactions induced by the durable polymers used in 1st gen des were suggested to contribute to the occurrence of lst.1820 recent advances in stent technology , with the introduction of more biocompatible polymers , have reduced the risk of this complication.21,22 chin - quee et al23 evaluated two different polymers currently available for des in rabbit iliac arteries where cocr stents were coated with pvdf - hfp or phosphorylcholine polymer ( without drug ) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy ( sem ) .
endothelialization was equivalent and near complete for pvdf - hfp versus phosphorylcholine polymer - coated stents ( > 80% by sem ) .
also , acute thrombogenicity was assessed in a chandler loop model using porcine blood ; thrombus adherence was similar for both polymers ( 0.940.23 versus 0.990.20 ) .
these results suggest that the polymers examined here did not impede endothelization.23 polymer coating defects can potentially change the drug elution kinetics of des , or they can lead to chronic inflammatory reactions.17 furthermore , the nonuniform coating of stent struts may impact platelet adhesion and endothelialization . finally , coating fragments may embolize downstream , resulting in myocardial ischemia .
yazdani et al24 evaluated 48 des for coating integrity in cocr - ees , zotarolimus - eluting stent ( zes ) , paclitaxel - eluting stent ( pes ) , and biolimus a-9-eluting stent ( bes ) in a rabbit iliofemoral stent model for durations of 7 days , 28 days , 90 days , and 180 days .
the cocr - ees and zes had the least amount of coating defects as compared to the pes and bes .
however , coating defects were shown to increase over time within the zes , whereas in the cocr - ees , the amount of irregularity remained constant over time .
newer generation des were designed to outperform first - generation devices in this regard . in our laboratory,25
we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees ( 64.0%27.5% ) , followed by zes ( 30.2%14.2% ) , pes ( 26.8%15.8% ) , and sirolimus - eluting stent ( ses ) ( 6.4%4.2% ) , with a statistically significant difference versus cocr - ees ( p<0.003 ) and bms ( p<0.0001 ) as a control stent ( figure 1 ) . at 28 days , all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees , but without statistically significant differences among groups .
furthermore , cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry , the greatest frequency of uncovered struts was observed in the middle stented segment , while proximal and distal segments showed overall greater coverage .
we also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule , pecam-1 , as a surrogate , and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts , which demonstrates a functionally and biologically active endothelium.25
the antiproliferative drug used is everolimus , a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant .
it induces cell cycle arrest in the g1 phase by inhibiting the mammalian target of rapamycin , a serine / threonine protein kinase that regulates cell growth , proliferation , motility , protein synthesis , and transcription , among others.8,9 the polymer is a thin ( 7.8 m ) bio - inert , non - erodible , and ultra - pure fluorinated copolymer ( poly - n - butyl methacrylate [ pbma ] and poly - vinylidene fluoride and hexafluoropropylene [ pvdf - hfp ] ) that provides both elasticity and stability .
pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp , which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus , respectively , and no top - coat layer is applied .
the polymer composition provides mechanical integrity after stent deployment , followed by the controlled release of everolimus at a total dose of 100 g / cm , delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability , and at the same time , increasing its radiopacity , radial strength , and fracture resistance.12 the xience v and xience nano share the same platform design , delivery system , drug , and coating materials .
the differentiating features of the xience nano pertain to a balloon diameter of 2.25 mm with a nominal inner stent diameter of 2.25 mm , as compared to xience v , which is available at diameters of 2.5 mm , 2.75 mm , 3.0 mm , 3.5 mm , and 4.0 mm .
there are several stent - related factors that have been associated with lst / vlst , such as strut thickness , polymer characteristics , coating integrity , and drug dose , among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard , des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model .
two main factors were reported to determine acute thrombogenicity : 1 ) strut thickness ; and 2 ) polymer coating .
thrombogenicity within the various bms designs correlated with strut thickness ; stents with thicker struts were 49% more thrombogenic than stents with thinner struts ( 0.880.38 for struts < 100 m versus 1.440.65 for struts > 100 m ; p=0.036 ) .
after 3 days of implantation in porcine coronary arteries , stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts ( 0.210.041 mm versus 0.130.019 mm ; p=0.004 ) ; also , neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts ( 1.560.40 versus 0.830.41 ; p=0.016 ) .
the authors also evaluated overlapping stents , which were more thrombogenic than single length - matched controls , more so for thicker than thinner struts stents ( 2.320.96 and 3.250.11 versus 1.000.17 ; p<0.001 ) .
moreover , overlapping thinner strut des ( 0.510.019 ) were less thrombogenic than overlapping bms ( p<0.001 ) and even the single bms controls ( p<0.001).17 in this landmark study , kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms ( 0.760.02 versus 1.000.15 ; p<0.002 ) , and clot mass was also significantly lower for des when compared with bms ( 0.670.35 versus 1.030.54 ; p=0.011 ) .
hypersensitivity reactions induced by the durable polymers used in 1st gen des were suggested to contribute to the occurrence of lst.1820 recent advances in stent technology , with the introduction of more biocompatible polymers , have reduced the risk of this complication.21,22 chin - quee et al23 evaluated two different polymers currently available for des in rabbit iliac arteries where cocr stents were coated with pvdf - hfp or phosphorylcholine polymer ( without drug ) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy ( sem ) .
endothelialization was equivalent and near complete for pvdf - hfp versus phosphorylcholine polymer - coated stents ( > 80% by sem ) .
also , acute thrombogenicity was assessed in a chandler loop model using porcine blood ; thrombus adherence was similar for both polymers ( 0.940.23 versus 0.990.20 ) .
these results suggest that the polymers examined here did not impede endothelization.23 polymer coating defects can potentially change the drug elution kinetics of des , or they can lead to chronic inflammatory reactions.17 furthermore , the nonuniform coating of stent struts may impact platelet adhesion and endothelialization . finally , coating fragments may embolize downstream , resulting in myocardial ischemia
. yazdani et al24 evaluated 48 des for coating integrity in cocr - ees , zotarolimus - eluting stent ( zes ) , paclitaxel - eluting stent ( pes ) , and biolimus a-9-eluting stent ( bes ) in a rabbit iliofemoral stent model for durations of 7 days , 28 days , 90 days , and 180 days .
the cocr - ees and zes had the least amount of coating defects as compared to the pes and bes .
however , coating defects were shown to increase over time within the zes , whereas in the cocr - ees , the amount of irregularity remained constant over time .
newer generation des were designed to outperform first - generation devices in this regard . in our laboratory,25
we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees ( 64.0%27.5% ) , followed by zes ( 30.2%14.2% ) , pes ( 26.8%15.8% ) , and sirolimus - eluting stent ( ses ) ( 6.4%4.2% ) , with a statistically significant difference versus cocr - ees ( p<0.003 ) and bms ( p<0.0001 ) as a control stent ( figure 1 ) . at 28 days , all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees , but without statistically significant differences among groups . furthermore , cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry , the greatest frequency of uncovered struts was observed in the middle stented segment , while proximal and distal segments showed overall greater coverage .
we also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule , pecam-1 , as a surrogate , and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts , which demonstrates a functionally and biologically active endothelium.25
randomized controlled clinical trials have established differential outcomes in the safety and efficacy of des used in distinct clinical settings , and stent - related factors may play an important role in the scenery of small vessel pci.13,14,26 cannon et al27 evaluated the safety of xience nano in vessels > 2.25 mm but < 2.5 mm at 1-year follow - up .
the authors established a performance goal ( pg ) at 20.4% for target lesion failure ( tlf ) based on clinical trials and registries , which evaluated 2.25 mm diameter des.14,28,29 the 1-year tlf rate was 8.1% , with an upper one - sided limit ( 95% confidence interval ) of 13.0% , meeting the pg of 20.4% ( p<0.0001 ) .
the 1-year tlf rate was mainly driven by low cardiac death and myocardial infarction ( mi ) rates .
the most important difference noted in this study was a higher tlf rate for the reference vessel diameter ( rvd ) 2.12 mm ( number [ n ] = 72 ) , which reached 13.89% , compared to 1.56% for a rvd > 2.12 mm ( n=64 ) . in a post
clinically , no statistically significant differences were found for tlf ( 5.7% versus 4.9% ; p=1.0 ) and major adverse cardiovascular event rates ( mace)/tlf rates ( 7.5% versus 8.5% ; p=1.0 ) in diabetics versus nondiabetics , respectively . also , angiographically , in - stent and in - segment late loss showed no difference between diabetics and nondiabetics ( 0.220.47 mm versus 0.190.36 mm , p=0.83 ; and 0.140.48 mm versus 0.170.38 mm , respectively , p=0.76).27 small vessel cad represents a challenge for interventional cardiologists , with higher restenosis and stent thrombosis ( st ) rates.30,31 hermiller et al32 evaluated the safety of cocr - ees in small and nonsmall vessels in a real - world scenario , applying a 2.5 mm diameter cut - off .
the mean rvd for small vessels was 2.550.36 mm and 3.250.46 mm for the nonsmall vessel group ( p<0.001 ) .
definite or probable st rates were low and not significantly different between the groups at 0.37% versus 0.40% ( p=0.88 ) for the small and nonsmall vessel groups , respectively
. the composite rate of cardiac death or mi was comparable for the small and nonsmall vessel group ( 4.5% versus 5.1% , respectively ; p=0.57 ) .
the 1-year tlr rate was also comparable in the small vessel group ( small group 3.8% versus nonsmall group 3.0% ; p=0.35 ) .
this study demonstrated the safety of cocr - ees in small vessels despite the fact that this group consisted of more females , those with a higher rate of diabetes , and those with more complex lesion characteristics.32 in a separate study , ito et al33 compared cocr - ees and pes for small vessel revascularization by pooling the data from the spirit iii and iv trials.34,35 from 4,689 patients , two groups were analyzed : the small vessel group ( rvd : 2.250.19 mm ; n=1,019 ) and the large vessel group ( rvd : 2.990.35 mm ; n=2,586 ) .
after 1-year follow - up , in patients with small vessels disease , the tlf ( cocr - ees 4.4% versus pes 7.9% ; p=0.03 ) and mace ( cocr - ees 4.5% versus pes 7.9% ;
the clinical endpoint , tlf , was composed of cardiac death , target vessel mi , and ischemia driven - tlr ( id - tlr ) . amid the others , only id - tlr showed a significant reduction at 1 year ( everolimus - eluting stents [ ees ] 2.4% versus pes 5.5% ; p=0.02 ) .
although , st showed higher rates in small vessel revascularization , the authors found that st was significantly lower in patients with small vessels treated with cocr - ees than in those treated with pes ( 0.2% versus 1.2% , respectively ; p=0.04).33 currently , the factors predictive of in - stent restenosis can be divided into patient - related , procedure - related , and lesion - related factors .
patient - related factors such as diabetes , a history of restenosis , and genetic factors have been reported as risk factors of in - stent restenosis.33 procedure - related factors include the number of stents implanted , the total stent length , and stent overlap .
lesion - related characteristics , which impact the rate of restenosis , include small vessel size , long lesion length , and the severity of pretreatment as well as posttreatment lesion stenosis , among others.36 claessen et al37 collected data from spirit ii , iii , and iv,34,38,39 and combined three groups : short lesions in large vessels ( group a ) ; long lesions in large vessels or short lesions in small vessels ( group b ) ; and long lesions in small vessels ( group c ) to evaluate the safety and efficacy of cocr - ees versus pes .
the mace rate after 2 years of follow - up was lower in group a , intermediate in group b , and highest in group c ( 5.6% versus 8.2% versus 10.4% , respectively ; p<0.0001 ) .
also , a similar trend was observed for mi ( 3.3% versus 3.0% versus 4.5% , respectively ; p=0.02 ) and id - tlr ( 2.9% versus 5.0% versus 6.3% , respectively ; p=0.0002 ) .
the authors also evaluated mace rates by stent type ( pes and cocr - ees ) , and found that the higher the lesion complexity , the greater the mace incidence ( 7.0% , 11.2% , and 12.8% for pes , respectively , p=0.007 ; versus 4.8% versus 6.6% versus 9.1% for cocr - ees , respectively , p=0.001 ) . on the other hand
, the 2-year rate of definite or probable st ( academic research consortium , arc definition ) also increased with greater lesion complexity after pes implantation ( group a 0.7% versus group b 1.9% versus group c 2.8% ; p=0.03 ) , but that relationship was not present after cocr - ees implantation ( 0.9% versus 0.6% versus 0.6% ; p=0.65 ) .
cocr - ees were associated with significantly lower rates of mace , mi , id - tlr , and st in groups b and c , but no statistical significance was found in the less complex group ( group a ) .
multivariate analysis found that the use of cocr - ees rather than pes was an independent predictor of freedom from mace in group b ( p<0.0001 ) and group c ( p=0.004 ) , but not in group a ( p=0.19).37
recently , we reported40 the pathologic findings of 2nd gen des and compared these to 1st gen des . a total of 204 lesions ( ses = 73 ; pes = 85 ; cocr - ees = 46 ) from 149 autopsy cases with implant duration > 30 days and 3 years were pathologically analyzed to determine differences .
the observed frequency of lst and vlst was less for cocr - ees ( 4% ) compared with ses ( 21% ; p=0.029 ) and pes ( 26% ; p=0.008 ) .
the prevalence of restenosis for cocr - ees ( 17% ) did not differ significantly from that observed in ses ( 14% ) and pes ( 12% ) .
the frequency of uncovered struts was markedly lower for cocr - ees ( 2.6% ) as compared to ses ( 18.0% ; p<0.0005 ) and pes ( 18.7% ; p<0.0005 ) .
the prevalence of des with > 30% uncovered struts was also significantly lower in cocr - ees ( 20% ) than in ses ( 60% ; p<0.0005 ) and pes ( 67% ; p<0.0005 ) . in terms of inflammation ,
the overall prevalence of neoatherosclerosis after cocr - ees implantation in native coronary arteries was 29% , which did not differ significantly from ses ( 35% ; p=0.62 ) and pes ( 19% ; p=0.47).40 complex lesion characteristics and unstable plaques are associated with a greater delay in arterial healing when compared to pci of a simple and stable plaque by pathology.41 therefore , we evaluated the prevalence of > 30% uncovered struts in the setting of off - label versus on - label clinical indications .
cocr - ees compared with ses and pes showed greater strut coverage for both on - label ( 14% versus 50% versus 57% , respectively ) and off - label ( 25% versus 68% versus 75% , respectively ) indications . when analyzing the cvpath stent database composed of 865 cases , 68 had cocr - ees implanted and 12 cases
were found with a stent diameter of 2.5 mm or less ( table 1 ) . from those 12 cases ,
the mean stent length was 35.624.9 mm and the mean stent diameter was 2.30.27 mm .
case 1 : a 45-year - old woman with obesity , hypertension , and diabetes who presented with mi secondary to involvement of the left circumflex artery , which was revascularized ; 5 days after the procedure , she died suddenly with st .
case 2 : a 58-year - old male with a history of obesity and cad presented with non - st segment elevation mi , and left anterior descending artery occlusion in the region of the left diagonal branch ; bifurcation stenting was performed .
the patient died suddenly 7 days after the procedure ; at autopsy , there was a large infarction and mild to moderate thrombus in the stented region .
the other three patients had stable cad and only mild inflammation was observed with moderate peristrut fibrin and platelet deposition ( figure 2 ) .
one of the three cases had thrombosis and stent fracture ( 20% ) ; the other two were nonstent - related deaths .
the remaining seven cases ( table 1 ) had a duration > 30 days ( 242.9222.6 days ) .
acute coronary syndrome was the indication in one case ( 14.3% ) , and the cause of death was stent - related a 72-year - old woman with obesity , hypertension , diabetes , atrial fibrillation , and a history of multiple revascularizations .
she died from st 210 days later with underlying restenosis . for the rest of the cases ( n=6 ) with duration > 30 days , there were no differences in the principal histopathological findings among those presenting with acute coronary syndrome versus stable cad .
overall , the histopathological analysis showed mild to moderate chronic peristrut inflammation consisting of monocytes , t - lymphocytes , and macrophages ( figure 3 ) without any significant eosinophils .
restenosis was observed in one of the six cases ; however , the xience case was sandwiched between two vision stents , both of which had total occlusion .
nevertheless , we must recognize the limited number of cases analyzed at autopsy . a greater number of cases and matched control groups will be required to understand the full scope of histopathological findings of cocr - ees in small vessel disease .
des have progressively improved clinical outcomes , but the potential risk of st is still a concern and limits the use of des , especially in small vessel cad .
overall , lst and vlst have been reported with an incidence of 0.2% and 0.4% per year , respectively.42 however , considering the large amount of stents implanted worldwide , those numbers are still high .
several reports of st have been associated with 1st gen des , especially after dual anti - platelet therapy termination.43,44 over time , great effort has been made to improve the technology , thus reducing strut thickness from 140 m to approximately 7080 m , resulting in a dramatic reduction of thrombogenicity in bench studies.17,45 advances in polymer technology have been enormous ; new biocompatible polymers ( pbma or pvdf - hfp ) result in less inflammation after stent implantation , with the consequence of more complete and functional endothelization.46,47 also , cocr - ees show fewer coating defects after implantation when compared to different des , and this result was maintained over time and may improve vascular biocompatibility.25 clinical data confirmed the outstanding performance of cocr - ees , with lower rates of definitive / probable st , tlr , and mace .
the pg for tlf was overperformed with cocr - ees when compared with a competitor des in small vessel disease .
mace and tlf rates were similar among diabetics and nondiabetics.27 at pathology , cocr - ees revealed less inflammation and greater strut coverage when compared to 1st gen des , while maintaining similar efficacy in reducing neointimal growth . specifically , in small vessel disease , cocr - ees have been shown to be less thrombogenic compared to 1st gen des ; however , inflammation and restenosis remain a problem in this setting , and further technological and procedural progress is needed to improve patient outcomes .
in the des era , small vessel cad remains a great challenge for interventional cardiologists .
stent design and the material combination may provide better outcomes , especially in small vessel disease .
cocr - ees with thinner struts , biocompatible polymers , reduced drug load , and better radiopacity and trackability have shown excellent results from preclinical , clinical , and pathological studies in small vessel cad . | coronary artery disease ( cad ) is the leading cause of morbidity and mortality worldwide .
the pathogenesis of cad relates to the presence of atherosclerotic plaques in the coronary arteries , which are most frequently treated today by percutaneous coronary intervention .
small vessel disease treatment represents one - third of all percutaneous coronary interventions with higher rates of restenosis and major adverse cardiac events .
initially , drug - eluting stents ( des ) were developed to reduce in - stent restenosis , improving clinical outcomes and reducing the need for target vessel revascularization .
however , late and very late stent thrombosis emerged as a new problem compromising des s long - term results .
the cobalt
chromium everolimus - eluting stent ( cocr - ees ) represents the results of an evolutionary process in des technology aimed at improving the shortcomings of first - generation des .
small vessel cad has historically been an obstacle to long - term patency following implantation of des .
antirestenotic efficacy has been shown to be of high relevance in small vessels . therefore , stent selection may play an important role in determining outcomes in this subgroup of patients .
this article will review the performance of cocr - ees in the treatment of small vessel cad from preclinical , clinical , and pathology perspectives , and it will highlight the most important findings in this regard . | Introduction
Device characteristics
Xience V
Preclinical findings
Clinical findings
Pathology findings
Summary
Conclusion |
odor from modern intensive pig production can cause serious nuisance to people living in the vicinity of the facilities .
volatile reduced sulfur compounds are considered to have a significant influence on odor from pig production due to their low odor threshold values and low solubility in slurry .
the most abundant sulfur compounds found in pig production are hydrogen sulfide , methanethiol , and dimethyl sulfide [ 2 , 3 ] .
the concentration of hydrogen sulfide in pig production is normally several hundred ppbv , whereas the concentration of methanethiol and dimethyl sulfide is in the low ppbv range ( 120 ppbv ) .
based on the reported odor threshold values , particularly hydrogen sulfide and methanethiol may have a large influence on odor from pig production .
different methods have been applied for analyzing sulfur compounds in sample air from animal production .
online measurements by proton - transfer - reaction mass spectrometry ( ptr - ms ) have been used to measure sulfur compounds and other odorants from pig production [ 2 , 3 ] and cattle production [ 4 , 5 ] .
gas chromatography combined with a sulfur - specific detector such as flame photometric detection ( gc - fpd ) , pulsed flame photometric detection ( gc - pfpd ) , and sulfur chemiluminescence detection ( gc - scd ) is often used to measure sulfur compounds in air .
measurement of sulfur compounds from pig production with sulfur - specific detectors requires the use of sampling bags or preconcentration of sample air on adsorbent tubes or adsorptive fibers .
sulfur compounds can be stored in tedlar bags for several hours with a recovery above 9095% [ 710 ] , but this sampling method requires a detector with a detection limit in the low ppbv range or a preconcentration step prior to analysis .
pre - concentration of sample air on adsorbent tubes or adsorptive fibers at the source can avoid the potential loss of sulfur compounds in sample bags , however , graphitized and carbon molecular sieve sorbents and to some extent tenax ta result in oxidation of thiols into disulfides [ 11 , 12 ] .
compared to graphitized and carbon molecular sieve sorbents and tenax ta , the use of silica gel for pre - concentration of sulfur compounds has been shown to have a higher recovery of hydrogen sulfide ( 78.7% ) and methanethiol ( 98.2% ) , but it requires drying of the sample air to avoid water sorption .
the advantage of silica gel for pre - concentration of sulfur compounds was used to develop a method with a single column for trapping / separation and chemiluminescence detection ( scts - cl ) .
sulfur chemiluminescence detection normally includes a conversion of sulfur compounds into so in a hydrogen / air burner , reaction with ozone , and detection of the chemiluminescent emission from excited so2*. however , even without the conversion step , reduced sulfur compounds react with ozone , thus resulting in a chemiluminescent emission that can be detected [ 16 , 17 ] .
this type of chemiluminescence detection was applied in the scts - cl , where separated sulfur compounds from the silica gel column and ozone were mixed , and the chemiluminescent emission was detected by a photomultiplier .
although other measurement techniques are available for measurement of sulfur compounds , the scts - cl is a portable and low - cost ( ca .
the scts - cl has previously been applied to atmospheric analysis of sulfur gases and isoprene and analysis of isoprene contained in breath , but the method has not been applied to ventilation air from pig production or other types of animal production . ventilation air from animal production is generally characterized by high levels of dust , moisture , and hydrogen sulfide , and the performance of scts - cl should therefore be tested under these specific conditions . the objective of the present study was ( i ) to optimize the scts - cl for measuring methanethiol and dimethyl sulfide under the conditions present in sample air from pig production and ( ii ) to perform an intercomparison between scts - cl , ptr - ms , and gc - scd with sample air from a pig production facility with growing - finishing pigs .
silica gel used for gas collection and separation was davison grade 12 , 60/80 mesh ( supelco , bellefonte , pa , usa ) .
the silica gel was packed in a ceramic tube ( 4 mm i.d .
the effective adsorbent bed was 6 cm long , and the ends were plugged with silanized quartz wool .
a nickel - chrome wire ( 0.2 mm 35 cm , nilaco , tokyo , japan ) was coiled around the ceramic tube and used to heat the column .
the linearity of the scts - cl was estimated based on gas mixture 1 ( table 1 ) .
desorption of compounds , trapping efficiency , and the interferences between sulfur compounds for the scts - cl were investigated by ptr - ms .
gas mixture 2 and 3 ( table 1 ) were used to investigate the desorption of methanethiol and dimethyl sulfide , and gas mixture 4 ( table 1 ) was used to investigate the trapping efficiency and the interferences between compounds .
the scts - cl for the sulfur gases was developed at kumamoto university , japan and was improved by toda et al . .
the scts - cl was constructed at kumamoto university by the lead author and was subsequently applied in denmark .
the instrument was composed of two parts which were ( i ) a silica gel column used for trapping and separation of sulfur compounds and ( ii ) a chemiluminescence detector .
the chemiluminescence cell was made of a stainless steel tube ( 32 mm i.d .
40 mm ) where one end was capped with a stainless steel plate with an inlet / outlet tube , and the other side was covered with an o - ring sealed glass plate for optical window .
sample air from the column and ozone was introduced to the reaction cell through stainless steel tubes .
an ozone generator ( 1000bt-12 ( 300 mg h ) , shanghai enaly m&e ltd . ,
shanghai , china ) with a flow of 150 ml min charcoal filtered atmospheric air was used to generate ozone .
a photomultiplier ( r3550a , hamamatsu photonics , hamamatsu , japan ) was used to detect the chemiluminescent emission from the reaction cell . the silica gel column and the chemiluminescence detector
were integrated in a system with four three - way solenoid valves ( vt307y-6g-01 , smc , tokyo , japan ) that were used to change between the trapping and the separation mode , see figure 1 .
each measurement cycle took 15 min and was divided into three steps , see figure 2 . in step 1 ,
sample air was collected on the column for 3 min with a sample flow at 200 ml min .
the sample air was passed through a 5 m teflon particle filter ( millipore , billerica , ma , usa ) and a nafion dryer ( md-110 - 48p-4 , perma pure , toms river , nj , usa ) prior to the collection on the column .
the countercurrent flow of dry air in the nafion dryer was set at 2 l min . during sample collection
, nitrogen carrier gas was passed through the column with a flow at 50 ml min , and the column was heated to release the trapped sulfur compounds .
the column temperature was held for 1 min at ambient temperature for purging , ramped up to 130c and held for 2 min to release methanethiol , ramped up to 240c and held for 2 min to release dimethyl sulfide , and ramped up to 270c and held for 1 min for cleaning of the column . in step 3
, nitrogen carrier gas was passed through the column with a flow at 50 ml min , and the column was cooled down to ambient temperature with a fan ( shicoh engineering co. ltd . ,
yamato , japan ) over a period of 6 min . during step 2 and 3
the detection limits of the scts - cl were estimated as three times the baseline noise .
a ptr - ms ( ionicon analytik , innsbruck , austria ) and a gc - scd ( gc 7890 a and scd 355 , agilent technologies a / s , horsholm , denmark ) were used to evaluate the results obtained by the scts - cl .
the ptr - ms was operated under standard ion drift tube conditions applying a total voltage of 600 v and maintaining the pressure in the range of 2.12.2 mbar ( e / n value ~135 td ) .
the temperature of the drift tube was controlled at 75c , and the inlet sampling flow was adjusted to ca .
instrumental background was measured on room air purified for hydrocarbon contaminants with a supelpure hc filter ( supelco , bellefonte , pa , usa ) .
the sensitivity of the sulfur compounds was estimated using the rate constant for proton transfer , the estimated drift tube residence time and the mass - specific transmission factor as described by de gouw and warneke .
the detection limits of the ptr - ms were calculated as three times the standard deviation on blank samples .
the gc - scd was equipped with a capillary column with a stationary phase of dimethylpolysiloxane ( db-1 , agilent technologies a / s , horsholm , denmark ) .
the column had a length at 60 m , an inner diameter at 0.53 mm , and a stationary phase at 5 m .
the gc oven temperature was held for 1 min at 60c , ramped up to 200c at 20c min and held for 1 min at 200c .
the gc - scd was equipped with a 1.0-ml sample loop . at each analysis ,
a 5 ppmv gas standard ( air liquide , horsens , denmark ) containing hydrogen sulfide , methanethiol , and dimethyl sulfide was used as a one - point calibration .
the detection limits of the gc - scd were estimated as three times the baseline noise . an experimental pig production facility with growing - finishing pigs
( pig research centre , danish agriculture & food council , grnhj , denmark ) was used for collection of field samples . the facility consisted of two pens with 16 growing - finishing pigs ( 32107 kg ) in each and
was equipped with dry feeding and negative pressure ventilation with a diffuse inlet through the ceiling .
the facility had one third drained floor ( slatted floor with less than 10% openings ) and two third slatted floor .
three air samples were collected in 10-l tedlar bags ( cel scientific corporation , santa fe springs , ca , usa ) from the ventilation outlet of the facility .
samples were aspirated into the bags by applying negative pressure around the bags in a vacuum container .
preconditioning of the bag inner surface was done by filling the bags with sample air and emptying them twice before the final filling .
the temperature in the ventilation outlet was 16.6 0.2c , and the relative humidity was 72.2 0.7% .
the tedlar bags were covered with black plastic to avoid photodegradation and transported back to the laboratory for analysis . within 3 h after collection
the samples were analyzed by scts - cl , ptr - ms , and gc - scd .
the first part of the study was concerned with the optimization and evaluation of the instrument in relation to the conditions present in sample air from pig production .
an advantage of the chemiluminescence detection is the linear response , which makes the calibration of the instrument more simple compared to other detectors such as the pulsed flame photometric detector ( pfpd ) that has a quadratic response . in figure 3 ,
calibration curves are shown for methanethiol and dimethyl sulfide in the range between 1 to 21 ppbv , which is the concentration range normally found in facilities with growing - finishing pigs [ 2 , 3 ] . according to figure 3 , the response from the scts - cl is linear for both methanethiol ( r = 0.997 ) and dimethyl sulfide ( r = 0.999 ) . due to the high linearity of the scts - cl ,
a one - point calibration with a gas mixture containing methanethiol ( measured by ptr - ms : 9.6 ppbv ) and dimethyl sulfide ( measured by ptr - ms : 12.6 ppbv ) was used for the measurements on field samples from pig production .
the trapping efficiency of the silica gel column was estimated based on trapping of ca .
the trapping efficiency of all three compounds was above 95% in accordance with previous studies on hydrogen sulfide and methanethiol .
although silica gel is suitable for trapping of sulfur compounds , it requires drying of the sample air prior to collection to avoid water sorption that can have a negative influence on the recovery [ 13 , 14 ] .
it has previously been shown that a nafion dryer [ 24 , 25 ] and a cacl2 column [ 26 , 27 ] can be used to dry sample air with only a limited influence on the recovery of sulfur compounds .
the nafion dryer was chosen for the scts - cl because it only requires a countercurrent flow of dry air , and the changing of the drying agent during the measurements was avoided .
10 ppbv ) measured by ptr - ms is presented in figure 4 .
figure 4(a ) shows that methanethiol was split into two peaks , whereas figure 4(b ) shows that dimethyl sulfide only resulted in one peak .
the second peak of methanethiol indicates that a fraction of the molecules is trapped more firmly by the silica gel and requires a higher desorption temperature .
a small peak from dimethyl disulfide is also seen in figure 4(a ) , which could origin from an impurity ( < 0.5 ppbv ) in the gas mixture or oxidation of methanethiol during the trapping and desorption process .
other studies have shown that graphitized and carbon molecular sieve sorbents and to some extent tenax ta result in the oxidation of thiols into disulfides [ 11 , 12 ] . in the study by lestremau et al . , it was shown that the formation of dimethyl disulfide on a carbon molecular sieve sorbent increased from 4% of the methanethiol peak to 60% when the desorption temperature was increased from 150c to 300c thereby indicating that it is a thermal oxidation . in the present study ,
the second peak of methanethiol and the dimethyl disulfide peak are desorbed close to the desorption temperature for dimethyl sulfide and could possibly interfere with the measurement of dimethyl sulfide .
the high trapping efficiency of hydrogen sulfide gives a problem in relation to sample air from pig production , because the desorption temperature of hydrogen sulfide from the silica gel column is close to methanethiol .
the intensity of the chemiluminescent emission from the direct reaction between ozone and hydrogen sulfide is 1/100 of methanethiol , but at 2030 times , higher concentrations which are normal in pig production , it will result in a detectable chemiluminescent emission that will interfere with the measurement of methanethiol .
figure 5 clearly shows that hydrogen sulfide is desorbed at the same time as methanethiol and that it will interfere with the measurement of methanethiol .
it was also found that the first peak of methanethiol was decreased when the hydrogen sulfide concentration in the gas mixture was increased from 50 ppbv to 300 ppbv ( results not shown ) , and it seems that a high hydrogen sulfide concentration also influences the trapping of methanethiol .
the second peak of methanethiol was only slightly increased when the hydrogen sulfide concentration was increased from 50 ppbv to 300 ppbv . during measurements on the field samples ,
hydrogen sulfide was removed from the sample air with a small column ( 13 mm i d 15 cm ) packed with glass wool treated with 5% na2co3/5% glycerin .
10 ppbv ) in the column was above 95% , and the removal efficiency of hydrogen sulfide ( ca .
ten samples , but the size of the column could be increased to handle more samples .
air samples were collected from a facility with growing - finishing pigs and an intercomparison was performed among scts - cl , ptr - ms , and gc - scd . to minimize the temporal variation between the three methods , sample air from the pig production
was collected in tedlar bags and transported to the laboratory for analysis within 3 h. the recovery of sulfur compounds in tedlar bags has previously been shown to be above 9095% several hours after collection [ 710 ] , and the air samples should more or less reflect the concentration levels in the pig production facility .
the measured concentrations of hydrogen sulfide , methanethiol , and dimethyl sulfide are presented in table 2 .
the difference between scts - cl , ptr - ms , and gc - scd in relation to methanethiol and dimethyl sulfide was below 10% .
the relative standard deviation for measurements by scts - cl on field samples was 5% for methanethiol and 2% for dimethyl sulfide , which is comparable with ptr - ms ( 4% and 6% ) and gc - scd ( 7% and 1% ) .
although the principle of the methods is quite different , it seems that all three methods can provide reliable measurements of methanethiol and dimethyl sulfide in sample air from pig production .
the measurements of hydrogen sulfide by ptr - ms and gc - scd were also comparable , although the measurements by ptr - ms were slightly higher ( ca .
in table 3 , the estimated detection limits for the scts - cl are presented along with the ptr - ms and the gc - scd .
the scts - cl and the ptr - ms demonstrated significantly lower detection limits for methanethiol and dimethyl sulfide compared to the gc - scd .
the gc - scd was based on injection of whole air samples through a sample loop and that was the main reason for the higher detection limits obtained for this method .
the gc - scd can also be combined with a pre - concentration step and in that case , it would also provide lower detection limits than shown in the present study .
the detection limits for the scts - cl and the ptr - ms were below the estimated odor threshold values for both methanethiol and dimethyl sulfide ( table 3 ) .
subthreshold detection limits give the opportunity to investigate the relation between odorants and the response from human noses under conditions with low concentrations such as the boundary line of an animal production facility or during the development of odor reduction technologies with a high odorant removal .
contrary to the other two methods , the ptr - ms has the advantage that it can include a wider range of odorants and provide online data with a high time resolution .
the scts - cl is a portable instrument , but it requires 15 min for each sample which gives a relatively high temporal variation between the measurements .
furthermore , field measurements with the scts - cl in pig production require a large column for removal of hydrogen sulfide , which limits the usability for long - term studies .
although the scts - cl can not be used as an online method such as the ptr - ms , it can still provide on - site measurements of methanethiol and dimethyl sulfide , so that storage and transportation of sample bags and adsorbent tubes or adsorptive fibers can be avoided .
it can be concluded that the scts - cl is a portable and low - cost alternative to the commercial methods that can be used to measure methanethiol and dimethyl sulfide in sample air from pig production , but it requires a removal of hydrogen sulfide to obtain reliable results for methanethiol .
the choice of method for analysis of gaseous sulfur compounds in future studies with pig production depends on the requirements for detection limit , time resolution , and the range of odorants that can be measured . | reduced sulfur compounds are considered to be important odorants from pig production due to their low odor threshold values and low solubility in slurry .
the objective of the present study was to investigate the use of a portable method with a single silica gel column for trapping / separation coupled with chemiluminescence detection ( scts - cl ) for measurement of methanethiol and dimethyl sulfide in sample air from pig production .
proton - transfer - reaction mass spectrometry ( ptr - ms ) was used to evaluate the trapping / separation .
the silica gel column used for the scts - cl efficiently collected hydrogen sulfide , methanethiol and dimethyl sulfide .
the measurement of methanethiol by scts - cl was clearly interfered by the high concentration of hydrogen sulfide found in pig production , and a removal of hydrogen sulfide was necessary to obtain reliable results .
air samples taken from a facility with growing - finishing pigs were analyzed by scts - cl , ptr - ms , and a gas chromatograph with sulfur chemiluminescence detection ( gc - scd ) to evaluate the scts - cl .
the difference between the concentrations of methanethiol and dimethyl sulfide measured with scts - cl , ptr - ms , and gc - scd was below 10% . in conclusion ,
the scts - cl is a portable and low - cost alternative to the commercial methods that can be used to measure methanethiol and dimethyl sulfide in sample air from pig production . | 1. Introduction
2. Materials and Methods
3. Results and Discussion |
cervical carcinoma is the most common malignancy in women in india and second only to breast cancer in women in worldwide data .
hematogenous dissemination usually occurs with more advanced disease , poorly differentiated tumors , and aggressive cell types such as adenosquamous or neuroendocrine tumors .
skeletal muscle metastases from cervical carcinoma are extremely rare , reported to be less than 1% of metastases of hematogenous origin of any cancer .
to our knowledge , from the existing literature search , only 11 cases of skeletal muscle metastases have been documented , of which ten have been from squamous cell carcinoma subtype of cervical cancer and one from the small cell carcinoma subtype .
we report an unusual case of adenocarcinoma cervix with extensive skeletal muscle metastases during staging work - up as detected by f - fluorodeoxyglucose positron emission tomography - computed tomography ( f - fdg pet / ct ) .
a 48-year - old female presented with a history of decreased appetite , episodic pain in lower abdomen associated with mild grade fever and chest pain for 1 month duration . clinical examination revealed a cachectic lady ( weight loss undocumented , body mass index - 15.6 kg / m ) with decreased air entry on the left side of the chest .
the lady was further investigated using contrast - enhanced ct scan ( cect chest and abdomen ) which revealed the following findings : ( a ) irregular nodular hypodense peripherally enhancing lesion in the cervix and right obturator internus muscle .
( c ) bilateral pleural effusion with a small suspicious pleural - based lesion and nodular thickening of interlobar fissures .
following the ct scan , a diagnostic pleural tap was done , which was negative for malignant cells . therefore , the lesions in the lung were inferred to be of infective pathology and the patient was treated with injectable antibiotics for the same .
female was further subjected to per vaginal examination for clinical correlation with the imaging findings , which revealed a puckered and nodular anterior and posterior vaginal wall mucosa .
a punch biopsy [ figure 1 ] was taken from the vaginal vault , which revealed a well - differentiated adenocarcinoma .
there was no clinical suspicion of distant metastasis to skeletal muscles . following this diagnosis ,
the patient underwent f - fdg pet / ct at our department for staging work - up [ figures 2,4 - 7 ] . the pet / ct scan revealed ( a ) an fdg avid ill - defined soft tissue thickening in the cervix and the upper part of vagina ( b ) multiple hypodense fdg avid muscle deposits in the following sites : semispinalis capitis , bilateral biceps and triceps brachii , bilateral paraspinal muscles , bilateral deltoid , left trapezius , bilateral supraspinatus , bilateral infraspinatus , bilateral gluteal muscles , bilateral pyriformis , right obturator internus , and left rectus femoris with the largest lesion ( metastatic deposit ) noted within the deltoid ( c ) non - fdg avid pleural effusion in bilateral hemithorax ( d ) fdg avid multiple bone marrow deposits . following
the study , a clinical diagnosis of metastatic adenocarcinoma cervix stage ivb ( international federation of gynaecology and obstetrics [ figo ] ) was made .
a fine needle aspiration cytology [ figure 3 ] from the representative focal deposit in the right deltoid revealed metastatic deposit of poorly differentiated carcinoma , thus confirming our findings .
after the final staging work - up , the patient was started on systemic chemotherapy by the treating onco - physician .
h and e - stained punch biopsy slide of vaginal growth revealing morphological features of well - differentiated adenocarcinoma with diffuse mixed inflammatory cell infiltrate whole body positron emission tomography / computed tomography images : ( a ) axial section showing hypermetabolic skeletal muscle deposits in both deltoid muscles .
( c ) axial section showing a metabolically active soft tissue density lesion in cervix and upper part of vagina along with a hypermetabolic muscle deposit in right obturator internus .
( d ) maximum intensity projection image in coronal section showing multiple focal muscle deposits and multiple bone marrow deposits romanovsky - stained slide of fine needle aspiration cytology taken from the right deltoid muscle showing atypical cells in clusters having large hyperchromatic nuclei with high nuclear - cytoplasmic ratio , prominent nucleoli and irregular nuclear membrane , suggestive of metastatic deposits of poorly differentiated carcinoma ( a ) plain computed tomography image showing hypodense soft tissue density deposits within bilateral deltoid muscle . ( b )
positron emission tomography / computed tomography image showing metabolically active soft tissue density deposits within bilateral deltoid muscle , right paraspinal and left trapezius muscle .
( c ) positron emission tomography / computed tomography image showing metabolically active soft tissue density deposits in right deltoid muscle and bilateral supraspinatus muscle .
( d ) positron emission tomography / computed tomography image showing metabolically active soft tissue density deposits in bilateral triceps muscle ( a ) axial maximum intensity projection image showing increased tracer uptake in the region of bilateral triceps muscle .
( b ) positron emission tomography / computed tomography image showing metabolically active soft tissue density deposits in bilateral infraspinatus muscle .
( c ) axial maximum intensity projection image showing increased tracer uptake in the region of bilateral infraspinatus muscle .
( d ) positron emission tomography / computed tomography image showing metabolically active soft tissue density deposits in left trapezius , bilateral deltoid and paraspinal muscles plain computed tomography image showing a soft tissue density mass lesion in the cervix along with a hypodense soft tissue density deposit in the right obturator internus muscle positron emission tomography / computed tomography image showing a metabolically active soft tissue density mass lesion in the cervix along with an fluorodeoxyglucose avid soft tissue density deposit in the right obturator internus muscle
we have reported an unusual case of adenocarcinoma cervix with extensive skeletal muscle metastases during staging work - up . based on initial clinical examination and investigations ( cect thorax and abdomen and punch biopsy vaginal vault ) , the patient was staged as a case of adenocarcinoma cervix stage iiib ( figo ) .
however , based on the f - fdg whole body pet /ct done at our center , the disease was upstaged to stage ivb ( figo ) .
skeletal muscle involvement from cervical carcinoma is a rare finding and is usually documented in the context of an advanced stage tumor [ as reported in literature appended in table 1 ] .
the likely reasons could be : ( a ) the constant movement of skeletal muscles , which represents a difficult condition for the implantation and growth of metastatic cells ; ( b ) the local production of lactic acid , which would create an unfavorable environment for metastatic cell growth ; ( c ) the inhibition of cell invasion by protease inhibitors located in the basement membrane ; ( d ) the antitumor activity of lymphocytes and natural killer cells within the skeletal muscle ; ( e)in vivo evidences that skeletal muscle delivered peptidic factors may negatively influence the process of metastatic spread .
characteristics of cervical cancer patients with skeletal muscle involvement , as reported in the literature our case was unusual for the fact that it was a case of multiple skeletal metastases from an uncommon histological subtype of cervical carcinoma .
this case further reiterates the importance of pet / ct in staging and further management of the carcinoma cervix .
skeletal muscle metastases from adenocarcinoma cervix are rare . however , the diagnosis of the same may alter disease staging , management , and prognosis
. it can be solitary or multiple ; therefore , fdg pet / ct has an added benefit of diagnosing advanced disease and extent of metastases by providing whole body scan information . in the above - mentioned case report ,
f-18 fdg pet / ct gave information of multiple skeletal muscle deposits in clinically obscured metastases . | adenocarcinoma cervix is an uncommon histological subtype of carcinoma cervix ; further incidence of skeletal muscle metastases is even rarer .
we report the identification of extensive fluorodeoxyglucose ( fdg ) avid metastatic skeletal muscle deposits in a known case of adenocarcinoma cervix .
the largest lesion representative of muscle deposit in the right deltoid was histopathologically confirmed to be metastatic poorly differentiated carcinoma .
this report also serves to highlight the importance of 18f - fdg positron emission tomography / computed tomography ( ct ) as compared to conventional imaging modalities such as ct and ultrasonography and comments better over the description of invasiveness as well as the extent of disease in carcinoma cervix . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest |
the aim of this study was to measure the level of oncostatin m ( osm ) a gp130 cytokine in the gingival crevicular fluid ( gcf ) and serum of chronic periodontitis patients and to find any correlation between them before and after periodontal therapy ( scaling and root planing , srp ) .
60 subjects ( age 2550 years ) were enrolled into three groups ( n=20 per group ) , group i ( healthy ) , group ii ( gingivitis ) and group iii ( chronic periodontitis ) .
group iii subjects were followed for 68 weeks after the initial periodontal therapy ( srp ) as the group iv ( after periodontal therapy ) .
clinical parameters were assessed as gingival index ( gi ) , probing depth ( pd ) , clinical attachment level ( cal ) , and radiographic evidence of bone loss .
gcf and serum levels of osm were measured by using enzyme linked immunosorbent assay ( elisa ) .
it was found that mean osm levels had been elevated in both the gcf and serum of chronic periodontitis subjects ( 726.65 283.56 and 65.59 12.37 pgml , respectively ) and these levels were decreased proportionally after the periodontal therapy ( 95.50 38.85 and 39.98 16.69 pgml respectively ) .
however , osm was detected in gcf of healthy subjects ( 66.15 28.10 pgml ) and gingivitis - suffering subjects ( 128.33 22.96 pgml ) and was found as below the detectable limit ( 0.0 pgml ) in the serum of same subjects .
significant correlation has been found between clinical parameters and gcf - serum levels of osm .
increased osm level both in the gcf and serum , and the decreased levels after initial periodontal therapy ( srp ) may suggest a use as an inflammatory bio - marker in the periodontal disease . | aimthe aim of this study was to measure the level of oncostatin m ( osm ) a gp130 cytokine in the gingival crevicular fluid ( gcf ) and serum of chronic periodontitis patients and to find any correlation between them before and after periodontal therapy ( scaling and root planing , srp).methodology60 subjects ( age 2550 years ) were enrolled into three groups ( n=20 per group ) , group i ( healthy ) , group ii ( gingivitis ) and group iii ( chronic periodontitis ) .
group iii subjects were followed for 68 weeks after the initial periodontal therapy ( srp ) as the group iv ( after periodontal therapy ) .
clinical parameters were assessed as gingival index ( gi ) , probing depth ( pd ) , clinical attachment level ( cal ) , and radiographic evidence of bone loss .
gcf and serum levels of osm were measured by using enzyme linked immunosorbent assay ( elisa).resultsit was found that mean osm levels had been elevated in both the gcf and serum of chronic periodontitis subjects ( 726.65 283.56 and 65.59 12.37 pgml1 , respectively ) and these levels were decreased proportionally after the periodontal therapy ( 95.50 38.85 and 39.98 16.69 pgml1 respectively ) .
however , osm was detected in gcf of healthy subjects ( 66.15 28.10 pgml1 ) and gingivitis - suffering subjects ( 128.33 22.96 pgml1 ) and was found as below the detectable limit ( 0.0 pgml1 ) in the serum of same subjects .
significant correlation has been found between clinical parameters and gcf - serum levels of osm.conclusionincreased osm level both in the gcf and serum , and the decreased levels after initial periodontal therapy ( srp ) may suggest a use as an inflammatory bio - marker in the periodontal disease . | Aim
Methodology
Results
Conclusion |
interpersonal conflicts are emotionally difficult and require regulation of negative affect and behavior for successful resolution ( arriaga and rusbult , 1998 ; lopes et al . , 2011 ) .
these self - regulatory mechanisms are reliant on cognitive control processes mediated by the lateral prefrontal cortex ( lpfc ; heatherton and wagner , 2011 ; ochsner et al . ,
lpfc dysfunction in cognitive control is a well - established neural impairment in schizophrenia ( barch , 2005 ; manoach , 2003 ; minzenberg et al . , 2009 )
, 2001 ; nishimura et al . , 2011 ; perlstein et al . , 2001 ; van veelen et al . ,
2010 ) and global functioning deficits ( sanz et al . , 2009 ; yoon et al . , 2008 ) .
however , there is a paucity of research examining how lpfc dysfunction contributes to illness severity .
consistent with the diathesis stress model , one proposal is that lpfc dysfunction is a biological vulnerability that , in the presence of an interpersonal stressor , contributes to symptom exacerbation via impaired cognitive control of emotion ( hooker et al . , 2010 ;
cognitive control of emotion comprises the dual processes of explicit and effortful control of the experience and expression of internal emotional states ( emotion regulation ) and the implicit and more automatic control of how external emotional information influences behavior ( gyurak et al .
, 2011 ; gross and thompson , 2007 , ochsner et al . , 2005 ; quirk et al . , 2006 ) .
the lpfc , comprising both dorsolateral ( dlpfc ) and ventrolateral ( vlpfc ) regions , is consistently implicated in laboratory assessed cognitive control of emotion ( ochsner and gross , 2005 ; ochsner et al . , 2012 ; pessoa , 2008 ) , including paradigms assessing explicit emotion regulation ( e.g. reappraisal paradigms ) as well as paradigms assessing implicit cognitive control of external and/or irrelevant emotional information ( e.g. emotional stroop , emotional flanker tasks ) ( gyurak et al . ,
lower vlpfc activity during social exclusion predicts higher self - reported distress ( eisenberger et al . , 2003 ) .
similarly , lower vlpfc activity when viewing negative facial expressions predicts increased negative mood and maladaptive behavior following interpersonal conflicts ( hooker et al . , 2010 ) .
in schizophrenia , interpersonal conflicts , especially conflicts characterized by criticism , predict symptom exacerbation and higher relapse rates ( hooley , 2007 ) , and symptom exacerbation in response to interpersonal criticism is related to poor working memory and cognitive control ( rosenfarb et al . , 2000 ) neurocognitive processes known to be mediated by the lpfc ( aron et al .
, 2004 ; curtis and d'esposito , 2003 ) and to predict functional outcome ( milev et al . , 2005 ) . collectively , these data suggest that compromised lpfc function is a vulnerability for symptom exacerbation and functional difficulties in response to interpersonal conflict .
however , research attempting to connect lpfc activity , interpersonal conflict , and schizophrenia symptomatology is sparse . to date
, studies have primarily focused on the direct relationship between the lpfc and symptoms ( goghari et al . , 2010 ; macdonald et al . , 2005 ; menon et al . , 2001 ; nishimura et al . , 2011 ; perlstein et al . , 2001 ; van veelen et al . ,
, only one study has examined the interaction between lpfc activation , symptoms , and real - world social interactions . in a sample of healthy individuals characterized along the schizophrenia - risk dimension social anhedonia , individuals at high - risk for schizophrenia ( i.e. those with high social anhedonia ) with low lpfc activation had worse symptoms of paranoia on days with distressing interpersonal conflicts compared to days without distressing interpersonal conflicts ( hooker et al . , 2014 ) . to date , no study has examined this relationship in individuals with schizophrenia .
the scarcity of studies directly linking lpfc function to social interactions and subsequent symptoms in schizophrenia may result from limitations inherent in the currently available and commonly used methods .
first , the tasks traditionally used to assess lpfc function , such as response inhibition or working memory tasks ( barch , 2005 ) , although robust activators , may not be the most sensitive measures for assessing how lpfc activity relates to real - world social functioning because they do not directly capture cognitive control of emotional information . given the inherently affective nature of social interactions and the accompanying need for self - regulation ( arriaga and rusbult , 1998 ) , tasks assessing the interaction between lpfc mediated cognitive control and emotional information may provide a more accurate reflection of the inhibitory demands of real - world social contexts . by using
cold cognitive tasks and not assessing lpfc mediated cognitive control in relation to emotional information , previous studies may have lacked the sensitivity necessary to identify the role of the lpfc in individuals ' response to social conflict .
second , prior research has primarily assessed social interactions using laboratory - based one - time retrospective measures of functioning .
although these provide an overview of an individual 's general level of functioning , they are not well suited for capturing the multidimensional nature of social interactions in daily life , and rarely provide the context in which they occur ( trull and ebner - priemer , 2009 ) .
social interactions do not occur in a vacuum ; day - to - day changes in social behavior may be prompted at a specific time , in a specific setting , or in the context of a particular interpersonal relationship .
one - time retrospective evaluations of social functioning miss these nuances , calling into question their ecological validity ( yager and ehmann , 2006 ) .
experience sampling methods ( esm ) , a technique in which assessments are collected in the person 's natural environment and repeated over time , have revealed a nuanced relationship between changes in the social environment , particularly social stressors , and symptoms ( myin - germeys et al . , 2009 ; oorschot et al . , 2009 ) ,
thus , using esm in conjunction with neuroimaging techniques may provide a more sensitive and ecologically valid approach to understanding the contribution of lpfc dysfunction to social deficits in schizophrenia .
the present study addressed these prior limitations by combining fmri and esm to test whether people with schizophrenia have lpfc deficits in cognitive control of negative emotional information , and , if so , whether these lpfc deficits are related to changes in mood and symptoms following interpersonal conflict .
individuals with schizophrenia and demographically - matched healthy controls completed an adapted version of the multi - source interference task ( msit ) , a cognitive control task specifically designed to activate the cingulo frontal
parietal cognitive control network ( bush and shin , 2006 ) . in our adapted version , the msit - emotion ,
msit stimuli are superimposed on a negative emotional scene that is irrelevant to the central task demand .
thus , rather than requiring explicit manipulation of emotional material and/or the explicit regulation of internal emotional states ( i.e. emotion regulation through reappraisal / suppression ) , the task requires participants to engage cognitive control mechanisms to inhibit the effect of irrelevant emotional information in the external environment on task performance .
this process is thought to more accurately reflect the interaction of emotion and cognitive control in real - world social contexts ( silbersweig et al . , 2007 ) , and considered to be a form of implicit cognitive control of emotion ( gyurak et al . , 2011 ) .
our measure of cognitive control of emotional information was lpfc activity when inhibiting the effect of irrelevant emotional information during high interference trials ( when cognitive control skills are most challenged ) .
stimuli were also superimposed on neutral pictures , included to test the specificity of schizophrenia participants ' lpfc deficits for controlling emotional information . following the scan
, schizophrenia participants completed an online , structured daily - diary questionnaire of mood and symptoms every evening for 3 weeks .
end - of - the - day reports provide data on daily events and day - to - day symptom fluctuations whilst minimizing interference with participants ' daily experience .
participants rated the extent to which they experienced mood and schizophrenia - spectrum symptoms , as well as the occurrence of interpersonal conflict and associated distress .
we hypothesized that : 1 ) schizophrenia participants would show reduced lpfc activity during cognitive control of emotional information compared to healthy participants ; and 2 ) among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict .
specifically , we expect that schizophrenia participants with low lpfc activity will have an increase in negative mood and psychotic symptoms the day after highly distressing interpersonal conflict .
23 individuals with schizophrenia or schizoaffective disorder and 24 healthy controls were recruited from the greater boston area .
groups were matched for gender , age , education , and iq ( table 1 ) .
inclusion criteria for all participants were : age 1865 , primary english speaker , no neurological or major medical illness , no head trauma history , no substance abuse within 6 months , and no current / past substance dependence .
inclusion criteria for schizophrenia participants were : diagnosis of schizophrenia or schizoaffective disorder , no comorbid axis i disorders , and no history of electroconvulsive therapy .
inclusion criteria for healthy participants were : no current / past axis i disorders , no first - degree relative with a psychotic disorder , scores within 1.5 standard deviations of the population mean on five measures of schizotypal personality ( perceptual aberration scale ( chapman et al . , 1978 ) , magical ideation scale ( eckblad & chapman , 1983 ) , referential thinking scale ( lenzenweger et al . , 1997 ) , physical anhedonia scale ( chapman et al . , 1976 ) , and revised social anhedonia scale ( eckblad et al . , 1982 ) ) .
psychopathology was assessed with the structured clinical interview for dsm - iv axis i disorders ( first et al . ,
2002 ) ; symptoms were assessed using the positive and negative syndrome scale ( panss ; kay et al . , 1987 ) ; social functioning was assessed with the social adjustment scale self - report ( sas - sr ; weissman et al . , 1978 ) and the global functioning : social scale ( gf : s ; auther et al . , 2006 ) ; positive and negative mood were assessed using the positive and negative affect schedule ( panas ; watson et al . , 1988 ) .
clinical assessments were conducted by trained phd - level clinical psychologists ( lmt , shl ) and supervised by a licensed clinical psychologist ( cih ) .
participants completed behavioral assessments , returned a separate day for the scan , and were subsequently oriented to the daily - diary .
1 ) , an adaptation of the msit , a standard cognitive control paradigm that combines the flanker ( eriksen and eriksen , 1974 ) and simon ( simon and berbaum , 1990 ) effects to robustly activate the cingulo frontal
parietal cognitive control network within individual subjects ( bush and shin , 2006 ) . in the msit , subjects see sets of three numbers ( 1 , 2 , or 3 ) and report the identity of the number that differs from the other two . during control trials ,
position of the target number is always congruent with its position on the button box and the other two distracter numbers are zeros ; thus there is no spatial or semantic interference . during interference trials ,
position of the target number is incongruent with its position on the button box and the distracters are other numbers .
this creates two sources of interference : spatial incongruence between the target and response ( simon effect ) and semantic incongruence between the target and distracter numbers ( flanker effect ) . in the msit - emotion ,
task stimuli ( i.e. the numbers for interference and control trials ) are shown on a background of either a neutral or negative picture , resulting in four conditions : neutral control ( neucon ) , neutral interference ( neuint ) , negative control ( negcon ) , and negative interference ( negint ) .
48 neutral pictures and 48 negative pictures were selected from the international affective picture system ( iaps ; lang et al . , 2005 ; see supplemental methods ) .
trials were presented in an event - related design so that correct and incorrect trials could be analyzed separately .
each trial was presented for 1.75 s followed by a variable inter - trial interval ( central fixation - cross ) for 410 s. pictures were randomly assigned to conditions ; trial types were presented in a fixed , pseudo - random order . before entering the scanner , participants completed a practice version of the task ( 10 trials for each of the 4 conditions ) .
participants were required to achieve 80% accuracy or higher before entering the scanner to ensure task competency ; thus any group differences would not be due to task difficulty / performance differences .
after the scan , participants rated the valence of each neutral and negative picture ( see supplemental methods ) .
images were acquired on a siemens 3 t timtrio scanner and analyzed using spm8 within the general linear model ( glm ) framework . for single - subject glms , vectors of onset times with 0 s duration were defined for each event in each condition ( neucon , neuint , negcon , negint ) and convolved with the canonical hrf .
the data were high - pass filtered ( cut - off period : 128 s ) . artifact detection and movement correction were conducted using the artifact detection tools software package ( art ; whitfield - gabrieli , 2009 ) .
regressors were created to exclude volumes with gross motion ( > 3 mm relative to previous time frame ) or spiking artifacts ( global mean image intensity greater than 3sd from the mean of the entire time series within a scan ) from analysis .
there were no group differences in number of outliers identified ( sz max = 30 ; hc max = 28 ) .
contrasts were calculated for each of the four conditions relative to fixation periods , and for interference versus control for neutral and negative pictures ( i.e. neuint > neucon ; negint > negcon ) .
we tested our central hypothesis that , compared to healthy controls ( hc ) , schizophrenia ( sz ) participants would show reduced lpfc activity during cognitive control of emotional information using a 2 2 2 full factorial anova with group ( hc / sz ) , emotion ( negative / neutral ) and condition ( interference / control ) as factors .
the predicted group emotion condition interaction was that hc would have greater cognitive control related activity ( interference control ) than sz when inhibiting irrelevant negative emotional information on interference trials versus control trials compared to inhibiting irrelevant neutral information on interference versus control trials .
clusters showing the predicted group emotion condition interaction within the lpfc were identified and contrast values for each condition for each subject were extracted from the peak voxel . the difference between cognitive control activation on negative trials and cognitive control activation on neutral trials ( i.e. ( negint neg con ) ( neuint neucon ) ) was calculated and used in the analysis with the daily - diary data .
group maps were thresholded at t = 3.18 , p < 0.001 ( uncorrected ) with 10 voxels/270 mm cluster size .
, 2004 ; maldjian et al . , 2003 ) to create our lpfc region of interest mask , comprising brodmann 's areas ( ba ) , 9 and 46 ( dorsal lpfc ; middle frontal gyri extending into portions of the superior frontal gyri ) and ba 44 , 45 , and 47 ( ventral lpfc ; inferior frontal gyri ) .
activation clusters surviving small volume corrections within this mask ( fwe , p < 0.05 ) are reported . the daily - diary consisted of a structured questionnaire completed online at the end of each day ( i.e. right before bed ) for 21 consecutive days .
participants rated the extent to which they experienced mood and psychotic symptoms , including positive and negative mood , paranoia , hallucinations / odd experiences , negative symptoms , and disorganized symptoms .
questions were rated on a 1-to-5 scale ( 1 = not at all ; 5 = extremely ) .
participants also reported whether they had an interpersonal conflict ( yes / no ) , and if so , the extent to which the conflict caused distress and was resolved .
mood items were selected from the profile of mood states - short form ( poms - sf ; curran et al . , 1995 ) .
diary questions for psychotic symptoms were adapted from previous literature using esm in schizophrenia ( myin - germeys et al .
, 2002 ; myin - germeys and van os , 2007 ; thewissen et al . ,
2008 ) as well as commonly used diagnostic interviews and questionnaires ( e.g. schizotypal personality questionnaire ; psychotic - like experiences scale ) .
consistent with prior esm literature on stress , we used the subjective rating of conflict distress as the measure of social stress .
if more than one conflict occurred , distress ratings were summed . if no conflict occurred that day , distress was coded as 0 .
research staff monitored diary entries daily and sent a reminder email if a day was missed .
participants could not miss more than 6 days.hypothesis 2among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . among schizophrenia participants ,
lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict .
analysis of daily - diary responses and lpfc activity used a hierarchical linear modeling ( hlm ) approach with the mixed procedure in sas ( sas institute , cary , nc ) .
the daily diary has a hierarchical structure in which the 21 daily assessments are nested within participant .
we expected that the relationship between conflict distress and symptom severity within each participant would vary as a function of the between - subject variable , lpfc activity .
thus , we conducted two - level models with the mixed procedure in sas , which allows for the simultaneous analysis of within and between subject variables .
the repeated assessments over 21 days allowed us to examine the longitudinal effect of conflict distress on the change in mood and symptoms the following day .
the main analyses tested whether daily symptom - severity is predicted by the interaction of lpfc activity and the previous day 's conflict distress .
thus , by accounting for previous day symptom - severity , we are able to test the effect of lpfc activity on the change in symptom - severity the day after conflict .
we expected that the interaction of lpfc activity and previous day conflict distress would significantly predict symptom - severity , such that sz participants with low lpfc activity would have an increase in symptoms the day after high conflict distress .
all variables were grand - mean centered . if the interaction between yesterday 's conflict distress and lpfc activity was significant , the effects of the interaction were further examined using simple slope analysis as described by aiken and west ( 1991 ) .
simple slope analyses for high and low groups were tested at 1sd above and below each centered mean . for completeness
, we also report the relationship between symptom severity and : 1 ) lpfc activity ; 2 ) same day conflict distress ; and 3 ) the interaction of same day conflict distress and lpfc activity . test
retest reliability ( i.e. stability of the diary ratings ) was examined by correlating average diary ratings for the first and second halves of the diary period .
internal consistency of diary items in each symptom construct was measured with standardized alpha coefficients .
construct validity was examined by correlating diary variables with corresponding mood and symptom constructs assessed with standard laboratory - based instruments .
one sample t - tests of interference versus control conditions ( i.e. neuint > neucon and negint > negcon ) confirmed that the msit - emotion task activates the expected cognitive control network . both hc and
sz participants demonstrated greater activity for interference versus control conditions in cognitive control regions , including inferior , middle and superior frontal gyri ( lpfc ) , and anterior cingulate cortex ( acc ) ( fig . 2 ; supplementary tables 1 and 2 ) .
all participants responded slower and were less accurate on interference trials compared to control trials ; participants also responded slower on negative picture trials compared to neutral picture trials .
a significant condition emotion interaction in the sz group indicated that individuals with schizophrenia were slower and less accurate on negative interference trials relative to neutral interference trials and negative and neutral control trials .
these data indicate that the negative interference trials , posited to be the most taxing on cognitive control resources , are particularly challenging for sz participants .
compliance was high ; average number of diary days completed was 20.0 ( sd 1.8 ) . on average , participants had 11.6 conflicts over the diary period and conflicts occurred on 28.8% of diary days .
the consistent daily responses and number of conflicts indicate sufficient data to analyze the relationship between conflict and symptoms .
retest reliability shows that diary estimates are stable ( i.e. correlations between the first 10 days and last 11 days had rs > .75 for all variables ) .
the items included in each mood and symptom construct were chosen according to existing literature and a priori face validity .
after the data were collected , standardized alpha of each construct was examined to verify internal consistency , and items that were not correlated with the construct were removed .
this analysis revealed that reverse - coded items were not effective for assessing symptoms and were removed ( i.e. two items were dropped from the negative symptom construct and one item from the disorganized symptom construct ) .
alpha was low for the negative symptom construct ( = .58 ) but acceptable for all other mood and symptom constructs ( > .70 ) .
analysis of construct validity showed that 21-day average daily - diary ratings for negative mood significantly correlated with laboratory measures of trait negative affect ( panas negative affect , r = .55 , p < .05 ) . daily - diary ratings of positive mood were not correlated with panas positive affect ( r = .01 ) ; however , there is no overlap between the panas and poms positive mood items , so a different lab - based assessment of positive mood may be necessary to test construct validity .
daily diary measures of positive , negative , and disorganized symptoms were not correlated with positive and negative symptom scale ( panss ) measures of positive , negative , and disorganized symptoms .
this does not necessarily indicate that the diary measures are inaccurate or invalid , but it does suggest that different factors may be influencing symptom assessments from the diary and the panss .
research comparing multiple methods of assessment may provide more information regarding the validity of different methods .
in addition , there were significant correlations between daily - diary measures of interpersonal conflict and lab - based measure social functioning .
for example , participants with worse social impairment as measured by the social adjustment scale had more interpersonal conflicts over the 21 day diary period ( r = .50 , p = .03 ) and worse conflict distress ( r = .50 , p = .03 ) .
a similar pattern was observed for the global functioning : social scale but the correlations were not significant .
panss measures of interpersonal functioning were also related to daily - diary measures of interpersonal conflict .
higher ratings on panss hostility were associated with more interpersonal conflicts ( r = .58 , p = .008 ) and more conflict distress ( r = .66 , p = .002 ) . a higher degree of panss uncooperativeness was also associated with more interpersonal conflicts ( r = .50 , p = .02 ) and more conflict distress ( r = .69 , p = .001 ) .
together these data indicate that the daily - diary assessments of mood and interpersonal conflict are measuring the intended construct . since prior studies have found a relationship between social stress and symptoms , we conducted simple regression analysis to look at the relationship between conflict distress and symptom severity .
consistent with prior findings , daily ratings of high conflict distress were associated with worse symptom severity , including worse negative mood , paranoia , negative symptoms and disorganized symptoms ( table 3 ) .
we also conducted simple regression analysis to examine the relationship between lpfc activity and symptoms regardless of interpersonal conflict : lpfc activity did not relate to any of the symptom measures .
we used hlm to investigate the interaction of right dlpfc activity with conflict distress on the same day of the conflict ( i.e. same day conflict distress ) .
no significant interactions were found ; that is , on the same day as an interpersonal conflict , individuals ' dlpfc activation during cognitive control of emotion did not interact with conflict distress in predicting symptom severity that day ( table 3 ) .
analysis revealed a single cluster of activation in our lpfc region of interest mask , in the right superior frontal gyrus ( ba9 ; dlpfc ) , with a significant group emotion condition interaction(hc > sz ) that survived small volume correction ( fwe p < 0.05 ; see fig .
3 barplots illustrate that hc participants consistently demonstrate the expected pattern of activation in the right dlpfc : increased activation in negint compared to negcon conditions ; sz participants show the opposite pattern , deactivating in negint compared to negcon conditions .
activation clusters with significant group emotion condition interaction(hc >
sz ) were also present in the right lateral orbital frontal gyrus extending into the insula ( lofg ; ba11 ) and the left dorsal anterior cingulate ( dacc ; ba32 ) , but these did not survive small volume correction in either the lpfc region of interest mask ( i.e. the lofg cluster ) or an anterior cingulate mask comprising ba24 and ba32 ( see supplemental table 3 ) .
no regions showed an interaction in the opposite direction ( i.e. sz > hc ) .
the cluster in the right superior frontal gyrus ( dlpfc ; ba 9 , peak : x = 27 , y = 53 , z = 40 ) was the only cluster that survived multiple comparison correction within our lpfc region of interest mask . all subsequent analyses investigating lpfc activity and diary data were conducted using the difference score ( negint negcon ) ( neuint neucon ) , i.e. our measure of cognitive control of emotional information , calculated from the contrast values in each condition extracted from the peak of this cluster in the right dlpfc.hypothesis 2among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . among schizophrenia participants ,
lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict .
we used hlm to examine the interaction of dlpfc activity with conflict distress from the previous day ( i.e. previous day conflict distress ) in predicting symptom severity on days following interpersonal conflicts .
dlpfc activity significantly interacted with previous day conflict distress in predicting change in positive mood [ f = 8.03 , b = 0.02 , p = 0.01 ] and negative mood [ f = 4.49 , b = 0.01 , p = 0.047 ] the following day .
4 . the interaction of previous day conflict distress and dlpfc activity did not significantly predict any other symptom ( see table 3 ) .
simple slope analyses were conducted to better understand the interactions predicting positive and negative mood .
analysis of effects influencing positive mood showed the following : the effect of dlpfc activity on positive mood was examined separately for days when previous day conflict distress was low and days when previous day conflict distress was high . on days when previous day conflict distress was low
0.02 ) , t(19 ) = 1.51 , p = 0.30 ] ( blue line plotted in fig .
however , when previous day conflict distress was high , dlpfc activity significantly predicted positive mood the following day [ b = 0.07(s.e .
0.02 ) , t(19 ) = 3.1 , p = 0.006 ] , such that schizophrenia participants with higher dlpfc activity had higher positive mood compared to participants with lower dlpfc activity ( red line plotted in fig .
next we examined the effect of conflict distress on positive mood for schizophrenia participants with high dlpfc activity and low dlpfc activity . for schizophrenia participants with low dlpfc activity ,
previous day conflict distress was not related to positive mood [ b = 0.02(s.e .
0.02 ) , t(19 ) = 1.0 , p = 0.35 ] . however , for schizophrenia participants with high dlpfc activity , previous day conflict distress had a significant effect on positive mood the next day [ b = 0.07(s.e .
0.02 ) , t(19 ) = 3.6 , p = 0.002 ] , such that positive mood was higher on days following high conflict distress .
these results indicate that , among schizophrenia participants with high dlpfc activity , positive mood is significantly higher on days preceded by high conflict distress as compared to days preceded by low conflict distress .
no relationship to positive mood was found among individuals with low dlpfc activity , regardless of level of previous day conflict distress .
analysis of effects influencing negative mood showed the following : on days when previous day conflict distress was low , dlpfc activity had no relationship to negative mood [ b = 0.02(s.e .
0.02 ) , t(19 ) = 1.54 , p = 0.14 ] ( blue line plotted in fig .
4b ) . similarly , on days when previous day conflict distress was high , dlpfc activity had no significant influence on negative mood [ b = 0.02(s.e .
0.01 ) , t(19 ) = 1.52 , p = 0.15 ] ( red line plotted in fig .
we then examined the effect of previous day conflict distress for schizophrenia participants with high and low dlpfc activities . for schizophrenia participants with low dlpfc activity ,
previous day conflict distress was a trend - level significant predictor of the change in negative mood the next day [ b = 0.03(s.e .
0.01 ) , t(19 ) = 2.0 , p = 0.06 ] , such that when previous day conflict distress was high , participants with low dlpfc activity had worse negative mood the next day .
however , for schizophrenia participants with high dlpfc activity , previous day conflict distress was not related to the change in negative mood the next day [ b = 0.02(s.e .
0.01 ) , t(19 ) = 1.2 , p = 0.26 ] .
these results indicate that , among schizophrenia participants with low dlpfc activity , negative mood is significantly higher on days preceded by high conflict distress as compared to days preceded by low conflict distress .
no relationship to negative mood was found among individuals with high dlpfc activity , regardless of level of previous day conflict distress .
this study combines fmri and experience sampling methods to investigate whether people with schizophrenia have lpfc deficits in the cognitive control of emotional information , and , if so , whether these lpfc deficits prospectively predict the influence of real - world interpersonal conflict on changes in mood and psychotic symptoms . two key findings emerged .
first , analysis of the msit - emotion task revealed a group emotion condition interaction in the right dlpfc ( superior frontal gyrus , ba9 ) .
inspection of activity in this region indicates that , compared to healthy participants , individuals with schizophrenia had lower dlpfc activity during cognitive control of task - irrelevant negative emotional information .
this is consistent with prior literature demonstrating that schizophrenia is associated with lpfc deficits in cognitive control of emotion ( ursu et al . , 2011 ; vercammen et al . , 2012
, schizophrenia participants showed deficient neural activity in the right dlpfc when controlling emotional information on trials with the greatest cognitive demand ( i.e. negative interference trials ) suggesting that schizophrenia participants ' ability to control the influence of irrelevant negative emotional information on task performance is particularly impacted during high cognitive load tasks .
this is consistent with research demonstrating a relationship between dlpfc dysfunction and cognitive load in non - emotional cognitive control tasks ( callicott et al . , 2003 ) , and
further indicates that tasks probing cognition emotion interactions may be a more sensitive tool for understanding the role of emotion processing in the pathophysiology of schizophrenia .
second , among schizophrenia participants , the interaction of right dlpfc activity during cognitive control of emotional information and the previous day 's conflict distress predicted changes in positive and negative mood the following day
. specifically , analysis of positive mood shows that schizophrenia participants with higher dlpfc activity had better positive mood the day after high conflict distress . however , dlpfc activity had no influence on days following low conflict distress , and for schizophrenia participants with low dlpfc activity , positive mood was no different on days following high or low conflict distress .
thus , higher dlpfc activity appears to convey a boost to positive mood following an interpersonal conflict , but only when that conflict is experienced as highly distressing .
conversely , analysis of negative mood showed that schizophrenia participants with lower dlpfc activity had worse negative mood when the previous day 's conflict distress was high .
however , on days when previous day conflict distress was low , dlpfc activation had no relationship to negative mood .
thus , lower dlpfc activity appears to contribute to an exacerbation of negative mood following an interpersonal conflict , but only when that conflict is experienced as highly distressing .
these data provide the first evidence tying lpfc dysfunction in schizophrenia to daily ratings of mood following real - world social interactions , and have substantive implications for understanding the specific role of lpfc in response to social stress .
first , our finding that low dlpfc activity during cognitive control of negative emotional information predicts increased negative mood on days following highly distressing interpersonal conflicts is consistent with our previous finding in healthy individuals ( hooker et al . , 2010 ) , and prior literature demonstrating reduced lpfc engagement in response to social stress predicts higher self - reported social distress ( eisenberger et al . , 2003 ; eisenberger et al . , 2007 ; hooker et al . , 2010 )
together , these data support the well - documented proposal that lpfc control - related mechanisms act to down - regulate negative affect / mood ( kohn et al .
, 2014 ; ochsner and gross , 2005 ) , and suggest that adaptive response to social stressors requires lpfc control - related functions to down - regulate negative emotional information .
second , our finding that individuals with high dlpfc activity show an increase in their positive mood the day after highly distressing interpersonal conflicts is particularly interesting , especially given that the paradigm we used to probe lpfc - mediated cognitive control focused on control of negative emotional information .
this finding supports the proposal that lpfc - mediated control mechanisms are also important in the up - regulation of positive mood ( kim and hamann , 2007 ; mak et al . , 2009 )
even in the context of incidental / automatic cognitive control of emotional information , and replicates parallel findings in our previous study ( hooker et al . , 2010 ) .
in the context of explicit emotion regulation , one explanation of this finding is that people with high lpfc control - related activation use more effective cognitive strategies to reevaluate distressing interpersonal conflicts and reframe them in a positive light , possibly by thinking about what they have learned from the interpersonal conflict and how it makes them stronger / better ( i.e. positive reappraisal ) . in line with this interpretation
, the use of positive reappraisal is related to increased experience and expression of positive emotions ( gross and john , 2003 ) , and has consistently been shown to predict well - being ( helgeson et al . , 2006 ) and promote an upward - spiral of adaptive responding to negative events ( fredrickson , 2004 ) .
interestingly , several studies examining explicit emotion regulation in mood psychopathology ( e.g. depression , bipolar disorder ) have not found strong group differences ( e.g. dillon and pizzagalli , 2013 ; ehring et al . , 2010 ; gruber et al . , 2014 ) .
this suggests that , when given clear instructions , individuals with emotion regulation difficulties can effectively engage emotion control mechanisms , but that in the absence of such clear instructions as is typically the case in daily life they struggle to regulate emotional responses .
thus , using the msit - emotion to probe incidental / automatic lpfc - mediated emotion control processes may have enhanced the probability of finding group differences and detecting a relationship between lpfc activity and daily social interactions . finally , the results are in line with the diathesis stress model of neural mechanisms of social stress ( e.g. dillon and pizzagalli , 2013 ; ehring et al . , 2010 ; gruber et al . , 2014 ) and have implications for the development of treatment interventions .
low or compromised lpfc function may be a vulnerability that contributes to the increased negative impact of highly distressing interpersonal conflicts via impaired cognitive control of emotion .
conversely , high or intact lpfc function may be protective against the negative impact of interpersonal conflicts , and appear to convey a benefit in the form of increased positive mood . given that greater dlpfc engagement during our laboratory - based measure of control of emotional information predicted increased positive mood the day after a distressing interpersonal conflict , interventions that aim to improve lpfc function and/or cognitive control of emotional information may also improve real - world forms of emotion control , and consequently , adaptive response to social conflict .
consistent with this proposal , normalization / improvement of lpfc function , either in response to medication ( garnefski and kraaij , 2006 ; zlomke and hahn , 2010 ) , cognitive therapy ( heller et al . , 2013 ) , or computerized cognitive training ( edwards et al . ,
2010 ) , is associated with improved symptoms in depression , psychosis , and global functioning respectively .
this study is also consistent with mounting consensus that ecological measures of daily life can be combined with neuroimaging data to meaningfully connect neural indicators of psychological processes to real - world behavior ( subramaniam et al . , 2014 ) . in healthy individuals ,
lpfc activity predicts daily levels of social support ( berkman and lieberman , 2011 ) , successful smoking cessation ( eisenberger et al . , 2007 ) , and maladaptive behavior following a conflict with a partner ( berkman et al . , 2011 ) .
our findings extend this brain - as - predictor ( hooker et al . , 2010 ) approach to understanding the consequences of well - established lpfc deficits in schizophrenia in the context of social stress ( i.e. interpersonal conflict ) .
using a task that specifically assessed the interaction between cognitive control and emotion processing likely increased sensitivity to this brain behavior relationship , and provides further support for the proposal that the consequences of cognitive and emotion processing deficits in schizophrenia may be better understood in the context of cognition emotion interactions ( berkman et al . , 2011 ) .
by using these methods together , this study demonstrates the value of the brain - as - predictor approach to research attempting to delineate the mechanisms that contribute to the development and persistence of problematic mood and symptoms in schizophrenia .
first , mood and symptom measures on the daily - diary were self - report and thus subject to bias .
second , we designed the msit - emotion with the assumption that in order to meet task demands participants must engage cognitive control mechanisms to inhibit the influence of the negative picture on performance .
it is possible , however , that participants could use an alternative , non - control related strategy to counteract the influence of the negative picture ( e.g. squint their eyes to blur the picture and focus on the numerical stimuli ) .
future investigations should use a paradigm that precludes the use of such alternative strategies by incorporating the emotional stimuli into the response pattern of the task , for example , the face - emotion stroop ( kring and elis , 2013 ; pessoa , 2008 ) .
third , based on our previous findings regarding predictive value of lpfc activity ( etkin et al .
, 2006 ) , we chose to focus specifically on the role of the lpfc in social behavior .
next steps should investigate the contribution of other regions involved in cognitive control of emotional information ( e.g. acc , amygdala ) and cognitive control network connectivity ( e.g. cingulo fronto parietal connectivity ) to mood changes in response to social stress . in sum , this study integrates fmri and experience sampling methods to demonstrate a direct link between lpfc activation during cognitive control of emotional information and response to real - world social conflict in schizophrenia .
results indicate that lpfc activity during a laboratory - based measure of cognitive control of emotional information predicts changes in positive and negative mood on days following highly distressing interpersonal conflicts .
these findings suggest that treatment interventions targeting lpfc mechanisms of cognitive control of emotion could promote adaptive responding to real - world social conflict in schizophrenia .
this work was supported by harvard university research funds to christine i. hooker and a sackler scholar fellowship from the sackler scholar program in psychobiology to laura m. tully . | lpfc dysfunction is a well - established neural impairment in schizophrenia and is associated with worse symptoms .
however , how lpfc activation influences symptoms is unclear .
previous findings in healthy individuals demonstrate that lateral prefrontal cortex ( lpfc ) activation during cognitive control of emotional information predicts mood and behavior in response to interpersonal conflict , thus impairments in these processes may contribute to symptom exacerbation in schizophrenia .
we investigated whether schizophrenia participants show lpfc deficits during cognitive control of emotional information , and whether these lpfc deficits prospectively predict changes in mood and symptoms following real - world interpersonal conflict . during fmri
, 23 individuals with schizophrenia or schizoaffective disorder and 24 healthy controls completed the multi - source interference task superimposed on neutral and negative pictures .
afterwards , schizophrenia participants completed a 21-day online daily - diary in which they rated the extent to which they experienced mood and schizophrenia - spectrum symptoms , as well as the occurrence and response to interpersonal conflict .
schizophrenia participants had lower dorsal lpfc activity ( ba9 ) during cognitive control of task - irrelevant negative emotional information . within schizophrenia participants ,
dlpfc activity during cognitive control of emotional information predicted changes in positive and negative mood on days following highly distressing interpersonal conflicts .
results have implications for understanding the specific role of lpfc in response to social stress in schizophrenia , and suggest that treatments targeting lpfc - mediated cognitive control of emotion could promote adaptive response to social stress in schizophrenia . | Introduction
Material and methods
Results
Discussion
Funding
Supplementary data |
it is currently accepted that about 510% of ovarian cancers and 25% of breast cancers have a hereditary background arising in brca1/2 genes germline mutation carriers .
because of high costs and cumbersome procedure the mutation testing is offered to patients in whom family history indicates high probability of finding the mutation .
families with the increased risk of carrying a mutation can be identified by the large number of breast and ovarian cancer cases occurring among family relatives . however , some families with detected mutation have negative family history or limited family structure [ 14 ] .
the purpose of our study was to establish the frequency and spectrum of brca1 gene mutations in polish consecutive breast and ovarian cancer patients in two groups : with positive and negative family history of brca1-related cancers .
our choice of the four brca1 mutations : 5382insc ( c.5266dupc ) , 300t > g
( p.181t > g ) , 185delag ( c.68_69delag ) , and 3819del5 ( c.3700_3704del5 ) analysed in the present study reflects their high prevalence among polish breast and/or ovarian cancer families and is based on the results of previous research . as a strong founder effect
is noted in poland , these four brca1 mutations comprise 7090% of all brca1 pathogenic alterations [ 612 ] .
consecutive , newly - diagnosed female breast cancer cases were collected without regard to age or family history of breast and ovarian cancer in two provinces , malopolska and mazowsze , between 2003 and 2005 .
consecutive invasive epithelial ovarian cancer cases were collected independently of age and family history in pomorze and malopolska between 1995 and 2005 .
the patients enrolled in the study were invited to provide blood samples and to complete a questionnaire including information on breast and ovarian cancers in first- and second - degree relatives regardless of their age .
family history was regarded as positive when , apart from the patient , at least one first- or second - degree relative suffered from breast or ovarian cancer regardless of their age .
( p.181t > g ) , 185delag ( c.68_69delag ) , and 3819del5 ( c.3700_3704del5 ) mutations of brca1 gene was performed on groups of 1,845 breast and 363 ovarian cancer patients .
mutation analyses were done using a combination of different screening methods ( restriction fragment length polymorphism
sscp , denaturing high performance liquid chromatography dhplc , allele - specific pcr - asa - pcr ) followed by bidirectional sequencing of samples with abnormal patterns .
comparisons between groups for statistical significance were performed by the use of and fisher s exact tests , as appropriate .
in the group of 1,845 unselected breast cancers collected in two polish provinces , malopolska and mazowsze , the most frequent mutation was 5382insc ( 35/1845 , 1.9% ) followed by 300t > g ( 18/1486 , 1.2% ) .
we observed high frequency of negative family history in the group of carriers . in 21 out of 55 ( 38% )
brca1-positive breast cancer cases no case of breast and/or ovarian cancer was observed in first- or second - degree relatives ( table 1).table 1brca1 gene mutations in consecutive breast cancer cases unselected for age and family history of breast and/or ovarian cancer and in those selected with negative family history from two polish provincesnumber of breast cancer cases with brca1 mutation ( frequency % ) malopolskamazowszeunselectedselected*unselectedselected*185delag0/7550/5412/705 ( 0.28)0/453300t > g9/787 ( 1.14)3/573 ( 0.52)9/699 ( 1.29)5/449 ( 1.11)3819del50/5540/3410/6880/4485382insc9/787 ( 1.14)4/573 ( 0.70)26/1058 ( 2.46)9/775 ( 1.16)cumulative frequency of four mutations1/44 ( 2.3)1/182 ( 0.55)1/25 ( 4.3)1/44 ( 2.27 ) * with negative family history for breast and/or ovarian cancer in i or ii degree relatives brca1 gene mutations in consecutive breast cancer cases unselected for age and family history of breast and/or ovarian cancer and in those selected with negative family history from two polish provinces * with negative family history for breast and/or ovarian cancer in i or ii degree relatives in the group of 363 unselected ovarian cancers analysed in two provinces , malopolska and pomorze , the most prevalent molecular change was 5382insc mutation ( 17/363 , 4.7% ) , followed by 300t > g ( 12/363 , 3.3% ) . 19 out of 37 ( 51% ) of brca1-positive patients from these provinces had negative family history of breast and/or ovarian cancer among first- and second - degree relatives ( table 2 ) .
we also observed the difference in frequency of positive family history in brca1-positive patient groups depending on the type of the mutation .
negative family history was more frequent in ovarian cancer patients with 300t > g missense brca1 mutation than in truncating 5382insc mutation carriers ( p = 0.0025 ) .
g - carriers had no first- or second - degree relative with breast and/or ovarian cancers whereas in the group of 17 brca1 5382insc - carriers only four had negative family history.table 2brca1 gene mutations in consecutive ovarian cancer cases unselected for age and family history of breast and/or ovarian cancer and with negative family history from two polish provincesnumber of ovarian cancer cases with brca1 mutation ( frequency % ) malopolskapomorzeunselectedselected*unselectedselected*185delag2/205 ( 0.97)2/162 ( 1.23)1/151 ( 0.66)0/96300t > g11/212 ( 5.19)9/169 ( 5.33)1/151 ( 0.66)1/96 ( 1.04)3819del51/160 ( 0.63)1/117 ( 0.85)4/151 ( 2.65)2/96 ( 2.08)5382insc11/212 ( 5.19)3/169 ( 7.78)6/151 ( 3.98)1/96 ( 1.04)cumulative frequency of four mutations1/8 ( 12.5)1/11 ( 9.09)1/13 ( 7.69)1/24 ( 4.17 ) * with negative family history for breast and/or ovarian cancer in i or ii degree relatives brca1 gene mutations in consecutive ovarian cancer cases unselected for age and family history of breast and/or ovarian cancer and with negative family history from two polish provinces * with negative family history for breast and/or ovarian cancer in i or ii degree relatives
many centres undertook the screening programme to estimate the prevalence of brca1 mutations in breast cancer cases unselected for age and family history of cancer .
these investigations allowed us to evaluate the frequency of brca1/2-positive breast cancer cases at 25% [ 2 , 1318 ] .
as this frequency is not sufficient to offer molecular testing to each breast cancer patient , in most genetic centres qualification for genetic testing is based on family history of cancer . in our study
, almost 40% of brca1-positive breast cancers could be classified according to family history as not - familial .
the great fraction of brca1-positive breast cancers without family history described in our study confirms previous observations reported in polish and other populations [ 2 , 6 , 18 ] .
the computer probabilistic models based on family history of breast and ovarian cancers are a useful tool for calculating brca1/2 mutation probabilities but , for some small families with the mutation , will assign insufficient mutation probability .
misassignment of carriers to a test group can be a consequence of incomplete family information regarding ancestry , small family size or transmission through males .
the previous data indicate inaccuracy in carrier prediction using computer models for families with a single breast cancer diagnosis [ 1 , 19 ] .
the high frequency of mutations detected in probants lacking obvious family history indicates the necessity to provide wide criteria for qualifying breast cancer women for genetic testing including pathological ( high grade , medullary histology , negative oestrogen and progesterone receptors , and her-2/neu status ) and clinical ( age , mammography ) parameters of patients [ 2022 ] . in this study
, we revealed that , in polish population , 51% of brca1-positive patients with ovarian cancer had negative family history of breast and/or ovarian cancer among i and ii degree relatives . in the study provided by risch et al .
the rate of brca1-positive ovarian cancers with negative family history was lower ( 19/75 , 25% ) .
this discrepancy can be explained by different familial criteria and spectrum of mutation in these two studies . as the brca1 mutation can be detected in 1015% of unselected ovarian cancer cases ,
positive family history is not necessary to qualify the patient for genetic tests [ 3 , 23 ] .
this observation indicates that , even in ovarian cancer cases with unknown or limited family structure , genetic tests should be offered .
we observed that in polish population negative family history is more frequent in the group of ovarian cancer patients with 300t > g brca1 mutation than in 5382insc carriers .
this finding indicates the necessity to search 300t > g mutation in families with a single diagnosis of ovarian cancer in family member including first- and second - relatives . | it is estimated that about 510% of ovarian and 25% of all breast cancer patients are carriers of a germline brca1 or brca2 gene mutation .
most families with detected brca1 or brca2 gene mutation are qualified for molecular testing on the basis of family history of breast or ovarian cancers .
the purpose of our study was to establish the frequency of positive family history of cancer in a series of polish consecutive breast and ovarian cancer patients in two groups , with and without the brca1 gene mutations .
we analysed the prevalence of four of the most common brca1 mutations : 5382insc ( c.5266dupc ) , 300t > g
( p.181t > g ) , 185delag ( c.68_69delag ) and 3819del5 ( c.3700_3704del5 ) . the patient group consisted of 1,845 consecutive female breast and 363 ovarian cancer cases .
19 out of 37 ( 51% ) of brca1-positive ovarian cancer patients and 21 out of 55 ( 39% ) brca1-positive breast cancer had negative family history of breast and/or ovarian cancer among first- and second - degree relatives .
in ovarian cancer patients , negative family history was more frequent in those with 300t > g brca1 gene mutation than in 5382insc carriers .
this finding indicates the necessity of searching for 300t > g mutation in families with a single diagnosis of ovarian cancer in family .
the high frequency of mutations detected in breast cancer patients lacking obvious family history shows that breast cancer patients should be qualified for genetic testing on the basis of wide clinical and pathological criteria . | Introduction
Subjects and methods
Results
Discussion |
s6 kinases are members
of the agc serine / threonine kinases of the
rsk family , exhibit high homology within their catalytic domain , and
are activated by the phosphorylation of a critical residue within
the activation loop by phosphoinositide dependent kinase 1 ( pdk1 ) .
yeast contains one s6 kinase called sch9 , and humans contain two isoforms
called s6k1 and s6k2 .
s6 kinases act downstream of phosphatidylinositol
( 3,4,5)-triphosphate ( pip3 ) in the phosphatidylinositide
3-kinase ( pi3k ) pathway .
phosphorylation of serine and threonine residues
in the c - terminal regulatory domain leads to the phosphorylation of
a s6k activation loop residue by pdk1 ( residue 252 on the longer splice
variant of s6k1 ) .
in addition to pdk1 ,
mtor is also involved in the activation of s6k1 and phosphorylates s6k1 at residue t412 .
s6 kinases
are associated with many cellular processes , including
protein synthesis , mrna processing , cell growth , and cell survival .
s6k1 and s6k2 phosphorylate and activate the 40s ribosomal protein
s6 , which promotes protein synthesis through an increased rate of
mrna transcription .
s6k1 also regulates
cell size and progression through the cell cycle , in addition
to promoting cell survival by inactivating the proapoptotic protein
bad .
the aberrant activation of
s6 kinases has been shown to play a
role in many disease conditions , including diabetes , obesity , aging ,
and cancer .
many melanoma cells harbor constitutive activation
of the pi3k - akt pathway , which results in akt phosphorylation and
leads to activation of the downstream targets mtor and s6k1 .
treatment with rapamycin ,
an allosteric mtor inhibitor , leads to significant dephosphorylation
of s6k1 and decreased cell growth
. however ,
treatment with mtor inhibitors abrogates feedback inhibition of other
pathways , which in part leads to side
effects such as hyperglycemia , hypercholesterolemia , and hyperlipidemia .
because of this , inhibition of s6k1 represents
an alternative therapeutic strategy that may bypass the limitations
of mtor inhibition .
we have previously reported on the development
of atp competitive
organometallic kinase inhibitors with high potency and specificity .
these inhibitors are structurally inspired by the class of indolocarbazole
alkaloids , such as staurosporine , but use a transition metal ion that
coordinates up to six ligands to replace the carbohydrate moiety of
staurosporine .
the scaffold design includes
a bidentate ligand that is able to target the metal complexes to the
atp - binding site .
this mimics atp and conventional indolocarbazole
inhibitors , while the increased size of the bulky transition metal
complex allows for exploration of additional chemical space at the
edges of the atp binding site specific to each kinase . despite being
conventional atp - competitive inhibitors , the combination of unusual
globular shape and rigid characteristic of these complexes
it is worth
noting that the coordinative bonds to the transition metal are considered
to be kinetically stable and are expected to remain intact when exposed
to the biological environment , thus avoiding metal - related cytotoxicities .
however , druglikeness of such complexes , including metabolic stability ,
bioavailability , and pharmacokinetic properties , is not established
yet and is subject to current studies .
regardless , this strategy has
led to the development of specific and potent kinase inhibitors for
gsk3 , pim1 , pi3k , mst1 , and braf . here ,
we present data on the development of potent and specific
organometallic s6k1 inhibitors , em5 and fl772 .
we show that fl772
binds to s6k1 with an ic50 value in the single digit nanomolar
range at 100 m atp and that the more potent fl772 compound
has a greater than 100-fold specificity over s6k2 .
crystal structures
of the s6k1 domain bound to the pan - kinase inhibitor staurosporine ,
em5 , and fl772 reveal that the organometallic inhibitors bind in the
atp binding pocket in a way that is distinct from staurosporine , likely
explaining their more favorable potency and selectivity .
cellular
data demonstrate that fl772 is able to inhibit s6k phosphorylation
in yeast cells .
the data provide an important starting point for the
development of s6k inhibitors for possible therapeutic applications .
inhibitors
for s6k1 were initially identified through millipore kinaseprofiler
( supporting table 9 ) .
ten different staurosporine - inspired
organometallic ruthenium complexes were screened against a diverse
panel of 283 protein kinases .
this screen led to the identification
of em5 as a potential inhibitor of s6k1 , with 7% activity at a concentration
of 100 nm in the presence of 10 m atp .
em6 , a similar complex
replacing the isothiocyanate functional group with an isocyanate ( figure 1a ) , inhibited significantly less , exhibiting 54%
activity under the same conditions . in the kinase panel ,
the em5 inhibitor
inhibited only 41 kinases ( 16% ) to less than 10% activity , including
s6k1 and the related s6k family members rsk1 , rsk2 , rsk3 , and rsk4 . first
generation organometallic ruthenium inhibitors exhibit potency
and specificity for s6k1 .
( a ) em5 and em6 , two organometallic ruthenium
compounds , use a similar structure to mimic the pan - kinase inhibitor ,
staurosporine .
( b ) a radioactive kinase assay was used to determine
the activity of five different protein constructs of s6k1 .
( c ) staurosporine
( dotted line ) , em5 , and em6 were assayed against s6k1(1421 ,
t412e ) pdk1 activated in a radioactive kinase assay at 100 m
atp .
a radioactive kinase assay was used to determine the activity
of
s6k1 protein constructs prepared in baculovirus - infected insect cells
in order to identify a construct that would be suitable for inhibitor
testing .
initial tests showed that the full - length i isoform
of s6k1 ( s6k(1525 ) ) and the isolated kinase domain ( s6k(84384 ) )
had low kinase activity , although the full - length kinase showed more
activity than the kinase domain ( figure 1b ) .
we reasoned that the s6k1 protein constructs had low kinase activity
because the full - length kinase contained the c - terminal autoinhibitory
domain . to address this issue and express a more active kinase for
further inhibitor studies
, we prepared a s6k1(1421 ) construct
including both the t252 and t412 phosphorylation sites , based on previous
data from keshwani et al . indicating
that the catalytic domain of the s6k1 aii isoform ( residues 1398 )
analogous to s6k1(1421 ) of the i isoform was highly
expressed in insect cells . to further enhance the catalytic activity
of s6k1(1421 ) , we prepared the t412e mutant to mimic phosphorylation
at this position and coexpressed the protein with pdk1 to promote
phosphorylation of t252 .
preparation of the s6k1(1421 , t412e ,
pdk1 activated ) protein resulted in highly active kinase that was
suitable for inhibition studies in vitro ( figure 1b ) . both em5 and em6 were assayed against s6k1(1421 ,
t412e ,
pdk1 activated ) in a radioactive kinase assay and determined to have
ic50 values of 33.9 nm and 23.5 m , respectively ,
at 100 m atp ( figure 1c ) .
for comparison ,
we also determined the ic50 of the nonspecific kinase inhibitor
staurosporine , which had an ic50 value of 64.1 nm under
the same conditions .
given the apparent specificity and potency of
em5 , it became the lead structure for the development of second - generation
organometallic s6k1 inhibitors .
our initial
attempts to cocrystallize the s6k1 kinase domain ( s6k1kd ,
residues 84384 ) bound to em5 using several factorial screens
were unsuccessful .
however , we were able to reproduce the crystals
reported by sunami et al . of the s6k1
kinase domain in complex with staurosporine .
we then soaked these
crystals with high concentrations of the em5 inhibitor in the hope
of exchanging em5 for staurosporine in the crystals .
the em5-soaked
crystals diffracted to about 2.5 resolution and formed in space
group p21 with two molecules per asymmetric
unit .
the structure was refined to rwork and rfree values of 19.15% and 22.21% ,
respectively , with excellent geometry ( table 1 ) . during the refinement process
similar
to the previously published structures of the s6k1 kinase
domain , the kinase domain is bilobal , consisting
of an n - lobe composed largely of -sheet and a c - lobe that is
mostly -helical .
the crystal structure revealed that one protein
molecule in the asymmetric unit was bound to staurosporine , while
the other molecule was bound to em5 in the same atp binding site .
fc difference peak corresponding to the ruthenium
atom in the atp binding sites of one of the molecules before the inhibitor
models were built into the electron density map ( figure 2a ) .
the staurosporine and em5-bound molecules in the asymmetric
units are similar to each other with an overall rmsd of 0.68
for the shared atoms .
( a ) the electron
density
map of em5 ( contoured at 12 ) corresponds to the ruthenium atom
of the inhibitor .
hydrogen bonds are shown as a dashed line , while other interacting
residues are labeled .
( d ) notable
changes between the em5 and staurosporine bound structures are shown .
this includes a change to the activation loop due to em5 interactions
with g100 and v105 and a shortening of two turns to the d helix
resulting from an interaction with e179 .
( e ) close - up around the c
helix , which is more ordered in the em5-bound structure because of
an interaction between f237 and l147 , and a hydrogen bond between
k123 and e143 .
although both staurosporine
and em5 bind in the atp binding pocket ,
the more elaborate em5 compound makes more extensive interactions ,
correlating with its greater s6k1 potency than staurosporine .
staurosporine
forms hydrogen bonds to s6k1 through the backbone oxygen of glu-222
of the kinase with the nitrogen of the methylamine of staurosporine ,
and the backbone nitrogen of leu-175 and backbone oxygen of glu-173
of the kinase hinge region contact the pyrrolidone oxygen and nitrogen
of staurosporine , respectively .
the ring system of staurosporine also
makes van der waals contacts to leu-97 , lys-99 , gly-98 , val-105 , ala-121 ,
tyr-174 , glu-179 , and met-225 ( figure 2b ) .
the em5 compound retains two hydrogen bonds between the backbone
atoms of the hinge residues ( glu-173 and leu-175 ) and the maleimide
ring of em5 and all of the van der waals interactions with the em5
ring system ( figure 2c ) .
however , in addition
to these contacts , em5 makes additional protein interactions between
the ruthenium coordination sphere and the protein .
in particular ,
the additional isothiocyanate group of em5 makes van der waals interactions
with gly-100 and val-105 of the kinase p - loop while the trithiacyclononane
ligand makes van der waals contacts to gly-100 of the p - loop and glu-179
and glu-222 across from the p - loop where the protein substrate is
likely to bind and thr-235 and asp-236 of the activation loop .
notably ,
each of these residues ( glu-179 , glu-222 , thr-235 , and asp-236 ) undergoes
a dramatic movement toward the em5 inhibitor relative to their positions
in the staurosporine complex ( figure 2d ) .
the
binding of em5 to s6k1 also introduces significant structural changes
in the kinase relative to the staurosporine complex , and these structural
changes appear to be indirectly caused by the 1,4,7-trithiacyclononane
ligand of the em5 inhibitor .
the d helix of the staurosporine
complex is about two turns longer at its n - terminus than the corresponding
helix of the em5 complex where the corresponding segment takes on
a -strand conformation .
this structural difference appears
to be driven by the interaction of the tridentate ligand of em5 with
glu-179 . on the opposite side of the inhibitor
, the staurosporine
complex
contains an activation loop that is folded toward the atp active site
in an inactive conformation and does not have an ordered c
helix , as previously reported .
strikingly ,
the em5 complex contains a well - defined c helix of about two
turns .
the difference in disposition of the c helix in the
two structures appears to be nucleated around the n - terminal region
of the activation loop that undergoes about a 6 movement toward
the em5 inhibitor relative to staurosporine .
the movement of the activation
segment toward the em5 inhibitor appears to be mediated by the van
der waals interactions that are made between thr-235 and asp-236 with
the trithiacyclononane ligand of em5 ( figure 2d ) .
this in turn provides enough room for the c helix to form
and to be stabilized by van der waals interactions between phe-237
of the activation loop and leu-147 of the c helix and a hydrogen
bonding between lys-123 of the small domain and glu-143 of the c
helix ( figure 2e ) .
interestingly , these interactions
are characteristic of the active conformations of kinases , even though
the activation segment is in an inactive conformation .
in contrast ,
the more out conformation of the activation loop of the staurosporine
structure places phe-237 and asp-236 in positions that sterically
occlude formation of the c helix ( figure 2d ) . taken together , while staurosporine binding to s6k1 places it
in the inactive conformation , the s6k1/em5 complex has characteristics
of both the inactive and active kinase conformations .
the em5 inhibitor
bound to s6k1 with an ic50 value in
the mid - nanomolar range , and a cocrystal structure confirmed that
the inhibitor was binding in the atp pocket of the kinase domain .
as a result ,
em5 was a promising lead structure for the design of
more selective and potent s6k1 inhibitors .
previous data indicate
that modifications to the pyridocarbazole moiety or the coordination
sphere can have significant effects on binding affinities or kinase
selectivity , and the structure of the
s6k1/em5 complex indicated several positions where chemical elaboration
could enhance specificity for the kinase .
a series of 64 derivatives
of em5 were designed with modifications at the pyridcarbazole heterocycle
and the remaining ligand sphere and were tested for inhibition of
s6k1 activity using both a radioactive kinase assay ( supporting information figure 3a ) and an adp - glo assay with
1 m compound .
twenty - five of these
inhibitors were further screened using 250 nm compound ( figure 3a ) .
the eight compounds that inhibited s6k1 to less
than 25% activity , the equivalent of em5 , were assayed to determine
their ic50 values ( at 100 m atp ) .
this analysis
produced several compounds that inhibited s6k1 similarly or more potently
than em5 including sek-222 ( ic50 = 18.9 nm ) , sek-243 ( ic50 = 15.9 nm ) , sek-214 ( ic50= 14.8 nm ) , sek-220
( ic50= 7.7 nm ) , and fl772 ( ic50 = 7.3 nm ) ( supporting information figure 3b ) . compound fl772
( figure 3b ) showed the most potent inhibition
with an ic50 of 7.3 nm , ( 100 m atp and 2 nm of enzyme )
( figure 3c ) .
( a ) the 25 top derivatives of em5 were assayed against
s6k1 in a radioactive kinase assay , using 100 m atp and 250
nm s6k1 .
( b ) structure of the fl772 inhibitor , which showed the most
potent inhibition of s6k1 .
the 1,4,7-trithiacyclononane ligand of
em5 is replaced by a 1,4,7-trithiacyclodecane bearing a methylated
amino group in fl772 .
( c ) radioactive kinase assay of fl772 against
s6k1 reveals an ic50 of 7.3 nm .
( d ) radioactive kinase
assay of fl772 against s6k2 shows an ic50 of 975 nm .
( e )
radioactive kinase assay of pf-4708671 against s6k1 indicates an ic50 of 142.8 nm .
panels c , d , and e employed 100 m atp
and 2 nm of the respective kinase .
testing
the fl772 inhibitor at a range of concentrations from 1 m atp
to 500 m atp resulted in an increase in the ic50 value concurrent with increasing atp concentrations from 3.91 nm
at 1 m atp to 25.79 nm at 500 m atp , confirming that
fl772 is an atp competitive inhibitor ( supporting
information figure 4 ) .
to
assess the specificity of fl772 for the s6k1 isoform , we also assayed
the fl772 compound against recombinant s6k2 ( figure 3d ) , which resulted in an ic50 value of 975.0 nm ,
more than 100-fold greater than the ic50 value for s6k1 .
this confirms that the fl772 is indeed specific for the s6k1 isoform
over the s6k2 isoform . for comparison , we tested the published s6k1
inhibitor pf-4708671 against s6k1 using
the radioactive kinase assay in order to compare the potency of our
compound with another specific s6k1 inhibitor ( figure 3e ) .
the ic50 of pf-4708671 against s6k1 was determined
to be 142.8 nm , nearly 20-fold higher than the ic50 of
fl772 against s6k1 . to establish the kinase selectivity profile
of fl772 , we submitted
the compound at a concentration of 100 nm to the kinomescan profiling
of lead hunter discovery services using 456 kinases ( supporting information table 10 ) .
fl772 demonstrated a high
degree of kinase selectivity , with only 10 of 456 ( 2.2% ) kinases showing
less than 10% activity and only 26 of 456 ( 5.7% ) kinases showing less
than 35% activity ( supporting information figure
2 ) .
like em5 , fl772 showed binding to the cam , dap , flt , pim ,
and rsk family member kinases .
unexpectedly , s6k1 itself exhibited
71% activity in the kinomescan set of lead hunter discovery services
with 70 of 456 ( 15.3% ) showing a higher degree of binding than s6k1 .
the potency of fl772 therefore appears to be greater against s6k1
prepared by us than s6k1 prepared by lead hunter discovery services .
we hypothesize that the different s6k1 kinase preparation and/or phosphorylation
state by lead hunter discovery services leads to the different fl772
potencies for s6k1 measured by us and lead hunter discovery services .
nonetheless , taking together our analysis of fl772 against s6k1 and
the kinase profiling results , we conclude that fl772 exhibits a high
degree of kinase selectivity .
fl772 is based on the em5 lead
structure and differs by a methylated
hydroxy group at the pyridocarbazole moiety and the thioether - containing
tridentate ligand .
the nine - membered ring of the symmetrical 1,4,7-trithiacyclononane
ligand is replaced by a prochiral 1,4,7-trithiacyclodecane bearing
a basic n - methylamino group in the 10-membered cyclic
tridentate ligand .
these structural changes in the tridentate ligand
significantly increase the structural complexity of the inhibitor
which is exemplified by the number of possible stereoisomers . to determine the molecular basis for the increased potency of fl772
over em5 , we determined the x - ray crystal structure of fl772 in complex
with s6k1 to 2.7 resolution ( table 1 ) .
the overall structure for the fl772-bound s6k1 is very similar
to the em5-bound structure , with an rmsd of 0.54 for all atoms .
in particular the p - loop and activation loop and d and c
helices take on nearly identical conformations , although the c - helix
is about one turn shorter at its n - terminal end ( figure 4a ) . in addition
, the fl772 inhibitor retains all of the interactions
made by em5 but makes some additional interactions including a hydrogen
bond between the backbone carbonyl of lys-99 of the kinase p - loop
with the amine ligand of the n - methyl-1,4,7-trithiacyclodecan-9-amine
ligand while the methyl group makes a van der waals interaction with
tyr-174 of the kinase hinge region ( figure 4b ) .
these additional interactions of fl772 likely contribute to the
greater potency of fl772 over em5 .
the protrusion of the amine ligand
into the region where protein substrate binds for phosphorylation
probably also contributes to the greater inhibitor potency .
( a ) comparison
of the s6k1 structures with em5 and fl772 . the c helix of s6k1
is one turn shorter at its n - terminal end of the fl772-bound structure
compared to the em5-bound structure .
hydrogen bonds are shown as a dashed line , while other interacting
residues are labeled .
fl772-specific interactions that are not made
on the em5-bound structure are highlighted in darker shade .
this includes
interactions between y174 and the ether methyl group and the k99 backbone
carbonyl with the amine group of the tridentate ligand .
after
establishing that
fl772 functions as a potent atp competitive s6k inhibitor in the in
vitro radioactive kinase assay , we carried
out studies to characterize its activity in cells .
we first tested
fl772 for overall cell cytotoxicity and downregulation of phosphorylation
of s6 in the 451lu ( braf mutant ) and 451lu - mr ( braf / mek - inhibitor
resistant ) melanoma cell lines .
cells were treated with vehicle or
a dose of inhibitor ranging from 0.001 to 10 m for 22 h ( figure 5a ) .
the 451lu or 451lu - mr cell lines exhibited neither
a significant decrease in s6 phosphorylation nor a decrease in cell
viability as indicated by the absence of cleaved parp .
there was also
no change in total s6 or peef2k levels , indicating that mtor was not
a target of fl772 .
fl772 does not inhibit s6 phosphorylation in human melanoma
cells
but does inhibit s6 phosphorylation in yeast cells .
( a ) 451lu or 451lu - mr cells
were treated with increasing concentrations of fl772 or vehicle for
22 h. cells were lysed and blotted for ps6 and other downstream effectors
of s6k1 .
( b ) 293 t cells were treated with fl772 , fl1324 ( a similar
organometallic ruthenium compound ) , a previously reported s6k1 inhibitor ,
pf-671 ( 4708671 ) , or a previously reported dual mtorc1 and mtorc2
inhibitor , azd8055 , for 3 or 16 h. ( c ) western blot of yeast cells
treated with fl772 .
by4742 budding yeast cells were treated with fl772
for 4 h. cells were lysed and blotted for ps6 .
quantitative western
blot signals were detected by li - cor , and the relative ps6 levels
were calculated by normalizing raw ps6 measurements to gapdh signals .
( ) p < 0.05 ( two - tailed student t test , n = 3 ) .
we also investigated the effect of fl772 in 293 t cells at
both
3 and 16 h of treatment ( figure 5b ) . as controls
azd8055
is an atp - competitive dual mtorc1 and mtorc2 inhibitor that inhibits
the phosphorylation of mtorc1 substrates s6k1 and 4ebp1 and the mtorc2
substrate akt .
pf-4708671 is a reported
s6k1 inhibitor that does not affect the phosphorylation of akt .
the
compound fl1324 is an fl772 analogue identified in our screen that
inhibited s6k1 with an ic50 of 11 nm ( figure 3a ) .
fl1324 replaces the fluorine of em5 with a hydroxyl group
and is thus a less polar molecule . since previous studies using pf-4708671
demonstrated a significant reduction in s6 phosphorylation in 293 t
cells in 30 min , we tried both a short
( 3 h ) and long ( 16 h ) time point for treatment .
as expected , the azd8055
mtor inhibitor showed a significant decrease in ps6 levels at both
the s235 and s240 sites , along with a decrease in pakt at t308 and
s473 .
the pf-4708671 compound showed a modest decrease in phosphorylation
of s6 at the 3 h time point , but this phosphorylation returned to
near basal levels by the 16 h time point .
notably , neither the fl772 nor the fl1324
compound inhibited phosphorylation of s6 or akt .
together , these results
suggest either that the fl772 inhibitor has poor cell membrane permeability
or that inhibition of s6k1 in cells does not significantly reduce
s6 phosphorylation . the latter possibility is consistent with the
fact that the structurally unrelated compound pf-4708671 also shows
poor inhibition of s6 phosphorylation in cells and the observation
that s6k2 also targets s6 for phosphorylation . to test if fl772 can inhibit s6 phosphorylation in
a setting where
s6k2 was not present , we investigated the ability of fl772 to inhibit
s6 phosphorylation in budding yeast where a single kinase , sch9p ,
is orthologous to human s6k1 .
we observed that treatment of wild - type
budding yeast cells ( by4742 ) with fl772 significantly decreased the
level of phosphorylated s6 in a dose - dependent manner ( figure 5c ) . at the highest dosage ,
these data
suggest that fl772 functions as an inhibitor of s6 kinases in vivo
in a yeast cellular system .
in this study , we developed an organometallic ruthenium compound
to inhibit s6k1 . using the millipore kinaseprofiler and radioactive
kinase assays
, we showed that the em5 lead compound was a potent and
selective s6k1 inhibitor , with 100 nm compound inhibiting 93% of s6k1
activity and only inhibiting 16% of 283 kinases by less than 90% .
we found that the em6 analogue in which an isocyanate group replaces
an isothiocyanate is about 1000-fold less potent , implying that potency
and specificity could be further optimized . a crystal structure of
em5 bound to s6k1 provided important molecular insights into em5 inhibition
of s6k1 and led to the development of fl772 , a compound containing
a novel ligand scaffold with an ic50 in the single digit
nanomolar range for s6k1 .
a crystal structure of fl772 bound to s6k1
revealed the molecular basis for the compound s potent and
selective inhibition of s6k1 . in order to investigate the efficacy
of the fl772 inhibitor in
cells
, we evaluated the inhibitor in both human 293 t and braf mutant melanoma cells and in budding yeast .
we found
that fl772 was only able to inhibit s6 phosphorylation in yeast cells ,
suggesting that either the compound is unable to enter human cells ,
a significant shift in the ic50 of the compound occurs
in the presence of physiological levels of atp , or the uninhibited
activity of s6k2 in human cells was sufficient to maintain s6 phosphorylation .
given that ruthenium compounds similar to fl772 have been used to
successfully target mst1 , pak1 , and pi3k in cells ,
we do not believe that the ruthenium compounds are unable to penetrate
cells .
the radioactive kinase assay prohibits measurements at physiological
levels of atp , but we were able to assay the activity of fl772 against
s6k1 using an atp range from 1 to 500 m .
this analysis revealed
that the ic50 values increased with increasing atp concentrations ,
consistent with fl772 binding competitively with atp , as also confirmed
with the crystal structure of the s6k1/fl772 complex .
interestingly ,
the ic50 ranged from 3.91 nm at 1 m atp to only
25.79 nm ( a 6-fold increase ) at 500 m atp , suggesting that
s6k1 binds atp relatively loosely and that fl772 is likely to displace
atp even at the higher physiological concentration of atp . on the
basis of these accumulated data
, we propose that fl772 is unable to
inhibit s6 phosphorylation in human cells because s6 is phosphorylated
by the uninhibited s6k2 .
s6k1 is closely related to s6k2 , sharing
83% sequence identity
in the catalytic domain .
a study involving
s6k1/2 knockdown in mice suggests that both s6k1 and s6k2 are required
for full phosphorylation of s6 but that s6k2 may be the more important
of the two for phosphorylation of s6 .
the mek inhibitor azd6244 showed additive effects on decreasing
the phosphorylation of s6 in vitro when treated in combination with
sirna inhibition of both s6k1 and s6k2 combined , indicating the importance
of s6k2 in the phosphorylation of s6 .
furthermore , while normal tissues often express low levels of s6k2 ,
overexpression of s6k2 is more common than overexpression of s6k1
in cancer cells .
taking these data together suggests that targeting s6k2 either alone
or in combination with s6k1 inhibition may be a more viable option
for direct s6 inhibition in melanoma and potentially other cancers . despite the similarities in the catalytic domain , homology modeling
between s6k1 and s6k2 indicates an important difference in residue
tyr-174 that plays an important role in fl772 binding and is a cysteine
in s6k2 .
this residue is located in the
hinge region of s6k1 and makes an important van der waals interaction
with the methyl group of the secondary amine , which would not be made
with a cysteine residue , suggesting that fl772 may not be a potent
inhibitor for s6k2 .
indeed , we confirmed in our study that fl772 inhibits
s6k2 more than 100-fold more poorly than s6k1 .
the lethality of s6k1/s6k2 knockout
mice implies that s6k2 targeting may
need to be selective for therapeutic value . to date , there are no
commercially available s6k2-selective inhibitors , indicating a potential
target for the next series of organometallic ruthenium inhibitors .
taken together ,
the studies reported here provide a potent and selective
s6k1 inhibitor that should be useful to probe s6k1 function and as
a starting point for the development of efficacious s6k inhibitors
for therapeutic use .
protein kinase profiling of
em5 was performed with the millipore kinaseprofiler in a panel of
263 human protein kinases .
percentage of kinase activities were determined
for em5 and em6 at 100 nm in the presence of 10 m atp .
kinase profiling
of fl772 was performed with the discoverx kinome screen using a panel
of 456 kinases .
active - site - directed competition binding was determined
in the presence of 100 nm fl772 .
em5 and em6 were synthesized
in analogy to related compounds reported . a detailed synthesis and characterization of fl772
full length human s6k1
cdna ( 1525 ) was purchased from epitope
( catalogue number ihs1380 - 97652397 ) .
s6k1 constructs ( 84384 ,
1421 , 1421 t412e ) were subcloned into the pfastbac
htb vector for protein expression .
sf9 cells were transfected with
the recombinant bacmid dna using cellfectin ( invitrogen ) .
cells were
harvested after being incubated for 48 h at 28 c and stored
at 80 c .
the 1421 t412e construct was coexpressed
with pdk1 to phosphorylate the t412e residue ( cloned from cdna purchased
from openbiosystems ) .
frozen pellets of the s6k1 kinase domain , s6k1(84384 )
used for crystallography were resuspended in sonication buffer ( 50
mm kpi , ph 7.0 , 250 mm nacl , 5% glycerol , 1:1000 pmsf ) and sonicated
at a power output of 5.5 for 120 s with 20 s intervals ( misonix sonicator
3000 ) .
lysates were cleared by high - speed centrifugation at 18 000
rpm for 35 min at 4 c .
equilibrated talon metal affinity resin
( clontech ) was added to cleared lysates and incubated at 4 c
for 1 h with gentle shaking .
the resin / lysate mixture was loaded into
a gravity flow column , and the resin was extensively washed with wash
buffer ( 50 mm kpi , ph 7.0 , 250 mm nacl , 5% glycerol ) .
protein was
then eluted with elution buffer ( 50 mm kpi , ph 7.0 , 250 mm nacl , 500
mm imidazole , and 5% glycerol ) in a single step .
pooled talon eluent
was diluted 3.5-fold in dilution buffer ( 50 mm kpi , ph 7.0 , 5% glycerol )
and loaded onto an sp anion exchange column pre - equilibrated with
buffer a ( 50 mm kpi , ph 7.0 , 50 mm nacl , 5% glycerol ) .
protein was
eluted with buffer b ( 50 mm kpi , ph 7.0 , 500 mm nacl , 5% glycerol )
in a single step .
elution after the q column was concentrated and
loaded to a superdex s200 column equilibrated with 50 mm na citrate ,
ph 6.5 , 300 mm nacl , 1 mm dtt , 5% glycerol .
the eluent was collected
and concentrated to 3 mg / ml before protein was flash frozen in dry
ice and stored at 80 c .
purification of the 1421
t412e construct was completed as above , with gel filtration on the
superdex s200 column using a buffer containing 25 mm tris , ph 7.5 ,
200 mm nacl , 1 mm edta , and 5% glycerol .
full - length human s6k2 cdna
was purchased from ge healthcare dharmacon
( rps6kb2 , clone identification number 2959036 ) .
the s6k2 1423
construct , equivalent to s6k1 1421 , was subcloned into the
pfastbac htb vector for protein expression .
each reaction mixture contained
5 l of 5 reaction buffer ( 100 mm mops , ph 7.0 , 150 mm
mgcl2 ) , 2 l of inhibitor in 50% dmso , 3.6 l
of s6k1 substrate peptide ( rrrlsslra ) , 1 l of bsa ( 20 mg / ml ) ,
3.2 l of s6k1 ( concentration as described in results ) , and 5 l of atp / atp * mix ( concentration as
described in results ) in a total reaction
volume of 25 l .
reaction mixtures were incubated for 1 h at
room temperature before being transferred to whatman paper and washed
with 0.75% phosphoric acid .
ic50 values were determined using sigmoidal dose response with a variable
curve in prism , version 5 .
the s6k1
kinase domain crystals were obtained using room temperature hanging
drop vapor diffusion by mixing equal volumes of protein ( 15 mg / ml )
preincubated with 1 mm staurosporine with 2025% ( w / v ) peg335 ,
0.1 m bis - tris ( ph 5.55.7 ) , and 0.2 m liso4 .
following
the growth of crystals , crystal soaking was carried out by incubation
with a final inhibitor concentration of 1 mm in cryoprotectant containing
the well solution and 15% ( w / v ) glycerol for 4 h to overnight and
flash frozen in liquid nitrogen .
the structures were
determined by molecular replacement using the reported s6k1/staurosporine
complex ( pdb accession code 3a60 ) as a search model with the staurosporine removed
from the coordinate file and refined with cns and coot .
simulated annealing
omit maps were employed to unambiguously confirm the modeled inhibitors .
for the em5-soaked crystals , this revealed that one protein molecule
in the asymmetric unit was bound to staurosporine while the other
protein molecule was bound to em5 . for the fl772-soaked crystals ,
the asymmetric unit contained a single
, domain - swapped monomer and
only the fl772 inhibitor was modeled in the binding site .
the structures
were refined to convergence with a final rwork = 19.15% and rfree = 22.21% for the
s6k1/em5 structure and a final rwork =
20.63% and rfree = 23.01% for the s6k1/fl722
structure with excellent geometry ( table 1 ) .
human cell lines
were cultured in rpmi ( 10 - 040-cm ; cellgro ) supplemented with 5% fetal
bovine serum and harvested at 70% confluence . for immunoblotting ,
cells were treated for the specified times with the indicated drugs ,
washed with cold phosphate buffered saline ( pbs ) containing 100 mm
na3vo4 , and lysed using tne buffer ( 150 mm nacl ,
1% ( v / v ) np-40 , 2 mm edta , 50 mm tris - hcl , ph 8.0 ) supplemented with
protease inhibitors ( 11697498001 ; roche ) .
page and transferred to nitrocellulose membranes ( 9004700 ;
biorad ) . after blocking for 1 h in 5% ( wt / vol ) dry milk / tris - buffered
saline ( tbs)/0.1% ( v / v ) tween-20
, membranes were incubated overnight
at 4 c with primary antibodies followed by incubation with alexa
fluor - labeled secondary antibodies ( irdye 680lt goat - anti - mouse or
irdye 800cw goat - anti - rabbit antibodies ( li - cor biosciences ) for 1
h. -actin ( a5441 ) and vinculin ( v9131 ) antibodies were obtained
from sigma .
p - akt ( 4056 , 4060 ) , s6 ( 2317 ) , p - s6 ( 4858 , 5364 ) , s6k1
( 2708 ) , p - s6k1 ( 9234 ) , p - eef2k ( 3691 ) , peif4b ( 3591 ) , and cleaved
parp ( 5625 ) were obtained from cell signaling technologies .
overnight cultures of
wild - type yeast cells ( by4742 ) were diluted in synthetic complete
( sc ) medium and regrown at 30 c to early log phase ( od600 of 0.2 ) .
fl772 was added to an aliquot of culture to the final concentration
of 1 , 10 , 100 , and 1000 nm .
the treated cultures were further grown
at 30 c for 4 h before harvesting .
a culture of sch9 cells ( ks68 ) was grown and harvested in parallel as a control .
yeast cell pellets were lysed by spinning down cultures at 3000
rpm for 3 min at 4 c , washing with ice - cold water , and broken
in lysis buffer as previously described .
whole cell extracts were separated on a 412% bolt gel with
mops running buffer ( life technologies ) , followed by transfer to a
pvdf membrane in a mini trans - blot cell ( bio - rad ) at 20 v overnight .
the blot was blocked with 3% bsa at room temperature for 2 h and then
at 4 c for 4 h , followed by incubation with primary antibodies ,
phospho - s6 ( cell signaling , catalog no .
incubation with secondary antibodies ( anti - rabbit - dylight-680
and anti - mouse - dylight-800 , pierce , 1:10000 dilution ) was carried
out at room temperature for 1 h before imaging with li - cor odyssey . | aberrant activation of s6 kinase
1 ( s6k1 ) is found in many diseases ,
including diabetes , aging , and cancer .
we developed atp competitive
organometallic kinase inhibitors , em5 and fl772 , which are inspired
by the structure of the pan - kinase inhibitor staurosporine , to specifically
inhibit s6k1 using a strategy previously used to target other kinases .
biochemical data demonstrate that em5 and fl772 inhibit the kinase
with ic50 value in the low nanomolar range at 100 m
atp and that the more potent fl772 compound has a greater than 100-fold
specificity over s6k2 .
the crystal structures of s6k1 bound to staurosporine ,
em5 , and fl772 reveal that the em5 and fl772 inhibitors bind in the
atp binding pocket and make s6k1-specific contacts , resulting in changes
to the p - loop , c helix , and d helix when compared to
the staurosporine - bound structure .
cellular data reveal that fl772
is able to inhibit s6k phosphorylation in yeast cells .
together , these
studies demonstrate that potent , selective , and cell permeable s6k1
inhibitors can be prepared and provide a scaffold for future development
of s6k inhibitors with possible therapeutic applications . | Introduction
Results
Discussion and Conclusions
Experimental Section |
transitional cell carcinoma ( tcc ) of the upper tract comprises less than 5% of all urothelial tumors . while open nephroureterectomy ( onu ) has been the gold standard for the management of localized upper tract tcc , the laparoscopic approach for this procedure ( lnu ) is rapidly gaining acceptance .
oncologic results with lnu in terms of bladder recurrences , metastatic incidence , and cancer - specific survival have been comparable to open surgery , while providing minimally invasive surgery ( mis ) benefits in terms of lower morbidity and quicker recovery .
the technique of nu is best considered as two separate procedures nephrectomy and the removal of lower end of ureter with surrounding bladder cuff .
the controversy of the nephrectomy component seems to have rested with laparoscopic management outscoring open surgery with its mis benefits .
the management of distal ureter , however , has remained the most challenging and controversial feature of both open and laparoscopic procedures due to highlighted risks of retroperitoneal , peritoneal , and port - site metastases .
the oncology purists dictate that the best nephroureterectomy procedure would do a complete en - bloc resection of the kidney and ureter with surrounding bladder cuff , and avoidance of tumor seeding .
there is no controversy regarding the fact that failure to completely remove the lower end of ureter or the surrounding bladder cuff risks high recurrence in the remnant , and thus is an essential part of the procedure .
opening the bladder to achieve this , however , risks the seepage of urine with potential implantation of viable cancer cells . a closed technique without opening the urinary system , theoretically , would be the best , but has not been accepted due to high incidence of positive margins and bladder recurrences , attributed to the method 's inconsistency in removing the complete intramural ureter and bladder cuff segments .
there is also an increased risk of injury to opposite ureter on using extravesical stapling device .
the classical open transvesical technique of securing the lower end has been the gold standard even though it transgresses urothelium and requires a second incision when combined with open nephrectomy .
there have been consistent attempts to innovate minimally invasive methods as alternative to open lower end management .
the pluck and intussusception techniques were popularized in onu settings to avoid the second incision .
although there are enthusiasts of pluck methods in combination with lnu , multiple reports of local recurrences over last two decades have fuelled newer innovations to deal with the lower end .
the failure of pluck technique can be explained due to difficulty to confirm total ureterectomy in pluck
methods due to an absence of an identifying tag at the lower end .
moreover , the lower end of ureter is unsecured and open , thereby allowing seepage of urine into the wound .
the minimally invasive methods described in the last decade have tried to emulate the open surgery steps with some combination of transurethral endoscopy , suprapubic transvesical ports , and hand assistance .
while all these methods succeed in removing the intramural ureter with bladder cuff en - bloc with the kidney specimen , the methods disagree in the level of control of ureter to prevent passage of viable tcc cells down to the bladder or the wound , and in sequence whether the kidney or the lower ureteric end is to be dealt with first .
we describe a , minimally invasive surgical technique adhering to basic oncologic principles to manage the lower end of ureter .
we use the described technique in patients with unilateral upper tract high - grade tcc .
initial imaging and endoscopic evaluation excludes the disease in bladder and the contralateral upper tract .
patients with tcc in the intramural part of ipsilateral ureter are not considered for this procedure .
the lower ureteric end is dealt first , and the procedure carried out in three steps .
glycine is used as irrigant with stand height of 60 cm above pubic symphysis , and the diathermy is set at 90 w pure cutting and 60 w coagulating .
the incision line , about a cm around the right ureteric meatus , is marked with coagulating current .
incision is then deepened with cutting current . while the bladder wall overlying the intramural ureter
is cut partly , rest of the bladder incision from 1 to 11 o clock is deepened to reach perivesical loose areolar tissue .
bladder wall thickness is judged in lower half of the circular incision , and then carried toward the upper end on either side .
the procedure is stopped short of completing the full thickness bladder incision over intramural part of ureter [ figure 2 ] .
dissection of bladder cuff around the ureteric meatus with collings knife via resectoscope placed transurethrally endoscopic view of the dissected bladder cuff with fibers still attaching it at 12 oclock step ii [ figure 3 ] : bladder is filled with the resectoscope outlet closed . a needle puncture just above symphysis pubis is seen entering bladder under vision .
it is followed with a 5-mm vertical skin incision at the same site , cutting the underlying rectus sheath also in the midline .
a 5-mm threaded laparoscopic trocar is placed in the bladder in line of the initial needle puncture and its outlet is connected to low - pressure suction .
the dissected bladder cuff is held with the grasper and retracted inferomedially and inferolaterally to stretch remaining bladder muscle fibers to be cut with collings knife [ figure 5 ] .
the freed lower end of ureter is now retracted into the bladder to release intramural attachments [ figure 6 ] , as done in transvesical mobilization for reflux surgery , till ureter is completely free .
complete mobilization of intramural ureter with resectoscope and collings knife , while ureter is retracted with grasper via transvesical port endoscopic view of grasper introduced via transvesical port ureter is retracted inferomedially to stretch remaining attached bladder fibers which are cut with collings knife dissection is done till ureter is completely free of all attachments step iii [ figure 7 ] : resectoscope is removed and replaced with cystoscope with a 30-degree telescope and the biopsy forceps .
flow and the suction are adjusted to keep the vision just clear enough to identify structures .
the end of the cystoscope is kept just below and lateral to the dissected ureter , looking up toward the suprapubic port .
end of the dissected ureter , held till now with the grasper through the suprapubic port , is transferred to the grip with transurethral biopsy forces .
the grasper is exchanged with a 5-mm hem - o - lock applicator , and the ureter is doubly secured just proximal to the held distal end [ figure 8 ] .
a 22f two - way foley 's catheter is placed via urethra and connected to gravity drainage .
resectoscope is exchanged for a cystoscope and forceps to grasp the bladder cuff end of ureter , while clip applicator via the transvesical port seals its lower end clips in place to seal lower end .
note the biopsy forceps being used to hold the bladder cuff at 3 oclock patient is , then , placed in standard lateral kidney position for transperitoneal laparoscopic radical nephrectomy . after completing the nephrectomy , the ureter is traced to the lower end with surrounding tissues .
some tissue bands in retrovesical part of the ureter are easily dissected by retraction on ureter , and the clips applied at lower end soon come into view .
the suprapubic small incision for the port in step ii is extended up for 68 cm .
the bagged specimen is removed via the midline incision [ figure 9 ] , and closure done after leaving retrovesical drain exiting just above right anterior superior iliac spine .
completely removed nephroureterectomy specimen with clips at lower end histopathology and disease characteristics are recorded .
the urethral catheter is removed as outpatient on seventh postoperative day after a cystogram under fluoroscopy .
the lower end in two other cases of upper tract tcc was managed with open extravesical technique during this period . while one of latter cases had involvement of intramural part of ureter and tumor protruding out of ureteric orifice , the second case had synchronous bladder tumors .
three cases managed with presented technique have been followed for 36 , 12 , and 9 months , respectively .
a 10-mm suprapubic port was used to access the bladder in the first case , but a 5-mm port sufficed in cases 2 and 3 after the availability of a 5-mm hem - o - lock applicator .
the management of the bladder end consumed 40 , 55 , and 30 minutes , respectively , in three cases .
postoperative hospital stay was 3 days in cases 1 and 3 , and 4 days in case 2 .
the first and third case had disease localized in the pelvicaliceal system on right and left side , respectively .
histopathology revealed pt3 ( invasion of renal pelvis ) in case 1 , and pt1 high - grade tcc in cases 2 and 3 .
there has been no regional or local residual or recurrent disease in cases 1 and 3 .
single low - grade cystoscopic recurrence at opposite bladder wall was noted in case 2 nine months postoperatively .
controversy regarding the lower ureteric management as part of nephroureterectomy has largely been due to the threat of implantation of tcc cells .
the risk is low but real . in a large collective experience with 10,912 laparoscopic oncologic procedures from 19 institutions , port - site metastases occurred in 0.5% of operated cases , and
the inefficacy of partial cystectomy for tcc bladder and high incidence of local recurrences and peritoneal carcinomatosis following rupture of the bladder during turbt are also indicators of the risk of tcc cell implantation .
detachment of the ureteric meatus and the surrounding bladder cuff from the bladder exposes the raw area and the retroperitoneum to the seepage of urine containing viable tcc cells , thereby risking implantation during lnu .
hence , the management of the lower end during lnu still remains a controversial topic in urological practice .
synchronous bladder or upper tract disease may occur in 39% of patients with ureteric tcc and 24% of those with renal tcc .
a metachronous bladder recurrence occurs in 12.537.5% of patients with an upper tract tcc treated with surgery .
it is , therefore , imperative to exclude the disease in the bladder and the opposite renal unit before planning nephroureterectomy . once the disease is confined to a single unit , the number of viable cells to reach a critical mass for implantation is dependent upon the grade of disease and the degree of manipulation of the diseased area before taking control of the distal end .
such manipulation may be intraluminal , as may occur with placements of ureteric catheters and stents , and during surgical handling in nephroureterectomy . during the attempted improvements over the earlier pluck and intussusception techniques ,
gonzalez et al . controlled the upper ureter in laparoscopic view in the technical description of the case with renal pelvic tumor .
there were no local pelvic or peritoneal metastasis in 49 lnus by kurzer et al . with low control of ureter in laparoscopic vision
however , this technique was confined to pelvic and upper ureteral tumors only , while the lower ureteral tumors were managed by open technique .
further improved on it with control of the ureter at the vesical end , with no retroperitoneal or port - site metastasis .
all cases of upper tract tcc were , thus , eligible except those with disease in intramural ureter , or distal to it .
the sequence whether the lower ureteric end or the kidney should be tackled first may be argued .
the gold standard open method deals with the kidney first , and the lower end is tackled in the end via an open cystotomy . lower end first approach has been used with minimally invasive methods like pluck technique .
although there is no raw area for implant at this time , this release of tcc cells may potentially explain some of the local recurrences in the bladder seen early postoperatively , and may find the wound for implant after the cystotomy .
the lower end first , as in gill 's technique , makes it possible to clip the ureter at its lowest end avoiding any possibility of cell spillage from the diseased unit while manipulating the kidney or ureter later during nephroureterectomy .
the upper tract manipulation with an open lower end has been blamed for failures seen after the pluck technique , the earlier lower end first method .
although the concept is theoretically sound , whether clipping the lower end before renal manipulation would translate into lower bladder recurrences is a question that may be answered only with randomized trials and longer follow - up .
manipulations like ureteric catheterization and barbotage may increase the number of shed tcc cells ( as done for cytological diagnosis of the disease ) .
whether intraluminal manipulations in the described techniques in literature may increase the shed load of tcc cells may also be debated .
had a double - j stent in18 of their 49 cases at the time of lnu , 7 of which developed bladder tumors postoperatively within a median follow - up of 10 months .
gill et al . precede the ureteric meatal dissection with placement of a ureteric catheter till pelvis , although they did not report any retroperitoneal or port - site metastasis .
it would , however , be desirable to avoid any intraluminal manipulations before taking control of the ureter at its lowest end , in order to avoid release of tumor cells , as is the case with extraluminal handling .
all minimally invasive procedures have been criticized for leaving cystotomy site without proper closure , allowing extravasation of urine to occur .
while the ureteric hiatus is closed during the gold standard open surgery , the bladder has been left with the catheter drainage for 7 days without primary closure with most minimally invasive methods with no added morbidity .
suprapubic ports , as in the present and some other recent described techniques , leave another bladder rent , albeit much smaller , adding to the criticism of these techniques .
there is no or little risk of leaking urine , or potentially viable tcc cells if the disease has been excluded in the bladder and the opposite kidney , and the ipsilateral ureter has been clipped watertight .
the conservative management of extraperitoneal bladder rupture with pelvic trauma has similar outcome to that of patients treated with primary closure .
all experiences with conservative management of cystotomy in lnu setting have also shown that the bladder shows no signs of leakage with radiologic tests within 7 days .
it has been , interestingly , observed that all extravesical recurrences or evidence of metastatic disease occurred when the perforation was managed with open surgery !
the ureteric catheter and stent in the bladder make the intravesical part of the procedure technically demanding .
emphasize the use of ureteric catheter via urethra , threaded through the endoloop via one of the suprapubic ports , and the resectoscope being passed by the side of the ureteric catheter to accomplish cuff excision .
there is no technical difficulty in separating the bladder muscle fibers and reaching perivesical plane from 1 to 11 oclock using collings knife without any assistance .
the need , then , is to retract the ureter to separate the muscle fibers overlying it near 12 oclock position , and then pull it inside bladder to release the intramural part .
single suprapubic port with a grasper suffices for this purpose in step 2 of our procedure , and later for step 3 to clip the lower ureteric end .
size of this single suprapubic port depends on the size of grasper and clip applicator available ; thus a 5-mm port should suffice in most settings .
the technique described by us achieves the oncologic objectives of complete excision of bladder cuff and lower ureter with early control of the lower end prior to upper tract manipulation .
it is a minimally invasive technique , which can potentially decrease patient morbidity and can be accomplished cost - effectively using routine endourological instrumentation .
the technique uses a single suprapubic port , and avoids transluminal and extraluminal manipulation of the tumor containing upper tract .
it seals the lowest end of ureter , thus making the procedure acceptable to all cases with unilateral tcc irrespective of the site , except rare cases where the disease is protruding out of ureteric orifice .
the disadvantage is that it leaves the patient with two cystotomy sites for conservative healing .
small numbers of upper tract tcc cases even at a referral center make any randomized trials difficult , and would require multi - institutional combined studies to prove , or disprove , the potential benefits of these aspects of newer minimally invasive methods to deal with lower end of ureter during nephroureterectomy .
the single suprapubic transvesical port approach has the potential of offering a minimally invasive approach for tackling the lower end of ureter in upper tract tcc while adhering to basic oncological principles of early control without prior manipulation of upper tract and complete excision of the lower end , as well as allowing en - bloc removal of the specimen with identification of the clipped lower end .
a further prospective analysis of this technique in comparison with other methods is warranted prior to recommending its widespread applicability . | context : various minimally invasive techniques laparoscopic , endoscopic or combinations of both - have been described to handle the lower ureter during laparoscopic nephroureterectomy but none has received wide acceptance.aims:we describe an endoscopic technique for the management of lower end of ureter during laparoscopic nephroureterectomy using a single suprapubic laparoscopic port.materials and methods : transurethral resectoscope is used to make a full thickness incision in the bladder cuff around the ureteric orifice from 1 oclock to 11 oclock .
a grasper inserted through the transvesical suprapubic port is used to retract the ureter to complete the incision in the bladder cuff overlying the anterior aspect of the ureteric orifice .
the lower end of ureter is subsequently sealed with a clip applied through the port .
this is followed by a laparoscopic nephrectomy and the specimen is removed by extending the suprapubic port incision .
our technique enables dissection and control of lower end of ureter under direct vision .
moreover , surgical occlusion of the lower end of the ureter prior to dissection of the kidney may decrease cell spillage .
the clip also serves as a marker for complete removal of the specimen.results:three patients have undergone this procedure with an average follow up of 19 months .
operative time for the management of lower ureter has been 35 , 55 and 40 minutes respectively .
a single recurrence was detected on the opposite bladder wall after 9 months via a surveillance cystoscopy .
there has been no residual disease or any other locoregional recurrence.conclusions:the described technique for management of lower end of ureter during laparoscopic nephroureterectomy adheres to strict oncologic principles while providing the benefit of a minimally invasive approach . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
several studies comparing emergency physicians ( ep ) skills at intubation with other practitioners , primarily anaesthesiologists , found similar proficiency [ 1 , 2 ] . complications of emergency department ( ed ) intubations have been studied , but most only examine the peri - intubation period .
while there are several studies that provide data as to rates of success of ed intubation and need for cricothyrotomy , there is little information as to the long - term prognosis of these patients .
patients who are intubated in the ed are some of the most critically ill patients in the ed ; the mortality after ed intubation has not been well studied .
previous studies have shown that the lay public have a lack of understanding of the dismal outcomes associated with cardiac arrests .
understanding the outcomes of ed intubation can be useful in discussion with families of patients as well as provide information for future research .
the purpose of this study is to identify the rate of ed intubation and the mortality associated with ed intubation .
we conducted a retrospective chart review for all ed intubations performed over a 1-year period between 1 january 2004 and 31 december 2004 at an urban level one trauma centre with approximately 50,000 ed visits annually .
ed intubations were identified retrospectively by reviewing the hospital financial databases for intubations for all ed patients .
patients that had critical care billing for an intubation procedure or for ventilator use in the ed were obtained .
ed patient logs were also examined to identify all patients who were admitted to the intensive care unit ( icu ) , the operating rooms or died .
patients who were intubated prior to arrival in our ed either by another hospital or by prehospital personnel were excluded from our cohort .
two investigators reviewed all charts and abstracted the following data : age , sex , final disposition from hospital , reason for intubation using a standardized list of terms and trauma versus non - trauma .
if all data were not available from the discharge summary , the original chart was reviewed to gather the necessary data .
de - identified data were entered into a microsoft excel 2003 ( redmond , wa , usa ) database and analysed using stata v8.2 ( college station , tx , usa ) .
confidence intervals were calculated and comparisons were made using unpaired t - tests and fisher s exact tests .
one hundred and sixty - three patients were intubated in our ed during the 1-year study period .
the total 163 patients , 44 ( 27.0% ) died prior to discharge from the hospital , and 72 ( 44.2% ) patients were discharged to home or left against medical advice ( ama ) ( 1 patient ) .
forty - two ( 25.8.% ) patients were discharged to a skilled nursing facility ( e.g. nursing home , rehabilitation and extended care facility ) for further care . dividing this patient population into trauma and non - trauma subgroups ,
6/37 ( 16.2% ) of the trauma patients died and 38/136 ( 30.2% ) of the non - trauma patients died .
the mean age for all patients was 61.3 years , with trauma patients being younger ( 49.0 versus 65.0 ) than the non - trauma subgroup .
the mean age of the survivors was lower than the non - survivors : 57.3 versus 72.2 ( p < 0.0001 ) .
no children were identified as having been intubated in our study , as our institution does not routinely care for paediatric patients .
table 1age and mortality of patients intubated in the ed overalltraumanon - trauman16337126age61.3 ( 58.064.7)49.0 ( 40.557.5)64.5 ( 61.768.3)mortality27.0% ( 20.033.9)16.2% ( 4.128.3)30.2% ( 22.238.3)table 2comparison of age of intubated patients survived vs died alltraumanon - traumasurvivor ( mean age)57.3 ( 53.361.3)44.0 ( 34.953.0)62.0 ( 58.166.0)death ( mean age)72.2 ( 67.476.9)74.8 ( 65.684.0)71.7 ( 66.477.1 ) age and mortality of patients intubated in the ed comparison of age of intubated patients survived vs died of the deaths , 38.6% occurred within 24 h of intubation . an additional 22.7 , 29.5 and 9.1% of deaths occurred at 72 h , 7 days and 30 days , respectively , after intubation . no deaths were found to occur beyond 30 days of hospitalization .
in our search of the literature no large studies reporting mortality after intubation in the ed were found .
the near database , the largest ed airway registry at present , provides insight into the ed course of these patients , but does not collect post - intubation data or outcomes on their patients .
most of the emergency medicine literature concentrates on the mechanics of the intubation and the time surrounding the event .
considerable emergency medicine research focuses on the tools of airway assessment and techniques to improve intubation success such as the lemon score and the burp maneuver [ 5 , 6 ] .
alternatively , there is a lack of information on how these patients do once they leave the ed , and how the various techniques impact on the mortality associated with ed intubation .
while the patients who require ed intubation certainly represent a sicker population , our study demonstrates a relatively high mortality rate .
now that we know the mortality rate of 27% , we can begin to examine those interventions that not only improve the success of ed intubation , but also ultimately improve mortality . as an initial step , we chose to do a retrospective chart review of all ed intubations for a 1-year period .
while recognizing the limitations of such an approach , we thought this initial study would provide some useful information and indications for future research .
the considerable mortality rate following intubation is not surprising as the need for intubation in the ed probably represents a marker for severity of disease . like mortality from sepsis or after a myocardial infarction ,
establishing a baseline mortality rate after ed intubation will provide us with an outcome measure we can use to test the benefit of specific interventions . due to the limited number of patients in our cohort , many questions remain unanswered .
stratification of the patients by disease entity could not be achieved in a meaningful way .
for example , should head injuries be classified as neurological or as traumatic ? the one major subdivision of patients we could perform analysis on was trauma versus non - trauma patients .
this finding could be explained by the lower age of trauma patients and their ability to respond to medical interventions . in both groups ,
this is most evident in the trauma group where the mean age was 44.0 years , but the mean age of those who died was 74.8 years as compared to the non - trauma group .
older patients with myocardial infarction and stroke have been demonstrated to have worse outcomes in the literature [ 7 , 8 ] .
older patients tend to have less physiological reserve so the same insult would be expected to have a greater deleterious effect upon prognosis .
yanagawa et al . , in a study comparing intubation without medications versus with medications for non - traumatic ich , reported the mortality of the two groups as 46.8 and 72.9% , respectively .
the study had 70 patients for their control and study group combined and the main outcome measure was the glasgow outcome score , not mortality . if we select non - traumatic ich from our patients , 9/16 ( 56.2% ) died .
the high mortality rate in the ich subgroup , and the differences in the trauma and non - trauma groups , suggest that with a large enough number of patients subgroup analysis may reveal differences in mortality by specific disease entity .
survival to hospital discharge was chosen over 30-day mortality since it eliminated the problem of having patients that could not be followed up while still providing a meaningful endpoint .
this study was conducted as a pilot study to provide background data as to what directions would be of interest in future studies . as a single - centre retrospective study we are limited in our ability to make general statements based on our study population . as we saw no paediatric patients , we can not make any inferences as to their mortality rate after intubation .
the small sample size limits our ability to provide subgroup analysis for outcomes based on the reason for intubation other than the fairly broad trauma / non - trauma division . since there is no central way to identify patients who were intubated in our ed , we used multiple methods to identify cases ; however , there is a small chance we still could have overlooked a particular group that could alter the outcome .
future studies should attempt to identify predictors of mortality among ed intubations as well as look at this question in a multicentre setting . | introductionthe purpose of this study is to identify the rate of emergency department ( ed ) intubation and the mortality associated with ed intubation.methodswe conducted a retrospective chart review of all patients intubated in the ed between 1 january 2004 and 31 december 2004 at an urban level one trauma centre with approximately 50,000 ed visits annually .
all ed intubations were identified and reviewed .
two investigators reviewed all charts and collected the following data : age , sex , and final disposition from hospital as well as reason for intubation .
the main outcome measure was survival to hospital discharge.resultsone hundred sixty - three intubations were reviewed . of
the total 163 patients , 44 ( 27.0% ) died prior to discharge from the hospital , 42 ( 25.8.% ) patients were discharged to a skilled nursing facility ( e.g. nursing home , rehabilitation and extended care facility ) and 71 ( 43.6% ) patients were discharged home . dividing our cohort into trauma and non - trauma subgroups , 38/126 ( 30.2% ) of the non - trauma patients and 6/37 ( 16.2.% ) of the trauma patients died .
the mean age for all patients in our study group was 61.5 years , with trauma patients being younger than the non - trauma subgroup .
the mean age for trauma patients was 50.1 while non - trauma patients had a mean age of 64.8.conclusionsthe mortality after an ed intubation in our study population was relatively high .
further studies need to confirm these findings and help identify predictors of mortality . | Introduction
Materials and methods
Results
Discussion
Limitations
Conclusion |
during 20022004 , many sick badgers were seen at the bases of mountains , on village roads , and within residential houses . at the same time , rabies in livestock was reported in the nearby villages .
local residents stated that dead animals were seen everywhere ; however , accurate numbers and distribution of affected animals in these areas were difficult to estimate . during 20052007 , ferret badger hunters were recruited to help capture the animals for further investigation ; 12 badgers were captured each week .
the badgers were no longer commonly seen in the fields , probably the result of depopulation by the disease . among the 58 specimens collected in linan , chunan , and jiande counties of hangzhou , none of the brain tissue samples were positive for rabies by standard direct fluorescent assay .
serum samples from the 63 animals captured in the 3 counties mentioned above did not have detectable rabies virus neutralizing antibodies according to the fluorescent antibody virus neutralization test ( table 1 ) . *
arbitrary cut - off value for seroconversion is 0 iu / ml . during 20072008 , the population of the ferret badgers in the same regions seemed to recover , and rabies infection in badgers began to increase . since the summer of 2008 , sick and dead badgers have been seen by local residents inside houses , in the fields close to the residential areas , and on the roads in hangzhou district . of the 71 brain samples collected in 2008 , 4 had positive direct fluorescent assay results . of 30 serum samples ,
in addition , a human rabies case was recorded in april 2008 in lishui county , zhejiang .
our most recent retrospective epidemiologic investigation of human rabies cases from the end of 2007 through 2008 showed that in wuyuan county , jiangxi , adjacent to hangzhou , zhejiang , 4 of 5 recorded human rabies cases were caused by badger bites .
phylogenetic analysis using the nucleoprotein and the glycoprotein genes ( table 2 ) demonstrated that the ferret badger rabies virus isolate ( zj - la , isolated from a badger in linan county of hangzhou , zhejiang ) had 89.0% homology with a local dog rabies virus isolate ( zhejiang wz0 ) and overall 86.5%95.9% homology with other isolates from china ( figure 2 )
. the zj - la strain had the highest homology with a dog rabies virus isolate ( gn 07 , from guangning county , guangdong province ) and a vaccine strain ctn-33 ( originally from a person who died of rabies in jinan , shandong province , in 1957 ) . because dog - associated human rabies has been reported only sporadically in zhejiang province , whether the ferret badger
associated rabies is a spillover event from dogs , or the animals now serve as a natural reservoir in the rabies - endemic area , needs further investigation . *
position of a ferret badger associated rabies virus isolate ( zj - la ) in a phylogenetic tree constructed based on the nucleoprotein genes from representative dog rabies virus isolates and common vaccine strains in china ( a ) and the glycoprotein genes from representative dog rabies virus isolates and common vaccine strains in china ( b ) .
this figure was drawn by mega 4 ( www.megasoftware.net ) with maximum composite likelihood model .
rabies in ferret badgers occurred during 2 alleged epizootics ( 19941995 and 20022004 ) in southeastern china ( figure 1 ) ( 13 ) .
rabies in ferret badgers is becoming a greater public health threat to humans in eastern anhui , middle to western zhejiang , and northern jiangxi provinces in china .
because no practical rabies vaccine has been developed for wildlife in china , a rabies epidemic in ferret badgers is almost inevitable without intervention , and the threat to public health is immediate .
lack of communication and cooperation among the chinese center for disease control and prevention , ministry of agriculture , and wildlife services from the bureau of forestry makes the situation more complicated than canine rabies control . whether rabies in ferret badgers is a spillover event from rabid dogs or whether ferret badgers serve as a natural reservoir remains to be addressed .
in addition to more detailed epidemiologic investigations , control and elimination of rabies in dogs is a primary suggestion to test the latter hypothesis . | ferret badger associated human rabies cases emerged in china in 1994 .
we used a retrospective epidemiologic survey , virus isolation , laboratory diagnosis , and nucleotide sequencing to document its reemergence in 20022008 . whether the cause is spillover from infected dogs or recent host shift and new reservoir establishment requires further investigation . | The Study
Conclusions |
loss of cardiac resynchronization therapy ( crt ) is a frequent problem that limits the potential benefits associated with this form of non - pharmacological treatment for patients with heart failure.1 it usually occurs in consequence of loss of left ventricular ( lv ) and right ventricle ( rv ) capture , loss of atrial sensing , or loss of atrial tracking due to atrial rates greater than the maximum tracking rate ; however , its presentation at rates lower than the programmed maximum tracking rate is also possible . in this case report , we present a 67-year - old woman with hyperglycemia - induced t - wave oversensing as a cause of crt failure .
a 67-year - old female with a history of diabetes mellitus and renal failure as well as a diagnosis of dilated cardiomyopathy and new york heart association functional classification iii , referred for cardiac resynchronization defibrillator therapy .
a crt - d device is an implantable cardioverter - defibrillator ( icd ) enabled with crt implantation .
echocardiography showed a left ventricular ejection fraction ( lvef ) of 20% and left venricular end - diastolic dimension ( lvedd ) of 60 mm .
basal electrocardiogram ( ecg ) showed a left bundle branch block ( lbbb ) pattern and sinus rhythm . additionally , the patient had serum creatinine ( cr ) of 1.8 mg / dl , blood urea nitrogen ( bun ) of 55 mg / dl , and serum potassium ( k ) of 5.4 mm / dl .
the patient underwent implantation of a dual - chamber insync sentry dr 7298 icd ( medtronic inc .
the icd was programmed to ddd , 60120 beat / min , with a single shocking zone and a rate cut - off of 188 beat / min .
three months later , she returned to the emergency ward due to frequent icd shocks and deterioration of dyspnea .
the results of the admission blood test were as follows : k = 5.2 mg / dl , blood sugar = 560 mg / dl , serum creatinine = 2.6 mg / dl , and bun = 65 mg / dl .
also , the serial cardiac markers levels were within the normal limits , and arterial blood gas measurement did not show acidosis or alkalosis .
the stored electrograms at supine position revealed a ventricular sensing threshold of 6 mv and t - wave oversensing during sinus rhythm ( ventricular sensitivity = 0.5 mv ) .
moreover , atrial and ventricular pacing , sensing thresholds , and leads impedances were unchanged in comparison with previous measurements .
the patient was admitted to the coronary care unit ( ccu ) and treated with insulin to control her diabetes .
after the patient s blood sugar was reduced to 187 mg / dl , another icd interrogation was carried out , which demonstrated no t - wave oversensing at a maximum sensitivity of 0.18 mv . with the patient s permission
, the serum glucose was allowed to increase through a temporary discontinuation of insulin therapy .
two days later at a capillary glucose of 531 mg / dl , intermittent t - wave oversensing was detected at a sensitivity level of 0.5 mv .
after a two - month follow - up period , the patient s serum glucose was well controlled with insulin therapy and she had no further icd shocks and t - wave oversensing , which conferred good biventricular pacing and good response to crt .
t - wave oversensing is the major cause of inappropriate icd shocks ( figure 1).1 different mechanisms having been implicated in the double counting of r and t waves include drugs , hyperkalemia , hyperglycemia variations in sympathetic tones , and alterations in the shape of the intracardiac electrogram.2 , 3 elimination of t - wave oversensing is possible through the correction of reversible causes and device programming , and the repositioning of the ventricular electrode to find a greater r - wave amplitude makes oversensing less likely.35 t - wave oversensing was avoided by considering the following options in the present study : ( 1 ) reprogramming the icd to lower sensitivity ; ( 2 ) implanting the lv epicardial lead for sensing and pacing ; ( 3 ) changing the detection counts ; ( 4 ) administering heart rate slowing drugs ; ( 5 ) repositioning the right ventricular lead to obtain ventricular electrograms with higher amplitudes ; ( 6 ) implanting another endocardial sense - pace lead ; and ( 7 ) prolonging postsens refractory periods . decreasing the sensitivity or programming longer refractory periods can sometimes treat t - wave oversensing , but these measures may interfere with the ability of the icd to correctly detect tachyarrhythmias .
the best option for the treatment of t - wave oversensing due to a reversible cause is the treatment of the underlying cause .
the sensitivity and refractory periods of the device were , therefore , not changed in the present study . in this type of medtronic icd , postsense refractory periods are not programmable .
another option for the prevention of inappropriate shocks due to t - wave oversensing is changing the detection counts ; i.e. , increasing the number of successive counts before the icd is triggered to call the arrhythmic ventricular fibrillation ( vf ) .
appropriate identification and correction of the underlying cause of t - wave oversensing is an essential management strategy . in the case
presented herein , the elimination of t - wave oversensing through a successful control of the patient s blood sugar led to the tracking of the sensed or paced atrial impulses and biventricular pacing .
crt , a technology that simultaneously paces both the left and right ventricles , has emerged as an important treatment tool for heart failure patients with reduced lv function and ventricular dyssynchrony .
there are several possible reasons why as many as 30% of treated patients remain unresponsive to crt .
when a non - responder is identified , the first thing that should be determined is whether the patient is actually receiving crt .
there are two reasons for this problem : first , possible loss of function of the lead in the coronary sinus ; and second , the rapid or ectopic intrinsic heart rhythm ( e.g. , atrial fibrillation or frequent premature ventricular complexes ) .
one should also take into account inappropriate programming or oversensing as the other possible causes of biventricular pacing failure leading to crt failure . | t - wave oversensing occurs when the counter starts giving dual beeps for every cardiac cycle instead of one .
this usually happens when the monitoring lead displays a tall t wave , which is also sharp .
r wave sensing algorithms of the devices do not sense t wave because the slow rate of the t wave is much less than that of the r wave .
but the slow rate of t waves may change with time and also because of parameters like potassium levels and hyperglycemia .
we present a 67-year - old female who underwent the implantation of cardiac resynchronization therapy ( cardiac resynchronization and implantable cardioverter defibrilator [ crt - d ] ) because of severe left ventricular systolic dysfunction and ventricular dyssynchrony experienced recurrent inappropriate implantable cardioverter - defibrillator ( icd ) shocks and crt failure .
device analysis showed that the crt failure was in consequence of t - wave oversensing due to hyperglycemia .
elimination of the t - wave oversensing after hyperglycemia control conferred good biventricular pacing and good response to crt during a 6-month follow - up period . | Introduction
Case report
Discussion |
normally , a diverticulum is present in fetal life as the vitello - intestinal duct remnant , which usually disappears later on the 7th week of gestational period .
md occurs in about 2% of the population , with a male - to - female ratio of 2:1 .
it is located within two feet from the ileocecal valve , and it can be 2 in . in length and may vary based on clinical practice .
most cases of md are difficult to diagnose and are found incidentally during a surgical procedure for another reason . however ,
we describe a case of an adult female patient who presented with clinical picture of acute bowel obstruction due to md .
a 39 year old female patient known for hypothyroidism on thyroxine presented to king abdulaziz university hospital , emergency department , with a complaint of abdominal pain for 3 days .
then , it shifted to the right iliac fossa on the day of her er visit .
it was associated with vomiting , greenish in color , three times on the same day of presentation , in addition to history of constipation for 2 days .
there was no history of fever , diarrhea , bleeding per rectum , or urinary symptoms .
the abdomen was mildly distended . during palpation , the abdomen was soft and lax , with guarding in the right iliac fossa .
routine laboratory investigations were within normal limits : wbc 8.7 k/l , hb 15 g / dl , plt 240 , na 129 mmol / l , k 4.1 mmol / l , urea 4.1 mmol / l , and creatinine 41 mol / l . urine analysis was unremarkable .
erect abdominal x - ray was performed , and it revealed multiple air fluid levels with dilated small bowel loops and no free air .
she was kept npo on intravenous fluid resuscitation , nasogastric tube was inserted , and urine output was measured . in order to identify the cause of the bowel obstruction , a ct scan of abdomen and pelvis was performed as well , and it revealed multiple dilated loops of small bowel , reaching the maximum diameter of 4 cm with few air fluid levels .
there was a transitional point in the right iliac fossa , with whirling of the mesenteric vessels .
there was a diffuse wall thickening and an enchantment of the cecal wall with possible pneumatosis .
mild surrounding inflammatory changes were seen with minimal free fluid in the abdomen and pelvis .
meckel 's diverticulum was found to be perforated and adherent to the right lower abdominal wall ( fig .
a limited resection of involved part of ileum with the md was performed with subsequent primary anastomosis , in addition to appendectomy .
it was named after johann friedrich meckel , a german anatomist who described the features of md .
it is a true congenital diverticulum as it consists of all the three layers of the bowel wall and is usually found on the anti - mesenteric border of the ilium .
the average distance of the meckel 's diverticulum from the ileocaecal valve is 67 cm in adult population .
it can either be asymptomatic or mimic common abdominal disorders such as appendicitis , peptic ulcer disease and crohn 's disease .
however , they have been known to cause complications , including diverticulitis , intussusception , severe gastrointestinal hemorrhage , perforation , peptic ulceration and intestinal obstruction . yet ,
obstruction can be caused by trapping of a bowel loop by a mesodiverticular band , intussusception , a volvulus of the diverticulum around a mesodiverticular band , or an extension into a hernia sac called littre 's hernia .
the case described above presented with signs and symptoms suggestive of acute intestinal obstruction and radiological findings suggestive of cecal volvulus .
this patient had partial intestinal obstruction caused by adhesions of the diverticulum with the abdominal wall , causing cecal volvulus as discovered intraoperatively .
ct scan , technetium-99 m pertechnetate scan , abdominal ultrasound , and barium studies are all useful in the diagnosis of md . after diagnosing md , the mainstay treatment would be diverticulectomy in all symptomatic cases .
resection should always include the diverticulum or the segment of the bowel affected by the pathology . for asymptomatic md , evidence does not support elective resection of meckel 's diverticulum . in a systemic review , zani et al .
stated that a large number of md resections would need to be performed to prevent 1 death from md .
hence , avoiding surgery in case of asymptomatic md reduces the risk of postoperative complications caused by unnecessary surgical intervention .
although , md is the most common congenital condition of the gi tract , the clinical diagnosis of md is difficult to make .
ct scan can help identify the cause of bowel obstruction , but it may miss the diagnosis of md . hence , md is mostly identified during the operation .
appropriate knowledge of various pathophysiologies by which a meckel 's diverticulum can cause complication must be kept in mind for a better management without recurrences .
he was informed that this case will be written for publication as a case report with the accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
dr . aref 's contribution includes writing the manuscript and collecting the relevant data and pictures . | highlightsalthough , md is the most common congenital condition of the gi tract , the clinical diagnosis of md is difficult to make.most cases of md are found incidentally during a surgical procedure for another reason.the mainstay treatment would be resection of the diverticulum or the segment of the bowel affected by the pathology . | Introduction
Case report
Discussion
Conclusion
Conflict of interest
Funding
Ethical approval
Author contributions |
the fourth diabetes atlas , published by the international diabetes federation ( idf ) , revealed that there were an estimated 50.8 million cases of diabetes in india in 2010 .
data on hypertension , available from several regional studies , indicate an increase of about 30 times among urban indians and 10 times among rural indians , within a span of three and six decades , respectively . to date , no nation - wide epidemiological study aimed at determining the prevalence of hypertension has been reported .
hypertension is 1.52 times more prevalent in diabetic as in non - diabetic individuals and about 3035% of hypertensives are detected to have diabetes .
the combination of diabetes and hypertension imparts an additive risk and accelerates progression of both microvascular and macrovascular complications .
hence , these two conditions contribute significantly to the growing load of cardiovascular diseases and lead to a high economic burden that accounts for 520% of the total indian healthcare expenditure .
this rapid increase in diabetes and hypertension in india is most likely to result in a twin epidemic .
the screening india 's twin epidemic ( site ) study was aimed at determining the number of diagnosed and undiagnosed cases of diabetes and hypertension in india and reporting the measures employed to manage and control these diseases in an outpatient setting .
the study was conducted in eight states maharashtra , delhi , tamil nadu , west bengal , karnataka , andhra pradesh , madhya pradesh , and gujarat .
although site is not a population - based study , it is one of the largest studies of its kind .
also , in lieu of poor existing information , site serves as a good proxy for providing valuable insights on the national prevalence and management of both diabetes and hypertension .
we present herewith the site study design , methodologies and highlight the amendments made as the study progressed .
site was a cross - sectional , national , multicentric , non - randomized , observational study .
the primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states .
the secondary objectives included estimation of the prevalence of other cardiovascular risk factors such as dyslipidemia , obesity [ body mass index ( bmi ) , waist circumference ( wc ) , and waist - hip ratio ( whr ) ] , metabolic syndrome , urine microalbuminuria , arrhythmia , diet , smoking and alcohol consumption ; estimation of the prevalence of abdominal obesity in sub - groups of patients ( hypertension , diabetes , dyslipidemia , smokers and alcoholics ) ; and understanding the management and the level of control of hypertension and diabetes .
the study was conducted in accordance with the guidelines for good epidemiology practices . the protocol complied with recommendations of the 18 world health congress ( helsinki , 1964 ) and the local laws and regulations of india .
the sample size depended on the expected prevalence of diabetes / hypertension ( whichever was lower ) .
based on the conservative expected prevalence of 15% for diabetes , it was determined that a sample size of 1225 patients was required per state . assuming a dropout rate of 39% , it was estimated that 2000 patients were required to ensure 95% confidence such that the prevalence of diabetes is between 13% and 17% , that is , 15% 2% in each state .
it was initially planned to conduct the study in 10 states across five zones , north , south , east , west and central , in waves
one state at a time and recruit 2000 patients from 100 centers per wave .
physician selection : the study was conducted in outpatient settings and the data were collected from general practitioners rather than specialists .
also , the source of any potential bias was much reduced , as the number of people presenting the disease would evidently be higher at a specialist clinic rather than a general practitioner 's clinic .
patient selection : patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study .
pregnant women are predisposed to gestational diabetes and weight gain , and their exclusion decreased eventual confounding effects on the results . the first 10 patients visiting the physicians clinic on two consecutive days , irrespective of the purpose of the visit , and satisfying the inclusion criteria , were selected for the study . during 20092010
, site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [ figure 1 ] . for the remaining two planned states ,
on the basis of the fact that the sample size was calculated per state and that a low dropout rate was observed among the patients enrolled , the national coordinators deemed the data adequate to pan out substantial results .
so , by the end of the 8 wave , the recruitment was declared completed .
because of a national multicentric approach of the study , the data obtained were representative of diverse demographic populations within india .
the states in which the screening india 's twin epidemic study was conducted the study was sponsored by sanofi - aventis ( india ) .
site management organization ( smo ) and data management services were provided by max neeman ltd .
the sponsor ensured that the central laboratory , smo , and data management personnel dedicated to study , as well as the participating physicians , received adequate training on various aspects of the study .
thus , the complete study team was well versed with the data collection form ( dcf ) and the study procedures were executed uniformly . the data collection process was carried out in two steps [ figure 2 ] .
data collection process of the screening india 's twin epidemic study the dcf used was designed in line with the study objectives .
it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . details on the patient 's socioeconomic , educational , psychosocial , and physical activity status were not collected .
anthropometric measurements : height was measured without shoes , in centimeters , using a standard stadiometer .
weight was measured with light clothes and without shoes , in kilograms , using a professional calibrated weighing scale , and rounded off to the nearest number .
since no standard weighing machine was employed , inter - instrument variations can not be ruled out .
body mass index ( bmi ) was calculated using the formula : weight ( kg)/height ( m ) .
the hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . the hip circumference ( hc ) was measured over light clothing .
the patient was made to stand erect with arms at the sides , feet placed together with weight equally distributed on each leg , and the gluteal muscles relaxed .
the tape was placed horizontally around the point of the maximum posterior extension of the buttocks .
the patient 's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements .
the patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg .
the wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest .
the measurer stood beside the patient and made the tape fit snugly , without compressing any underlying soft tissues .
the circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration .
the method and angles of measurement were adequately detailed to ensure limited variance in the technique .
whr was calculated by dividing the wc ( cm ) by the hc ( cm ) .
blood pressure ( bp ) measurement : a mercury sphygmomanometer was used to measure the bp , preferably from the right arm of the patient .
an average of two readings , recorded at 5-minutes interval , was taken to ensure accuracy . at the end of visit 1
, patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers , within 2 - 5 days .
fasting lipid profile , microalbuminuria , fasting plasma glucose ( fpg ) , and hba1c were determined for each patient presenting a valid coupon .
hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method .
urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol ( cholesterol esterase ) , high - density lipoproteins ( hdl ; cholesterol oxidase , esterase , and peroxidase ) , and triglycerides ( tg ; lipoprotein lipase , glycerol kinase , glycerol-3-phosphate oxidase , and peroxidase ) . low - density lipoprotein ( ldl ) was also calculated by using friedewald 's equation for tg < 300 mg / dl .
diabetes was diagnosed according to the american diabetes association ( ada ) guidelines , and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) .
metabolic syndrome was diagnosed on the basis of national cholesterol education program ( ncep ) .
bmi was categorized into normal ( < 23 kg / m ) , overweight ( 2325 kg / m ) and obese ( > 25 kg / m ) , according to the guidelines jointly suggested by the indian health ministry , diabetes foundation of india , all india institute of medical sciences , indian council of medical research , and national institute of nutrition and 20 other institutions .
normal and truncal obesity was categorized according to the whr , as suggested by the ncep .
fasting lipid profile revealed the total cholesterol , tg , hdl - cholesterol , and ldl - cholesterol levels .
of these , elevated tg ( 150 mg / dl ) and concomitant low hdl - cholesterol ( < 40 mg / dl ) were used to identify dyslipidemia .
the criteria used are ( 1 ) primarily attributed to atherogenic dyslipidemia , ( 2 ) linked to insulin resistance , ( 3 ) measurable during the initial phase of development of metabolic syndrome , ( 4 ) individually extremely inheritable , ( 5 ) simple to measure , and ( 6 ) determinants of the atherogenic index of plasma ( aip ) , a key prognostic marker of cardiovascular disease , calculated as log ( tg / hdl - c ) . urine microalbumin was defined according to the albumin creatinine ratio ( acr ) as normoalbuminuria ( acr < 30 g / mg ) , microalbuminuria ( 30300 g / mg ) , and macroalbuminuria ( > 300 g / mg ) .
monitoring visits were randomly conducted for at least 20% of the active sites ( enrolled at least one patient ) and the reports documented .
phlebotomy services , testing , processing , shipment of blood and urine samples , and preparation and distribution of reports were undertaken by the central laboratory ( metropolis healthcare ltd . ) .
data entry , verification , and validation were carried out using pheedit database system , version 3.1 ( sas institute inc . , cary , nc , usa ) .
a double - entry method was used to ensure that the data ( except comments ) were transferred accurately from the dcfs to the database . moreover , every modification in the database could be traced using an audit trail .
a data checking plan was established to define all automatic validation checks , as well as the supplemental manual checks .
all discrepancies were researched until resolved . additionally , a national steering committee comprising india 's leading endocrinologists and cardiologists was established to provide scientific direction for the study .
the total sample number , mean , standard deviation , and range ( minimum and maximum ) were calculated for all continuous variables ( age , height , wc , hc , and lipid values ) , and frequency and percentage were calculated for all categorical variables ( gender , presence of hypertension and diabetes and their treatment , smoking status , and urine microalbuminuria ) .
student 's t - test and chi - square test were used to make comparisons between groups , with p value < 0.05 considered statistically significant .
a multivariate analysis was performed to estimate the association of hypertension and diabetes with dyslipidemia , obesity , metabolic syndrome , microalbuminuria , arrhythmia , smoking , alcohol consumption , and diet .
a simple linear regression analysis was performed to estimate the contribution of individual variables to systolic and diastolic bp .
relative risk for developing diabetes and hypertension was calculated by assessing the following variables : age , gender , bmi , whr , family history of diabetes and hypertension , and smoking status .
site was a cross - sectional , national , multicentric , non - randomized , observational study .
the primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states .
the secondary objectives included estimation of the prevalence of other cardiovascular risk factors such as dyslipidemia , obesity [ body mass index ( bmi ) , waist circumference ( wc ) , and waist - hip ratio ( whr ) ] , metabolic syndrome , urine microalbuminuria , arrhythmia , diet , smoking and alcohol consumption ; estimation of the prevalence of abdominal obesity in sub - groups of patients ( hypertension , diabetes , dyslipidemia , smokers and alcoholics ) ; and understanding the management and the level of control of hypertension and diabetes .
the study was conducted in accordance with the guidelines for good epidemiology practices . the protocol complied with recommendations of the 18 world health congress ( helsinki , 1964 ) and the local laws and regulations of india .
the sample size depended on the expected prevalence of diabetes / hypertension ( whichever was lower ) .
based on the conservative expected prevalence of 15% for diabetes , it was determined that a sample size of 1225 patients was required per state . assuming a dropout rate of 39% , it was estimated that 2000 patients were required to ensure 95% confidence such that the prevalence of diabetes is between 13% and 17% , that is , 15% 2% in each state .
it was initially planned to conduct the study in 10 states across five zones , north , south , east , west and central , in waves
one state at a time and recruit 2000 patients from 100 centers per wave .
physician selection : the study was conducted in outpatient settings and the data were collected from general practitioners rather than specialists .
also , the source of any potential bias was much reduced , as the number of people presenting the disease would evidently be higher at a specialist clinic rather than a general practitioner 's clinic .
patient selection : patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study .
pregnant women are predisposed to gestational diabetes and weight gain , and their exclusion decreased eventual confounding effects on the results . the first 10 patients visiting the physicians clinic on two consecutive days , irrespective of the purpose of the visit , and satisfying the inclusion criteria , were selected for the study .
during 20092010 , site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [ figure 1 ] . for the remaining two planned states ,
on the basis of the fact that the sample size was calculated per state and that a low dropout rate was observed among the patients enrolled , the national coordinators deemed the data adequate to pan out substantial results .
so , by the end of the 8 wave , the recruitment was declared completed .
because of a national multicentric approach of the study , the data obtained were representative of diverse demographic populations within india .
site management organization ( smo ) and data management services were provided by max neeman ltd .
the sponsor ensured that the central laboratory , smo , and data management personnel dedicated to study , as well as the participating physicians , received adequate training on various aspects of the study .
thus , the complete study team was well versed with the data collection form ( dcf ) and the study procedures were executed uniformly .
the data collection process was carried out in two steps [ figure 2 ] . data collection process of the screening india 's twin epidemic study the dcf used was designed in line with the study objectives .
it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension .
details on the patient 's socioeconomic , educational , psychosocial , and physical activity status were not collected .
anthropometric measurements : height was measured without shoes , in centimeters , using a standard stadiometer .
weight was measured with light clothes and without shoes , in kilograms , using a professional calibrated weighing scale , and rounded off to the nearest number .
since no standard weighing machine was employed , inter - instrument variations can not be ruled out .
body mass index ( bmi ) was calculated using the formula : weight ( kg)/height ( m ) .
the hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . the hip circumference ( hc ) was measured over light clothing .
the patient was made to stand erect with arms at the sides , feet placed together with weight equally distributed on each leg , and the gluteal muscles relaxed .
the tape was placed horizontally around the point of the maximum posterior extension of the buttocks .
the patient 's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements .
the patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg .
the wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest .
the measurer stood beside the patient and made the tape fit snugly , without compressing any underlying soft tissues .
the circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration .
the method and angles of measurement were adequately detailed to ensure limited variance in the technique .
whr was calculated by dividing the wc ( cm ) by the hc ( cm ) .
blood pressure ( bp ) measurement : a mercury sphygmomanometer was used to measure the bp , preferably from the right arm of the patient .
an average of two readings , recorded at 5-minutes interval , was taken to ensure accuracy . at the end of visit 1
, patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers , within 2 - 5 days .
fasting lipid profile , microalbuminuria , fasting plasma glucose ( fpg ) , and hba1c were determined for each patient presenting a valid coupon .
hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method .
urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol ( cholesterol esterase ) , high - density lipoproteins ( hdl ; cholesterol oxidase , esterase , and peroxidase ) , and triglycerides ( tg ; lipoprotein lipase , glycerol kinase , glycerol-3-phosphate oxidase , and peroxidase ) . low - density lipoprotein ( ldl ) was also calculated by using friedewald 's equation for tg < 300 mg / dl .
it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension
. details on the patient 's socioeconomic , educational , psychosocial , and physical activity status were not collected .
anthropometric measurements : height was measured without shoes , in centimeters , using a standard stadiometer .
weight was measured with light clothes and without shoes , in kilograms , using a professional calibrated weighing scale , and rounded off to the nearest number .
since no standard weighing machine was employed , inter - instrument variations can not be ruled out .
body mass index ( bmi ) was calculated using the formula : weight ( kg)/height ( m ) .
the hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . the hip circumference ( hc )
the patient was made to stand erect with arms at the sides , feet placed together with weight equally distributed on each leg , and the gluteal muscles relaxed .
the tape was placed horizontally around the point of the maximum posterior extension of the buttocks .
the patient 's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements .
the patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg .
the wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest .
the measurer stood beside the patient and made the tape fit snugly , without compressing any underlying soft tissues .
the circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration .
the method and angles of measurement were adequately detailed to ensure limited variance in the technique .
whr was calculated by dividing the wc ( cm ) by the hc ( cm ) .
blood pressure ( bp ) measurement : a mercury sphygmomanometer was used to measure the bp , preferably from the right arm of the patient .
an average of two readings , recorded at 5-minutes interval , was taken to ensure accuracy . at the end of visit 1
, patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers , within 2 - 5 days .
fasting lipid profile , microalbuminuria , fasting plasma glucose ( fpg ) , and hba1c were determined for each patient presenting a valid coupon .
hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method .
urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol ( cholesterol esterase ) , high - density lipoproteins ( hdl ; cholesterol oxidase , esterase , and peroxidase ) , and triglycerides ( tg ; lipoprotein lipase , glycerol kinase , glycerol-3-phosphate oxidase , and peroxidase ) . low - density lipoprotein ( ldl ) was also calculated by using friedewald 's equation for tg < 300 mg / dl .
diabetes was diagnosed according to the american diabetes association ( ada ) guidelines , and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) .
metabolic syndrome was diagnosed on the basis of national cholesterol education program ( ncep ) .
bmi was categorized into normal ( < 23 kg / m ) , overweight ( 2325 kg / m ) and obese ( > 25 kg / m ) , according to the guidelines jointly suggested by the indian health ministry , diabetes foundation of india , all india institute of medical sciences , indian council of medical research , and national institute of nutrition and 20 other institutions .
normal and truncal obesity was categorized according to the whr , as suggested by the ncep . fasting lipid profile revealed the total cholesterol , tg , hdl - cholesterol , and ldl - cholesterol levels .
of these , elevated tg ( 150 mg / dl ) and concomitant low hdl - cholesterol ( < 40 mg / dl ) were used to identify dyslipidemia .
the criteria used are ( 1 ) primarily attributed to atherogenic dyslipidemia , ( 2 ) linked to insulin resistance , ( 3 ) measurable during the initial phase of development of metabolic syndrome , ( 4 ) individually extremely inheritable , ( 5 ) simple to measure , and ( 6 ) determinants of the atherogenic index of plasma ( aip ) , a key prognostic marker of cardiovascular disease , calculated as log ( tg / hdl - c ) .
urine microalbumin was defined according to the albumin creatinine ratio ( acr ) as normoalbuminuria ( acr < 30 g / mg ) , microalbuminuria ( 30300 g / mg ) , and macroalbuminuria ( > 300 g / mg ) .
monitoring visits were randomly conducted for at least 20% of the active sites ( enrolled at least one patient ) and the reports documented .
phlebotomy services , testing , processing , shipment of blood and urine samples , and preparation and distribution of reports were undertaken by the central laboratory ( metropolis healthcare ltd . ) .
data entry , verification , and validation were carried out using pheedit database system , version 3.1 ( sas institute inc . ,
a double - entry method was used to ensure that the data ( except comments ) were transferred accurately from the dcfs to the database . moreover
a data checking plan was established to define all automatic validation checks , as well as the supplemental manual checks .
all discrepancies were researched until resolved . additionally , a national steering committee comprising india 's leading endocrinologists and cardiologists was established to provide scientific direction for the study .
the total sample number , mean , standard deviation , and range ( minimum and maximum ) were calculated for all continuous variables ( age , height , wc , hc , and lipid values ) , and frequency and percentage were calculated for all categorical variables ( gender , presence of hypertension and diabetes and their treatment , smoking status , and urine microalbuminuria ) .
student 's t - test and chi - square test were used to make comparisons between groups , with p value < 0.05 considered statistically significant .
a multivariate analysis was performed to estimate the association of hypertension and diabetes with dyslipidemia , obesity , metabolic syndrome , microalbuminuria , arrhythmia , smoking , alcohol consumption , and diet .
a simple linear regression analysis was performed to estimate the contribution of individual variables to systolic and diastolic bp .
relative risk for developing diabetes and hypertension was calculated by assessing the following variables : age , gender , bmi , whr , family history of diabetes and hypertension , and smoking status .
screening studies play a very important role in determining the trends of disease prevalence ; such trends can provide valuable insight regarding necessary corrective measures that can be implemented in a given population .
india has witnessed a constant increase in the prevalence of diseases such as diabetes and hypertension .
however , most previous reports on the prevalence of both diabetes and hypertension are either regional or vary in methodologies and sampling frames .
site aims not only to indicate the most accurate prevalence data of diabetes and hypertension in an outpatient setting in india at the state , regional , and national levels , but also to raise awareness on the need for early diagnosis and on the management of these diseases to reduce complications .
after wave-1 and wave-2 , the protocol was refined and the laboratory investigations simplified and then employed for all other subsequent waves . during the first wave conducted in maharashtra , the investigations included : random blood sugar ( rbs ; in all patients ) , electrocardiogram ( ecg ; in patients 40 years of age ) , and hba1c ( in known cases of diabetes ) at visit-1 ; and the oral glucose tolerance test ( ogtt ; in patients with rbs 140 mg / dl ) and microalbuminuria test ( in patients with rbs 140 mg / dl and in patients with hypertension ) at visit-2 . the ogtt involved a lengthy procedure that was logistically difficult to perform in general clinics .
going forward , this attrition could have potentially introduced a bias in the study results .
thus , based on the experiences of the pilot study in maharashtra , certain protocol improvements were suggested for the second wave conducted in the national capital territory , delhi .
the ada recommends either ogtt or fpg and disease - related symptoms ( polydipsia , polyuria , and unexplained weight loss ) for the confirmation of diabetes . during wave-2 ,
the ogtt was not performed ; fpg was measured for all unknown cases of diabetes , as opposed to rbs in the pilot study .
though signs and symptoms of diabetes were recorded in this amended protocol , they did not play a decisive role in diagnosing new cases of diabetes .
additionally , specifications with regard to alcohol consumption and diet of patients were included in the dcf .
alcohol consumption and diet are established high - risk factors leading to the development and progression of diabetes and hypertension .
since significant regional differences exist in the indian diet , it would be interesting to assess if a correlation exists between the regional prevalence of these diseases and diet . to establish a connection between lifestyle factors ( diet and alcohol consumption ) and prevalence of diabetes and hypertension
this inclusion also broadened the scope of secondary analyses to be performed with the results of this study . during wave-2 , difficulties in maintaining uniformity in the ecg recordings and in dealing with logistical hurdles of organizing the ecg test at the sites surfaced .
the inability to standardize ecg testing in a study of this magnitude would lead to inaccurate data generation .
additionally , as an effort to further simplify the protocol , the fpg and hba1c testing was conducted in all patients from wave-3 onward .
however , by employing hba1c as a screening tool as per the newly proposed guidelines released in july 2009 , our approach to diagnosing diabetes was further simplified .
additionally , the correlation between fpg and hba1c for the diagnosis of diabetes could also be studied .
an important limitation of this study is the reliance on a single - day fpg and bp measurement to detect new cases of diabetes and hypertension , respectively .
also , owing to the cross - sectional nature of our study , the possibility of residual confounding can not be ruled out .
nonetheless , site is one of the largest prevalence studies on diabetes and hypertension conducted in outpatient settings in india . due to the national multicentric approach of the site study ,
the amendments to the site protocol were efforts made to maintain consistency and accuracy of the data collected as the study progressed through each subsequent wave .
the wave-3 protocol ( conducted in tamil nadu ) reflected the most adequate data collection process for this study and was employed for all other waves , permitting robust analyses . in conclusion , site will provide a real - world national perspective on diabetes and hypertension and their contributing risk factors .
it will identify practice variations across states and evaluate compliance to international guidelines for the management of diabetes and hypertension .
the standardization of the data collection process and the data analyses will justify various comparisons .
the study will raise awareness on the need for early diagnosis and management of disease to reduce complications .
site data will be useful for national recommendations , policies and guidelines , and will also support future exploratory researches . | objectives : the recent years have seen a surge in the prevalence of both diabetes and hypertension . significant demographic variations reported on the prevalence patterns of diabetes and hypertension in india establish a clear need for a nation - wide surveillance study . the screening india 's twin epidemic ( site ) study aimed at collecting information on the prevalence of diagnosed and undiagnosed diabetes and hypertension cases in outpatient settings in major indian states to better understand disease management , as well as to estimate the extent of underlying risk factors.materials and methods : during 20092010 , site was conducted in eight states , in waves
one state at a time .
it was planned to recruit about 2000 patients from 100 centers per wave .
each center enrolled the first 10 eligible patients ( 18 years of age , not pregnant , signed data release consent form , and ready to undergo screening tests ) per day on two consecutive days .
patient demographics , medical history , and laboratory investigation results were collected and statistically interpreted .
the protocol defined diabetes and hypertension as per the american diabetes association ( ada ) and seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) recommendations , respectively.results:after the first two pilot phases in maharashtra and delhi , the protocol was refined and the laboratory investigations were simplified to be further employed for all other states , namely , tamil nadu , west bengal , karnataka , andhra pradesh , madhya pradesh , and gujarat.conclusion:site's nation - wide approach will provide a real - world perspective on diabetes and hypertension and its contributing risk factors . results from the study will raise awareness on the need for early diagnosis and management of these diseases to reduce complications . | I
M
Study design
Sample size calculation
Sampling design
Study waves
Training
Methods
At visit 1
At visit 2
Definitions and diagnostic criteria
Quality control
Statistical considerations
R |
ageing is a worldwide recognized fact , implying social and economic aspects , wich lay in the way ageing is controled , because it could have an enormous impact regarding healthcare , hospitalization , and/or reconstructive surgery costs .
ageing is a degrading , progressive , inward - oriented , universal process , and the threshold between physiological and pathological is very thin . although the general approach towards elderly patients is to increase the quality of life ( free prescriptions , occupational therapy , medical healthcare at home ) , there are a lot of real and recognized situations when the ageing phenomenon may lead to discrimination that can range from providing poor medical healthcare to restricted accessibility to some healthcare programs ( e.g. , screening for breast cancer in women over 65 ) .
however , we must be aware that the ageing process is in steady growth in all developed countries for two main reasons : on the one hand due to reduced mortality and on the other due to decreasing tendency of birth rate and fertility .
aging is defined as being a slow , irreversible changing process that occurs within single cells the seat of some remarkable biochemical activity , whose development is genetically programmed in advance .
the notion of time in biology equals a measurement of motion in the form of rhythms and cycles , of which the circadian biorhythms of the endocrine functions are the most important .
variations in normal hormonal secretions differ from one hormone to another in the course of 24 hours , so acth and cortisol levels reach their peaks during early morning .
one of the most well - spread theories regarding aging is based on neuroendocrinology , and it well suits the global concept stating the lack of balance between degradation and repair , balance influenced by many internal and external factors . circadian rhythm of cortisol secretion is stable , with little influence from light and sleep , and it is also highly reproductibile . it
matures around the age of 4 , remaining unchanged into elder years , with no difference in the two sexes .
the rhythm of cortisol secretion is dependent on the circadian secretion of acth , its acrophase being around 4 am .
feedback on the hypothalamic - pituitary axis , is exerted by cortisol on the autonomous circadian rhythm .
. the rhythm is defined by a peak between the hours of 68 am , the lowest value being recorded in the evening .
if more determinations of hormone levels are analysed , an ultradian rhythm is revealed , with a daily pulsatile secretion which reflects the episodic secretion of the zona fasciculata , in four phases over night : before bed , low cortisol secretion ; during sleep , between 35 am , higher secretion levels ; from the last hours of sleep and the first hour after awakening , maximum discharge ; during activity periods , intermittent discharge .
clinical and paraclinical evaluation of the degree of impaired hormonal secretions in diseases general and endocrine disorders open new perspectives that will optimize chronomodulated therapeutic methods and means in relation to biological rhythms .
starting from these general considerations , we wanted to make some observations on the dynamics of cortisol secretion in healthy elderly people and those with known cardiovascular disease , especially those with hypertension .
the study was conducted in two locations : the municipal clinical hospital philanthropy and the emergency clinical county hospital in craiova during 20032010 , with both a prospective and a retrospective component .
case studies presented are based on a number of 135 patients , over 65 years of age , who were divided into two groups : group one ( subset 1 ) consisting of 66 healthy elderly people ( control group ) , without systemic , renal , endocrine , or cardiovascular known issues at the time of enrollment in the group and group 2 ( subset 2 ) consisting of 69 elderly patients who associate diagnosed hypertensive disease and other cardiovascular issues ( ischemic heart disease , degenerative valvular heart disease , or arrhythmias , all of the patients being in compensated stages of heart failure grade i or ii nyha , without any endocrine disorders ) . of these , only 41 patients can be considered as true hypertensive patients , because the hypertension was the dominant issue compared to the others associated problems .
the cases were investigated by means of clinical examination and cardiovascular evaluation carried out on standard protocol , supplemented by laboratory tests and further investigation ( hematological , biochemical , enzymatic , hormonal , immunological , and imaging tests ) .
blood pressure measurement was made under the same circumstances in all subjects in the morning and evening , in supine and standing positions .
blood pressure was also measured in both arms due to the presence of atherosclerotic lesions , which in the elderly patients can lead to variations in the levels of the blood pressure values .
measurements were recorded for three consecutive days , and each individual patient 's values was averaged to determine systolic and diastolic blood pressure . the who criteria for defining hypertension were commonly used .
we considered as significant for the inclusion in the category of hypertensive patients , values higher than 140 mm hg ( regarding systolic pressure ) , and also values higher than 80 mm hg ( regarding the diastolic values ) . in order to be able to correctly classify hypertension , in addition to blood pressure measurement values
, we aimed to assess whether or not target organ damage could be recorded , optic blood vessels were analysed , ecg and echocardiography ( for left ventricular hypertrophy ) were performed , as well as urinary protein dosage .
patients in both groups were put through further hematological and biochemical sorting investigations , along with serum cortisol determination .
the dosage system elecys 1010 found in the laboratory of the emergency clinical county hospital of craiova was used to measure cortisol levels .
the sorting and assessment of trial groups were completed by specific - cardiovascular oriented investigations ( echocardiography and electrocardiogram ) and the complex imaging explorations ( radiology , ultrasound , and mri ) .
the results were processed by statistical methods ( student 's t - test , bartlett 's test , kruskal - wallis ' test , chi - squared test , the arithmetic mean , standard deviation , anova test , and simple linear regression ) , using the data analysis subpackage module microsoft excel program and also the ep12000 program , specialized in the execution of graphs , tables , and statistical tests . in this study , we will use data from the measurements of cortisol levels , with normal values admitted to this study for cortisol serum levels in the morning ( measured between 710 am ) ranging between 171 and 536 nmol / l ( cortisol 1 ) , while cortisol values in the evening ( measured between the hours of 68 pm ) range between 64 and 327 nmol / l ( cortisol 2 ) .
in this study , we analyzed two groups of patients in order to observe the dynamics of hormonal secretion in elderly patients : a control group ( subset 1 ) consisting of 66 healthy elderly people and a study group ( subset 2 ) consisting of 69 elderly patients diagnosed with hypertensive disease associated with various cardiovascular diseases .
the percentage analysis of cases grouped by age showed that the largest share of older people in both groups belonged to age group of 7584 years .
the average values of biochemical and hematological parameters were within normal limits of age in both groups , except for some values of glucose or lipid fractions .
subset 1 or the control group was represented by healthy elderly , who had been submitted for routine clinical and laboratory tests . following the recollection of past medical history , anamnesis , clinical examination , and laboratory tests ,
the subset was composed of people over 65 years of age ( limits between 65 and 92 years ) , mean age being 78.31 in men and 78.53 in women .
three age groups have been made : first age group ranging between 65 and 74 , second one ranging from 75 to 84 , and the last group for patients older than 84 .
the group 's structure included more men ( 40 male cases ) than women ( 26 cases ) , sex ratio equaling 1.53 in the studied group .
mean cortisol measured at this time among the cases in subset 1 was 336.92 nmol / l ( limits at 172537 nmol / l , ci95%
a statistically significant difference between mean values of cortisol 1 was not recorded in subjects from subset 1 regarding age differences ( p = 0.168 ) , but mean cortisol 1 was higher in women ( mean 396.38 nmol / l ; limits 266536 nmol / l , ci95% 360.16432.61 nmol / l ) than in men ( mean 298.27 nmol / l , limits 172537 nmol / l , ci95%
t - test to compare mean values identified a high statistical significance for the difference between the two areas ( p = 0.0012 ) .
an average value of 205.58 nmol / l ( limits 67327 nmol / l ; ci95% 186.27224.88
measurements in the evening recorded no significant variation regarding age groups in the healthy eldery ( p = 0.09 ) , but mean cortisol 2 was higher in women ( mean 238.81
ci95% 217.27260.34 nmol / l ) than in men ( mean 183.97 nmol / l , limits 67327 nmol / l ,
ci95% 156.76211.19 nmol / l ) , with high statistical significance for the difference between the two areas ( p = 0.0047 ) .
subset 2 consisted of elderly patients with hypertension and cardiovascular disease , but not endocrine issues .
these patients were admitted for previously diagnosed cardiovascular issues or newly diagnosed disease . following the anamnesis , physical examination , and laboratory findings , a total of 69 patients were selected for the study . similar to subset 1 , subset 2 was composed of elderly over 65 years of age ( ages ranging from 65 to 90 years ) ; the patients were also divided into three age groups the same as group 1 .
men were at a slight advantage ( 40 cases58% ) over women ( 29 cases42% ) , sex ratio equaling 1.38 in the studied group .
women taken into study from this group had ages between 65 and 89 years of age ( mean age 77.69 ) ; the men had ages ranging from 65 to 90 years old ( mean age 78.22 ) .
mean cortisol 1 measured at 8 am , in subjects included in group 2 , was 392.93 nmol /
ci95% 363.66422.19 nmol / l ) , while average cortisol 2 measured at 8 pm , among the cases in group 2 , was 228.55 nmol / l ( limits 73326 nmol / l , ci95%
210.58246.52 nmol / l ) . there was no statistically significant link between the age of the subjects of subset 2 and cortisol values 1 or 2 .
knowing that , in the elderly multiple pathology is very frequent ; it was quite difficult to select patients with one cardiovascular disease .
thus , we have selected for the study the most frequently encountered heart diseases in clinical practice : high blood pressure ( 59% ) , ischemic heart disease ( 58% ) , heart failure ( 29% ) , and fibrillation ( 28% ) .
the associations between the above - mentioned issues are as follows : first pairing was represented by hypertension
ischemic heart disease ( 17 cases , 24.6% ) , followed by the association of hypertension - heart failure ( 11 cases , 15.9% ) , hypertension and atrial fibrillation ( 5 cases , 7.2% ) , and finally hypertension , ischemic heart disease , heart failure , and atrial fibrillation ( 3 cases , 4.3% ) . for these reasons we have formed a group of patients called subset 2 , with cardiovascular issues , but with clinical dominating hypertension symptoms , consisting of 41 subjects .
mean cortisol 1 , measured within the hypertension dominant group ( subset 2 ) , was 483.73 nmol / l ( limits 321536
471.46496.01 nmol / l ) . comparing the mean morning cortisol in the control group ( 336.92 nmol / l ) to
that obtained in patients from subset 2 , we noticed that the average value is higher for the latter ( 483.73
nmol / l ) ; the difference between the two values being highly significant in statistical terms ( p < 0.001 ) ( table 1 ) . by groups of age ,
mean cortisol 1 values were over 130 nmol / l higher in hypertensive subjects compared with the subjects in group 1 , regardless of age pairings .
nmol / l in group 2 , while the subjects in group 1 did not record a value higher than 346 nmol / l ( figure 1 ) .
regardless of the grading of the hypertensive disease , patients with hypertension in group 2 had an average value of cortisol 1 higher than the one recorded in the control group . also , as the severity of the hypertensive disease was greater , mean cortisol 1 was higher , the difference being highly significant ( p < 0.001 ) . thus , the subjects in subset 1 had a mean cortisol 1 of 336.92 nmol / l , while patients ' cortisol 1 value ( from subset 2 ) increased proportional to the grading of the hypertensive disease ( grade 1450 nmol / l , grade 2482.31 nmol / l , and grade522.17 nmol / l ) ( figure 2 ) .
cortisol 2 measured at 8 pm averaged at 205.58 nmol / l in subset 1 ( limits 67327 nmol / l ; ci95% 186.27224.88
the average value of cortisol measured at 8 pm was higher228.55 nmol / l ( limits 73326
the difference between the 2 obtained values was not significant in statistical terms . comparing the average value of cortisol 2 in subset 1 ( 205.58 nmol / l ) with the one obtained from the subjects of subset 2
nmol / l ) is higher than the average value of cortisol 2 in the control group , this being highly significant ( p < 0.001 ) ( table 2 ) . if we analyse by age group , mean values of cortisol 2 ( measured in the evening )
were about 70 nmol / l higher in group 2 compared to the subjects of the control group in all age groups ( figure 3 ) .
regardless of the grade of the hypertensive disease , mean cortisol 2 values were higher than in group 1 , and the more severe the hypertension was , the higher the mean value of cortisol 2 was .
the difference recorded to the control group is highly significant in statistical terms ( p < 0.001 ) ( figure 4 ) . for group 1
nmol / l , and for cortisol 2 a mean value of 205.58 nmol / l was identified .
we can clearly say that in control group subjects , for all age groups , mean cortisol 2 values were about 130 nmol / l lower than those recorded during the mornings ( figure 5 ) .
nmol / l , and mean cortisol value measured at 8 pm in the same group was 228.55
just like in the case of the control group , we can clearly state that the patients with cardiovascular issues , in all age groups , have mean values of cortisol 2 lower than the morning value with over 150 nmol / l ( figure 6 ) .
patients in group 2 had a more pronounced decrease in cortisol 2 values than in cortisol 1 ( over 150
nmol / l ) , compared with the decrease recorded in the control group ( about 130
the patients in group 2 recorded a mean cortisol 1 value higher ( 392.93 nmol / l ) than the one of the control group ( 336.92 nmol / l ) , the difference between the two values being statistically significant ( p = 0.009 ) . although average values of cortisol 2 were higher in patients with cardiovascular disease ( 228.55 nmol / l ) compared with the values obtained in the control group ( 205.58 nmol / l ) , the difference was not significant in statistical terms . for group 2 (
cardiovascular afflicted patients , with dominating hypertension symptoms ) , mean morning cortisol was 483.73
nmol / l and the evening cortisol in the same patients was 281.42 nmol / l . in hypertensive patients ,
mean cortisol 1 values , by age groups , were similar , ranging between 481 and 486 nmol / l , to cortisol 2 values which ranged between 275 and 289 nmol / l ( figure 7 ) .
decrease in cortisol 2 values in patients with hypertension was around 200 nmol / l to morning cortisol in all age groups .
comparing , the biggest difference between values of cortisol 1 and 2 , in patients in group 2 , was recorded in the age groups of 75 to 84 and over 84 years ( 42.7% and 42.8% ) ( figure 8) .
the present study confirms some existing data in the literature , namely that , in the elderly , normal levels of cortisol are recorded . because of purely economic reasons
, multiple measurements of cortisol levels could not be performed in the same patient . in some studies , normal values of cortisol in elder patients have been recorded , but with a decrease in the amplitude of its diurnal variations ; slightly elevated values have also been recorded , especially in men with cardiovascular diseases , resistance to cortisol suppression also being noticed in other studies . in this study ,
the dynamics of cortisol was within normal reference limits , both in healthy elderly and in elderly hypertensive patients , with occasional narrowing or widening of the range of obtained results .
we noted that healthy women over 65 years in group 1 had higher mean cortisol levels than average elderly men from the same set .
elderly hypertensive patients had mean cortisol values greater than those obtained in healthy people aged over 65 years .
it is highly important that the mean values of cortisol , regardless of when they were determined ( morning or evening ) , varied along with the severity of blood hypertension : the higher the degree of hypertension was , the higher the mean cortisol value was .
patients with cardiovascular disease , especially hypertensive patients , showed a more pronounced decrease of evening determined cortisol values , compared with healthy elderly .
mean morning and evening cortisol measured in both groups were within normal limits , approaching the upper limit of reference .
mean morning and evening cortisol determined were higher in healthy women compared with men from the same group .
the mean values of morning and evening cortisol recorded in hypertensive patients were higher than the values obtained from healthy patients .
the severity of blood hypertension correlated with cortisol values : the higher the grading of hypertension was , the higher mean cortisol determined during morning or evening was . we take into account the need to introduce the screening of hormonal levels ( specifically cortisol values ) in elderly patients , because the modified values of certain hormones may become predictive factors for the evolution and prognosis of cardiovascular diseases . | ageing is defined as a slow , irreversible process of cellular changes , that are due to a lack of balance between degradation and repair , a continuous interaction between physiological and pathological processes .
physiological aspects in elderly people are often confused with disease . given these general considerations , we would make observations about the dynamics of cortisol secretion in healthy elderly subjects and patients with a diagnosed cardiovascular disease , more precisely hypertension .
the study was conducted during 20032010 , on a number of 135 patients older than 65 years of age , who were divided into two groups : one group counting 66 patients and consisting of healthy elderly controls ( without systemic disease , renal , endocrine , or cardiovascular known issues ) and group 2 who consists of 69 elderly patients who associate known hypertensive and other cardiovascular issues . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion |
lymphatic filariasis is common in tropical countries and is endemic in india with most infections caused by wuchereria bancrofti .
the microfilaria are not just confined to the lymphatic system , but also associated with other organs , subcutaneous tissues , and serous cavities like pleura and pericardium .
demonstration of microfilariae ( mf ) in the peripheral blood smear or in the tissues is the mainstay of diagnosis .
filariasis acutely presents with fever , adenolymphangitis , funiculitis , epididymitis , orchitis , or pericarditis .
lymphedema , hydrocele , elephantiasis and chyluria , and pleural or pericardial effusion are features of chronic infection .
our case was a rare as recurrent hemorrhagic pericardial effusion and tamponade , which was first treated with diethyl carbamazine and finally albendazole .
she complained of cough and breathlessness on exertion and low grade fever since 3 weeks and generalized weakness since 1 week . on examination , she was severely dyspneic and had pulsus paradoxus . echocardiogram showed massive pericardial effusion with cardiac tamponade [ figure 1 ] .
apical four - chamber view showing large pericardial effusion with right atrial collapse ( left ) and parasternal short axis view showing large pericardial effusion with right ventricular outflow tract ( rvot ) diastolic collapse ( right ) pericardiocentesis was done and 1,300 ml of hemorrhagic fluid was collected .
results of complete blood picture were : total count - 6,800/mm , polymorphs - 67% , lymphocytes - 23% , eosinophils - 06% , monocytes - 04% , platelet count - 160,000/mm , hemoglobin - 11 g% , erythrocyte sedimentation rate ( esr ) - 40 1 h. , human immunodeficiency virus ( hiv)-nonreactive , and hepatitis b surface antigen ( hbsag)-negative .
complete urine examination , blood sugar , urea , creatinine , and electrolytes ; all were in normal range .
cytological analysis of fluid showed - 7,000 cells / mm , ( neutrophils - 35% , lymphocytes - 65% , plenty of red blood cells ( rbc ) ) , negative for malignant cells , sugar - 61 mg% , proteins - 6 g% , albumin - 2.5 g% , adenosine deaminase ( ada ) - 23 u / l , acid - fact bacilli ( afb ) - negative ; microscopy and routine cultures were negative for bacteria . while screening the smears , a few large , long , smooth - walled , refractile unstained worm - like structures were observed .
the organisms were identified as mf of w. bancrofti in the smears stained by leishman 's stain [ figure 2 ] .
cytology showed plenty of rbc , occasional pus cells , and microfilaria of w. bancrofti [ figure 3 ] .
the patient improved rapidly by next day and effusion was grossly reduced by the 2 day .
pericardial fluid cytology smear showing microfilaria of wuchereria bancrofti with occasional pus cells ( leishman stain , 1000 ) pericardial fluid cytology smear showing microfilaria of w. bancrofti with occasional pus cells ( leishman stain , 40 )
it is transmitted by the culex mosquito and is caused by two closely related nematodes , w. bancrofti and brugia malayi , which are responsible for 90 and 10% of the cases , respectively .
microfilaria have been confined not only to the lymphatic system but also associated with other organs , subcutaneous tissues , and serous cavities like pleura and pericardium .
pericardial effusion is a rare manifestation of filariasis , and recurrent effusion with tamponade is even more rare .
our case was of massive pericardial effusion established as filarial in origin on demonstration of mf in pericardial aspirate .
it might have lodged itself through blood stream subsequent to development of pericarditis . as mf are capable of penetrating the tissues of the vector , they can freely move in and out of small , delicate blood , and lymphatic capillaries and tissues of the vertebrate host .
peripheral blood smears may be negative for mf in cases of secondary manifestations of filariasis . in this case
patient developed effusion despite being treated with dec suggests that it may not be 100% effective in all cases as dec does not act on microfilaria residing in lymphatics , and they might have migrated to pericardial space causing recurrent effusion .
she was finally cured with alternate combination of ivermectin and albendazole , which makes it even more interesting .
although mf in unusual sites are considered incidental findings , on the basis of existing evidences we suggest that some of the cases of recurrent pericardial effusion , especially those from endemic areas , might be of filarial origin .
it is therefore emphasized that a thorough examination of the aspirated fluid for mf is warranted in all patients with unexplained pericardial effusion . | filariasis presenting with pericardial effusion with tamponade is rare .
we report a case of a 30-year - old female who was admitted with severe dyspnea and chest pain since 2 days .
echocardiogram showed massive pericardial effusion with tamponade .
pericardial fluid aspiration drained 1.2 l of hemorrhagic fluid .
cytology examination revealed microfilaria of wuchereria bancrofti .
she was treated with diethyl carbamazine and discharged .
six weeks later , she presented again with massive pericardial effusion with cardiac tamponade .
pericardiocentesis was done .
cytology examination revealed microfilaria of w. bancrofti .
this time she was treated with ivermectin and albendazole and cured .
hemorrhagic effusion resolved completely . though relatively uncommon , tropical diseases must always be considered in the etiological diagnosis of recurrent pericardial effusion . | INTRODUCTION
CASE REPORT
DISCUSSION |
lymphatic filariasis ( lf ) , a mosquito - transmitted parasitic disease caused by the filarial nematodes wuchereria bancrofti , brugia malayi , and brugia timori , is classified as a neglected tropical disease ( ntd ) that is endemic in many parts of the world including the south pacific [ 13 ] .
the global elimination programme to eliminate lf ( gpelf ) began in the late 1990s , and the pacific counterpart of gpelf , formed in 1999 under the auspices of the world health organization ( who ) , was named the pacific programme for the elimination of lymphatic filariasis ( pacelf ) [ 4 , 5 ] .
pacelf resolved to eliminate lf as a public health problem in the pacific using mass drug administrations ( mdas ) .
elimination of lf was defined as < 1% circulating filarial antigen ( cfa ) prevalence of the population and < 0.1% cfa prevalence in children born after the implementation of mdas .
sixteen of the 22 countries falling under the jurisdiction of pacelf were classified as endemic for lf following baseline prevalence surveys .
they were american samoa , the cook islands , the federated states of micronesia , fiji , french polynesia , kiribati , the marshall islands , new caledonia , niue , palau , papua new guinea , samoa , tonga , tuvalu , vanuatu , and wallis and futuna .
the cook islands , niue , vanuatu , and tonga have reached < 1% cfa prevalence of the population following five rounds of mda and are now implementing activities to ensure that transmission has been interrupted and to detect any remaining and/or new foci of transmission . the challenge for this phase of the lf programme is to find a suitable strategy for surveillance in those countries that have reached the target threshold .
recently , child transmission surveys ( cts ) have been proposed as a potential method for surveillance in the south pacific as part of the draft lf active surveillance strategy for the pacific island countries and territories ( pict ) .
this method relies on detecting cfa positive children in either school - based or community - based surveys .
the strategy was based on the preapproved lf diagnostic assays available and took into consideration resource and funding constraints . upon detection of a cfa positive child ,
using the field immunochromatographic test ( ict ) , surrounding residents ( 200 m radius or 24 houses ) would be tested to identify the microfilaraemic ( mf ) positive case from the child 's house of residence . since drafting the surveillance strategy , three south pacific countries have finished their cts , either alone or in conjunction with post - mda prevalence surveys .
the cts was completed in two countries which had entered surveillance mode , tonga and vanuatu , and samoa , where transmission was ongoing . as the prevalence of lf declines ,
the sensitivities of the gold standards , which measure mf and cfa , also decrease [ 913 ] , potentially making these assays less useful during this stage of the lf elimination programme [ 1416 ] .
antibody responses to lf exposure have been suggested as an ideal immunological measurement for identifying areas of residual endemicity in future surveillance campaigns [ 9 , 14 , 1719 ] .
previous studies demonstrated that antibody production in response to lf exposure occurs during the first few years of life .
therefore , children born after the cessation of lf transmission should be antibody negative [ 17 , 2127 ] .
one standardised diagnostic tool for the detection of antibodies is the filariasis cellabs enzyme linked immunosorbent assay ( celisa ) .
this diagnostic assay measures antifilarial igg4 and is adaptable for both serum samples and filter paper samples with little interlaboratory variation [ 28 , 29 ] .
antibody serology can be used in conjunction with seroprevalence mapping , which has been demonstrated to be useful for visualising problem areas where exposure occurs .
filariasis control began in tonga in 1977 , and by 1999 , when tonga joined pacelf , the cfa prevalence was recorded as 2.7% ( http://www.wpro.who.int/pacelf/programmes/ton/pacelf+activities+to+date.htm ) .
mda rounds were completed annually in tonga for five years from 2001 to 2005 with reported coverage rates of 79% , 84% , 91% , 86% , and 85% , respectively , . following the fifth round of mda
, tonga recorded an overall cfa prevalence of 0.4% and , therefore , completed its cts in 2007 .
the annual rounds were from 2000 to 2004 with reported coverage rates of 83% , 84% , 84% , 87% , and 85% , respectively , .
following the final round of mda in 2005 , countrywide cfa prevalence was 0.32% , with no detectable mf positives ( vanuatu country report , unpublished data ) .
consequently , vanuatu completed its cts in 2007 , the results of which are presented in this paper .
samoa has a long history of filariasis control ; initial filariasis surveys began as early as the 1920s , with attempts at control programmes in the 1940s . in 1966 , mdas began and samoa completed 10 rounds of mda before the establishment of pacelf [ 31 , 32 ] . in 1999 , samoa was the first country to implement the new mda regimen using a combination of diethylcarbamazine citrate ( dec ) and albendazole .
the reported mda coverage for the five rounds conducted from 1999 to 2003 was 90% , 57% , 68% , 60% , and 80% , respectively , . in 2004 ,
a stratified cluster nationwide survey demonstrated an overall mf prevalence of 0.4% with a cfa prevalence of 1.1% .
this corresponded to a 75.6% reduction in cfa positive individuals since the beginning of the new national programme in 1999 [ 4 , 5 ] .
the results from the follow - up survey in 2007 are presented in this paper in conjunction with the 2007 cts .
the aim of this research was to determine if the presence of cfa positive children alone accurately reflected a cessation of lf transmission , as assumed in the draft surveillance strategy , or if it was necessary to complement cfa testing with antibody serology using the filariasis celisa . by doing so
, we help contribute to defining a cessation of lf transmission and early detection of possible resurgence as part of active surveillance .
this will assess the potential of the filariasis celisa to be incorporated into the repertoire of available lf diagnostic assays currently used in lf elimination programmes .
serological mapping could then be used to easily identify and visualise areas where exposure is ongoing and where further investigations could be required by the country lf programme managers .
three south pacific countries , samoa , tonga , and vanuatu , participated in the study in 2007 .
the study was conducted under human ethics approval number h1423 , as approved by the james cook university research human ethics committee .
the post - mda prevalence survey was a national survey conducted by health district , across both savai'i and upolu using the stratified cluster method .
stratified cluster sampling was performed by dividing the country into implementation units ( ius ) within which villages were chosen randomly . within the village , a minimum of five households were randomly chosen .
all participants were bled for ict testing and those that tested ict positive were rebled for mf testing .
filter paper samples were also collected from children aged 5 to 10 years for antibody serology .
cts carried out in tonga was in accordance with the draft lf active surveillance strategy for the pict using the school - based approach , but with the addition of antibody serology .
blood collection occurred in primary schools across the islands of eua , ha'apai , and vava'u .
all participants were bled for ict testing and those that tested ict positive were rebled for mf testing .
a total of 3840 children aged 5 to 6 years were bled across the country in accordance with the cts outlined in the draft lf active surveillance strategy for the pict using the community - based approach , but with the addition of antibody serology .
registration and informed verbal consent , approximately 220 l of blood was collected by fingerprick .
one hundred microlitres was used for cfa testing in the field . if the ict test was positive , 60 l
the remaining blood was soaked onto the six protrusions of a tropbio filter paper disc , each holding 10 l of blood ( tropbio pty ltd , qld , australia ) .
the filter paper disc was dried , placed in ziplock bags , and transported back to australia for storage at 20c until testing for antifilarial antibodies .
the field test to detect cfa was the now filariasis immunochromatographic test ( ict ) and performed according to the manufacturer 's instructions as previously described ( binax , portland , me , usa ) .
briefly , the collected 100 l of blood was transferred onto the absorbent pad using the capillary tube and the result was read at exactly 10 minutes , according to the manufacturer 's instructions , and recorded as positive , negative , or invalid .
positives ( referred to as cfa positive ) were rebled for mf examination . blood taken from a fingerprick
was smeared into three lines , approximately 20 l thick , onto a microscope glass slide using a capillary as previously described .
the slides were left to dry for 48 hours then wrapped for transport . in the laboratory of each country , each slide was stained in 10% giemsa stain ( 20 minutes ) , washed in water , dried , then coverslipped .
the slide was examined under the microscope ( 200 ) , and mf were recorded .
the number of mf per ml of blood was calculated based on the initial 60 l volume .
blood collection for mf testing in vanuatu and tonga occurred between 2200 and 0100 hours .
blood collection for mf testing in samoa occurred between 0800 and 2000 hours according to peak levels of mf and biting tendencies of aedes polynesiensis .
antifilarial igg4 antibodies were detected using the filariasis celisa kit ( cellabs pty ltd , manly , australia ) .
one protrusion of filter paper was eluted overnight at 4c in 500 l of sample diluent .
the following morning , the elution was thoroughly vortexed and assayed in duplicate , according to the manufacturer 's instructions .
the washing steps were performed with an automated plate washer ( multidrop combi nl , pathtec , vic , australia ) using 200 l per well .
plates were read at a dual wavelength of 450 nm and 650 nm with a multiskan ex type 355 primary v.2.1 - 0 ( pathtec , vic , australia ) using the software labsystems genesis version 3.00 ( pathtec , vic , australia ) .
negative samples were defined as od value < 0.260 , and positive samples were defined as od value 0.400 .
samples with values between these od values were repeated , in accordance with the manufacturer 's instructions , and if the od value was < 0.400 , they were considered negative . antibody prevalence was based on positive / negative reactivity of the samples .
prevalence rates were calculated using the descriptive options in spss , and 95% confidence interval ( ci ) was calculated using the javastat binomial software ( http://statpages.org/confint.html ) and included in the figures .
chi - squared test was used to investigate the difference in prevalence rates among countries and health districts in samoa .
the kruskall - wallis test for nonparametric data was used to compare the od absorbance values ( antibody titres ) among the three countries .
in vanuatu and tonga , all children tested negative for cfa , whereas in samoa there were mf and cfa positive children detected ( figure 1 ) .
this was coupled with a significantly lower antibody prevalence recorded for both vanuatu ( 6.0% ) and tonga ( 6.3% ) when compared to samoa ( 30.7% ) ( p < 0.001 , figure 1 ) .
in addition , of those children who were antibody positive , there was a significantly higher average antibody titre in samoa than vanuatu and tonga ( p = 0 , figure 2 ) . consequently in samoa , the overall significantly higher antibody prevalence and titre , coupled with mf and cfa positive children , was further analysed by health district to identify potential residual areas of endemicity .
when analysing the data by health district , leulumoega 1 , on upolu , had a significantly higher mf prevalence ( 2% ) ( p < 0.001 ) and cfa prevalence ( 7.3% ) ( p < 0.001 , figure 3 ) . furthermore , including palalui , leulumoega 1 had a significantly higher antibody prevalence in children ( 44.9% ) ( p < 0.001 , figure 3 ) .
there were three health districts with detectable mf positive adults , but no cfa positive children ( safotu ( 0.2% ) and tuasivi ( 0.3% ) on savai'i and leulumoega 2 ( 0.4% ) on upolu ) .
in these areas , mf prevalence exceeded 0.1% , the defined threshold for ongoing transmission , and the antibody prevalence in children exceeded 20% .
prevalence for the three parameters was easily visualised on the geographical maps , including residual endemic areas in the aforementioned districts ( figure 4 ) . when analysing the data for children only , upolu maintained a significantly higher cfa prevalence ( p = 0.043 ) , yet
no differences were observed for mf prevalence between the islands ( p = 0.218 , figure 5 ) . for the health districts ,
mf positive children were found only in leulumoega 1 , which had a significantly higher antibody prevalence ( figure 3 ) and the highest cfa prevalence , although not significantly higher than the other villages with cfa positive children ( p = 0.271 ) .
lufilufi and palalui were the other 2 villages with cfa - positive children ( figure 5 ) .
active lf surveillance requires accurate and sensitive diagnostic assays to detect residual areas of endemicity and resurgence early .
the current draft lf active surveillance strategy for the pict relies solely on cfa testing in children , since the use of antibody serology is not yet currently approved .
this is due to the need for standardisation , which can now be achieved using the commercially available filariasis celisa ( cellabs pty ltd ) .
it was the aim of this research to assess the validity of the current surveillance strategy and if incorporating antibody serology would be advantageous in the pict .
the first aim of the research was partially addressed , but until long - term monitoring of the outcomes of the elimination programme occurs , the strategy will continue to be dynamic to meet individual country needs .
the data indicated the cessation of lf transmission in both tonga and vanuatu and ongoing lf transmission in samoa . in tonga and vanuatu , there were no detectable cfa positive children coupled with a significantly low antibody prevalence and titre .
identification of mf and cfa positive children in samoa was coupled with a significantly higher antibody prevalence and titre . for vanuatu and tonga ,
the higher value of antibody prevalence ( 6% ) versus the cfa prevalence ( 0% ) is a reflection of the sensitivity and specificity of the filariasis celisa for filter paper sampling .
when using filter paper samples , the filariasis celisa has a positive predictive value ( ppv ) of 60% ( 95% ci = 4375 ) .
this means that a false positive is expected at a rate of approximately 40% ( 95% ci = 25% to 57% of the time ) .
filter paper samples have inherent higher od values , but it would be disadvantageous to use serum for wide - scale programmatic sampling because of the difficulties with collection and storage .
the higher ppv of the filariasis celisa does not impact on the usefulness of the diagnostic assay from a programmatic perspective , since recent research has demonstrated the use of the assay for identifying clusters of exposed children .
it could be suggested that it would be appropriate for the country programme managers to follow up any identified clusters of exposed children with a more in - depth serological study using the gold standard serum .
previous studies in egypt , where the vector culex sp . is endemic , suggest a < 2% antibody prevalence in first - year primary school children as a threshold for elimination .
this study did indicate the potential necessity for different threshold targets for other endemic areas .
the data presented here does not concur with a 2% antibody prevalence threshold for interruption of transmission in the pict since both vanuatu and tonga recorded prevalence rates of 6% and transmission was interrupted .
however , the studies in egypt used serum samples with laboratory - based elisas , not filter paper samples with the commercial filariasis celisa .
the detection of ongoing transmission in samoa was further analysed by each health district to assess if the current surveillance strategy could identify problem areas .
cfa testing alone in children ( cts ) was not adequate for identifying all of the key residual areas of endemicity in samoa .
there were three health districts with detectable mf positive adults , but no cfa positive children , where transmission would likely be occurring .
this is because in these health districts mf prevalence exceeded 0.1% , the recommended threshold by who for the interruption of transmission and spatial clustering of lf has been previously identified .
the health districts identified were safotu and tuasivi on the island of savai'i and leulumoega 2 on the island of upolu . in these health districts ,
very importantly , it must be emphasised that these health districts would not have been detected as areas of residual or reemerging infection based on cfa testing in children alone and would have been missed if the proposed who surveillance programme using ict testing was in place .
these significant foci were only identified because antibody serology was included in the testing protocol .
therefore , it would be beneficial to strengthen the current strategy surveillance strategy by including antibody serology . in samoa
, the identification of mf and cfa positive children is indicative of ongoing transmission , which is further evidenced by a significantly higher antibody prevalence and titre in children .
these findings support previous studies whereby samoa exceeded 1% cfa prevalence following five rounds of mda .
the reasons for ongoing transmission in samoa are yet to be ascertained , but it is crucial that these are addressed in order for lf elimination to be successful . it has been suggested that lack of directly observed therapy ( dot ) , lack of compliance , and lack of vector control could all be contributing factors [ 8 , 31 , 41 , 42 ] .
recent research has indicated the potential for vector control in samoa which will have a positive impact on the samoan lf elimination programme .
the seroprevalence maps in the current study could provide the samoan lf programme managers with a starting point to further investigate residual endemic areas and potentially incorporate vector control .
in addition , the results from the current study indicate that antibody prevalence thresholds need to be complemented with analysing antibody titres , since the low antibody prevalence in tonga and vanuatu was coupled with a significantly low antibody titre in positive children . collectively , low prevalence and low titre ( with no detectable antigen - positive children ) suggest an interruption of transmission . during active surveillance mode , the detection of an area with low antibody prevalence but high antibody titre could require further investigation
. in conclusion , transmission of lf has been interrupted in tonga and vanuatu as evidenced by no detectable cfa positivity in children and significantly low antibody prevalence and titres .
ongoing transmission of lf is evident in samoa based on the presence of mf / cfa positive children and a significantly high antibody prevalence and titres .
most importantly , key residual areas of endemicity , which can also be observed by seroprevalence mapping , would have been overlooked when testing for cfa positivity alone in children .
this holds promise for future incorporation of antibody prevalence mapping in the current surveillance strategy for the pict . | elimination of lymphatic filariasis ( lf ) in the pacific island countries and territories ( pict ) has been defined as < 0.1% circulating filarial antigen ( cfa ) prevalence in children born after the implementation of successful mass drug administrations ( mdas ) .
this research assessed the feasibility of cfa and antibody testing in three countries ; tonga , vanuatu , and samoa .
transmission is interrupted in vanuatu and tonga as evidenced by no cfa positive children and a low antibody prevalence and titre .
transmission is ongoing in samoa with microfilaraemic ( mf ) and cfa positive children and a high antibody prevalence and titre .
furthermore , areas of transmission were identified with mf positive adults , but no cfa positive children .
these areas had a high antibody prevalence in children . in conclusion ,
cfa testing in children alone was not useful for identifying areas of residual endemicity in samoa .
thus , it would be beneficial to include antibody serology in the pict surveillance strategy . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion and Conclusions |
piroxicam ( pir ) ( 4-hydroxy-2-methyl - n-(pyridine-2-yl)-2h-1,2-benzothiazine-3-carboxamide-1,2-dioxide ) is a well - known nonsteroidal anti - inflammatory and analgesic drug , indicated for acute or long - term treatment of inflammation associated with musculoskeletal and joint disorders , such as osteoarthritis , rheumatoid arthritis , and ankylosing spondylitis .
nonsteroidal anti - inflammatory drugs ( nsaids ) are the group of analgesics and anti - inflammatory drugs most often used around the world , mainly to treat pain , inflammation , and fever in human .
these pharmaceuticals are weak acidic compounds because of their carboxylic groups or keto - enol tautomeric structure with their pk values between 3 and 7 .
several analytical methods have been described for the determination of pir , including spectrophotometry [ 39 ] , spectrofluorimetry [ 1012 ] , thin - layer chromatography ( tlc ) [ 13 , 14 ] , liquid chromatography ( lc ) [ 9 , 1323 ] , and capillary electrophoresis ( ce ) [ 7 , 13 , 2427 ] .
ce has emerged as a powerful analytical technique in the analysis of pharmaceutical compounds , such as nsaids .
the ce methods developed for the determination of pir in the previous studies were presented without validation except the study of bartsch et al . which indicated only precision data .
donato et al . reported also only linearity , precision , and accuracy in injectable formulation of pir and no further validation was shown .
one of them was a micellar electrokinetic chromatography ( mekc ) method for tablet analysis .
linearity range was not very well specified for the determination of pir ; only the quality control ( qc ) standards compared to pharmaceutical tablets were also presented without validation .
another method reported by chen and wu was a capillary zone electrophoretic ( cze ) method for the simultaneous determination of seven drugs such as pir , sulindac , ketoprofen , indomethacin , nimesulide , ibuprofen , and naproxen .
linearity range was 13.24165 g / ml and lod was 3.31 g / ml with 13 min of migration time .
. demonstrated pir photodecomposition using three different concentrations ( 40 g / ml , 250 g / ml , and 2 mg / ml ) by three methods including high performance lc ( hplc ) , high performance tlc ( hptlc ) , and ce . the quantification was evaluated only by external calibration without use of internal standard ( is ) and only precision data was presented .
boone et al . simultaneously separated six acidic drugs such as hydrochlorothiazide , pir , ibuprofen , phenobarbital , salicylic acid , and chlorothiazide in spiked serum and urine extracts by using cze , mekc , and nonaqueous ce ( nace ) techniques as well as basic drugs .
this study reports only separation of analytes by cze with 90 mm sodium tetraborate ( ph 8.4 ) , by mekc with 20 mm sodium phosphate and 50 mm sds ( ph 7.5 ) , and by nace with 20 mm ammonium acetate in methanol / acetonitrile ( 9 : 1 , v / v ) containing 1% acetic acid . pir ( 1 g / ml ) eluted up to 4 min in serum and urine by cze , up to 12 min with ibuprofen as unresolved peaks by mekc and nace application were not reported for pir .
separated 11 nsaids including pir in a background electrolyte solution of 100% methanol and 13 nsaids in a second background electrolyte solution of acetate in methanol and acetonitrile ( 70 : 30 , v / v ) by using nonaqueous system showing pir resolution from other drugs and its migration time appeared up to 8 min in both systems but the reported study did not cover any method validation or pharmaceutical application .
therefore there is a need for the determination of pir in pharmaceutical tablets by cze with full validation .
the aim of this study is to develop a validated , simple , and rapid cze method for the analysis of pir in pharmaceutical tablets .
the proposed method was linear over wide range of 0.2328.79 g / ml and was validated in relation to precision , accuracy , selectivity , and sensitivity ( with lod of 0.07 g / ml for pir ) in qc standards and tablet matrix .
pir and naproxen sodium ( nap ) as is were purchased from sigma chemical co. ( usa ) .
all other chemicals were of analytical grade and were purchased from merck gmbh company ( germany ) .
ultrapure water was purified with a milli - q system of millipore ( usa ) .
commercial pir tablets ( felden flash , pfizer , turkey ) containing 20 mg pir were obtained from a local pharmacy .
ce ( thermo separation products , spectra phoresis 100 , usa ) was performed with spd-10a model uv detector ( shimadzu , japan ) and data was processed by cr-7a model ( shimadzu , japan ) integrator .
fused - silica tubing of 53.6 cm effective and 68.2 cm total length ( agilent technologies , usa ) .
the ph of the solutions was measured by a model of m-822 ph meter ( electro - mag , turkey ) .
all solutions were sonicated in a b-220 model ultrasonic bath ( branson , usa ) before injection .
the final concentration of the is was always 6.76 g / ml . background electrolyte ( bge ) consisted of 10 mm borate buffer containing 10% ( v / v ) methanol adjusted to ph 9.0 . for uv spectrophotometric experiments 0.36 ,
0.72 , 1.08 , and 1.44 g / ml pir solutions were prepared in methanol and methanol was used as blank .
fused - silica capillary used for the first time was conditioned by flushing with 1.0 m naoh for 30 min followed by 0.1 m naoh , ultrapure water , and bge for 10 min , respectively .
each day , the capillary was washed and conditioned by rinsing for 10 min with each of 0.1 m naoh , ultrapure water , and bge , respectively .
the samples were then injected into the fused - silica capillary filled with bge , by vacuum injection for 1 s. between each run the capillary was rinsed with 0.1 m naoh ( 2 min ) , distilled water ( 2 min ) , and bge ( 2 min ) . at the end of each working day
, it was washed with 0.1 m naoh and ultrapure water for 10 min and left with aspirated air . during analysis
the precision of the method was determined by the measurement of repeatability ( intraday ) and intermediate precision ( interday ) .
standard solution of pir 1.79 g / ml was injected on three consecutive days , six times in a day .
the linearity of the method was investigated with 8 concentrations in the range of 0.2328.79 g / ml . linearity was evaluated by linear regression analysis using the least square regression method .
calibration plots were chosen in this range and six concentrations were injected for three consecutive days .
0.23 g / ml , 1.79 g / ml , and 14.41 g / ml pir solutions were used for accuracy studies .
the accuracy in matrix was determined by adding pir onto matrix to give the final concentrations of 0.26 , 2.56 , and 25.58 g / ml .
the matrix was prepared as common tablet excipients such as hydroxypropyl methylcellulose ( 7% ) , lactose monohydrate ( 60% ) , magnesium stearate ( 1% ) , polyethylene glycol 4000 ( 5% ) , povidone ( 5% ) , maize starch ( 5% ) , talc ( 1% ) , and titanium dioxide ( 1% ) .
quantification was accomplished on the basis of pir to nap normalized peak area ratios ( rpn ) [ i.e. , ( peak area of pir / migration time of pir)/(peak area of nap / migration time of nap ) ] .
the method was applied to a pir tablet as in the pharmacopeial rules . for the application of the method , 10 felden flash ( each containing 20 mg pir ) tablets were weighed to calculate the average weight of a tablet .
the supernatant was diluted with methanol to the appropriate concentration to measure the amount of pir .
buffer ph has an influence on the degree of ionization of the molecules , their electrophoretic mobilities , and electroosmotic flow .
therefore , alkaline conditions were considered to be suitable for the determination of pir according to its molecular structure .
initially , borate buffer was investigated in the concentration range of 1025 mm and 15 mm borate provided the optimum results .
methanol was preferred for the modifier of the bge because it decreases the mobility of the components in capillary column .
it was found out that better migration times and good peak shapes were obtained with the use of 10% ( v / v ) methanol .
, the electroosmotic flow has reached a maximum , fairly constant mobility and pir is completely ionized , so that its corresponding electrophoretic mobility was not affected by any slight variations in ph .
pir was in an anionic form in the bge of ph 9.0 and eluted after electroosmosis .
well - shaped peaks of pir appeared at + 25 kv and the applied potential was kept at + 25 kv . despite higher voltages which are preferred in ce , voltages higher than 25 kv caused problems such as current generation , poor separation , and resolution between pir and is .
an injection time of 1 s was used because longer duration caused zone broadening .
the optimum conditions determined were as follows : 10 mm borate buffer at ph 9.0 , containing 10% ( v / v ) methanol for bge .
+ 25 kv applied potential and 1 s of injection time were used as the instrumental parameters .
then nap was injected as is , and it appeared in appropriate time and resolution in the electropherogram . under these analytical and instrumental conditions ,
the migration times were 8.11 0.03 min for pir and 8.60 0.04 min for nap ( n = 6 ) as seen in figure 2(a ) .
electrophoretic mobilities were calculated as 1.5 10 cm / sv for pir and 1.66 10 cm / sv for nap . the electrophoretic mobility for pir
10 cm / sv in only one of the ce studies which used nonaqueous system and it is not comparable with this study .
the precision of the method was examined as individual days ( intraday ) and intermediate precision ( interday ) of rpn , expressed as a rsd% of a series of measurements .
statistical evaluation of the precision results showed that rsd% values both intraday and interday were below 2% .
the linearity of the method was examined in the range of 0.2328.79 g / ml and the calibration plots were chosen in this range .
good correlation fit the equation as y = 52511x 0.0027 with correlation coefficient of 0.9999 .
the percentage error was calculated by use of the formula [ ( found concentration spiked concentration)/spiked concentration ] 100% .
the acceptance criteria are not higher than 2% deviation from the nominal value for accuracy .
the percent recoveries were found almost 100% for drug substance and drug product , and accuracy was much less than the acceptable criteria .
lod and loq were estimated as [ ( standard deviation of regression equation)/(slope of the regression equation ) ] by multiplying with 3.3 and 10 , respectively .
the lod was 0.07 g / ml and the loq was 0.19 g / ml . specificity was performed using tablet inactive ingredients to assure that these common tablet dosage form ingredients could interfere with the peaks of interest .
the data indicated that these ingredients did not interfere with pir and is peaks indicating the specificity of this method as seen in figures 2(b ) and 2(c ) .
the method was applied to pharmaceutical tablets ( felden tablet ) containing 20 mg active material .
the peaks of tablet samples carried the characteristics of standard pir , and there was no interference originating from the matrix as seen in figure 2(d ) .
the values of rsd% were below 2% and the percent recoveries were almost 100% as seen in table 4 .
for the determination of pir in commercial tablets , to compare the results of cze method , uv spectrophotometric method was developed .
analysis was performed with standard addition method to show the selectivity parameter . a good linear relationship fitting the equation [ y = 84691x + 0.3974 ] , with high correlation coefficient ( 0.9999 ) , was obtained between added amounts to tablet and absorbance values .
also the common tablet dosage form ingredients did not interfere with pir peaks shown by standard addition technique , so the method was selective for pir .
uv spectrophotometry and cze , used for the determination of pir in tablets , were statistically compared with t- and f tests .
the calculated t value for the methods was 1.60 and was less than the table t value ( 1.73 ) at 95% confidence level .
calculated f values were 0.77 and 2.14 for cze and uv spectrophotometry , respectively . at 95% confidence level ,
the ratio of two f values was calculated as 2.77 and was less than the table f value ( 5.05 ) .
the difference between the two methods was found to be insignificant as a result of t- and f tests .
although some of the chromatographic methods [ 1420 , 22 , 23 ] seemed to have lower lods and shorter analysis times , cze methods need small amount of analyte volumes , and it takes shorter time to condition the capillary .
this study has lower sensitivity than a previous chromatographic one . among the ce studies the sensitivity of this method either is better than in [ 7 , 24 , 27 ] or could not be compared because the sensitivity is not declared [ 13 , 25 , 26 ] .
some of the ce studies [ 13 , 26 , 27 ] have shorter analysis time than this study .
but also the developed method here has shorter analysis time with better resolution than a ce study and could not be compared with some of them [ 7 , 25 ] because the analysis time is not declared . according to the best of our knowledge , the specificity of the proposed method was not shown in the tablet matrix in previous ce studies and the method sensitivity is the best one .
therefore , it could be a promising method for the possible application of pir in biological matrix because of low detection limit .
in this study , a simple and rapid capillary electrophoretic method for the determination of pir in tablets was developed and validated .
validation of the method has been shown with parameters of linearity , precision , lod and loq , accuracy , and specificity .
it was found that all the results of percent coefficient of variation are below 2% showing the method was valid . the cze method developed here | simple and rapid capillary zone electrophoretic method was developed and validated in this study for the determination of
piroxicam in tablets .
the separation of piroxicam was conducted in a fused - silica capillary by using 10 mm borate buffer ( ph 9.0 ) containing 10% ( v / v ) methanol as background electrolyte .
the optimum conditions determined were 25 kv for separation voltage and 1 s for injection time .
analysis was carried out with uv detection at 204 nm .
naproxen sodium was used as an internal standard .
the method was linear over the range of 0.2328.79 g / ml . the accuracy and precision were found to be satisfied within the acceptable limits ( < 2% ) .
the lod and loq were found to be 0.07 and 0.19 g / ml , respectively .
the method described here was applied to tablet dosage forms and the content of a tablet was found in the limits of usp-24 suggestions . to compare the results of capillary electrophoretic method ,
uv spectrophotometric method was developed and the difference between two methods was found to be insignificant .
the capillary zone electrophoretic method developed in this study is rapid , simple , and suitable for routine analysis of piroxicam in pharmaceutical tablets . | 1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion |
heart rate variability ( hrv ) is the variation in the time interval between heartbeats , and an hrv assessment is used for examining the interaction between the sympathetic and parasympathetic nervous systems . because of the convenience and noninvasiveness of the examination ,
measurement of hrv includes a time domain and a frequency domain , which generally represent the autonomic nervous activity and balance , respectively .
hrv is reported to be related to age , gender , obesity , hypertension , diabetes , and hyperlipidemia .
a decreased hrv reflects an imbalance of the autonomic system and is an important predictor for poor prognosis in patients with heart disease and in the general population and in a medical preparation before surgery .
hrv may also be influenced by alterations in respiration , smoking habits , and the circadian rhythm and should be performed under a careful procedural protocol to minimize artifacts and achieve more reliable results .
colonoscopy is considered to be the standard procedure in the diagnosis and treatment of colonic diseases and is commonly administered worldwide .
adequate bowel preparation is essential for optimal performance and visualization of mucosal lesions and details , contributing to the success of a colonoscopy .
patients undergoing bowel preparation may experience gastrointestinal discomfort associated with nausea , vomiting , perianal irritation , dizziness , weakness , fatigue , thirst , and headache .
these pharmacological effects in healthy individuals generally do not cause severe complications and subside quickly . in a modern health examination setting
, participants frequently undergo colonoscopy and hrv analysis on the same day . however , information regarding whether bowel preparation before colonoscopy affects hrv is lacking .
if bowel cleansing affects hrv , the derived hrv result might reveal an acute status rather than the basal status , possibly resulting in misinterpretation of the autonomic nervous condition .
a total of 1755 participants , aged 45 to 65 years submitted to health check - ups at a medical center in taipei city from january 2012 to december 2013 .
we retrospectively retrieved participants profiles from the health check - up database and conducted a cross - sectional observational study .
this study was approved by the institutional review board of the tri - service general hospital , national defense medical center ( tsghirb103 - 05 - 106 ) .
all of the participants received health examinations , which included a physical examination , biochemical analysis , and hrv examination . among these participants ,
bowel preparation before colonoscopy included oral administration of 2 bottles ( 45 ml each ) of oral sodium phosphate , 12 hours apart , 1 day before the health examination .
this study analyzed the data on the participants demographic characteristics ( age and gender ) , weight ( kg ) and height ( cm ) , body mass index ( bmi ; kg / m ) , waist circumference ( cm ) , systolic blood pressure ( sbp ; mm hg ) and diastolic blood pressure ( dbp ; mm hg ) and biochemical values , including triglyceride ( tg ; mg / dl ) , high - density lipoprotein ( hdl ; mg / dl ) , low - density lipoprotein ( ldl ; mg / dl ) , fasting plasma glucose ( mg / dl ) , sodium ( mmol / l ) ,
potassium ( mmol / l ) , calcium ( mmol / l ) , phosphorus ( mmol / l ) , blood urea nitrogen ( mg / dl ) , and creatinine ( mg / dl ) . participants with a fasting blood glucose level above 126 mg / dl were considered to be diabetic .
hrv was assessed using a measuring system ( sa-3000p ; medicore co. , ltd , korea ) to acquire , store , and process electrocardiography ( ecg ) signals for 5 minutes .
the participants were examined in a seated position in a quiet and dark room with a regular temperature ( 2224c ) , and the ecg recording was performed in the morning ( 8:00 am12:00 noon ) to minimize the diurnal fluctuation .
the time and frequency domains were calculated automatically . for the time domain , standard deviation of all normal to normal intervals ( sdnn , ms ) , and root mean square of successive differences ( rmssd , ms ) were measured . for the frequency domains , low - frequency component of spectral hrv ( lf , 0.040.15 hz , ms ) , high - frequency component of spectral hrv ( hf , 0.150.4 hz , ms ) , and lf / hf ratio were measured using the fast fourier transform system .
hf reflects the vagal modulation of the sinus node , and lf represents the sympathetic modulation or a combination of the sympathetic and vagal influences .
the lf / hf ratio is considered to reflect the sympathovagal influence on the heart rate control . the time and frequency domain parameters
the statistical significance of the differences between the respective groups was evaluated using student t test for the continuous data and pearson test for the categorical variables .
the association between hrv and bowel preparation was derived by a multivariable linear regression with adjusted factors , including age , gender , diabetes mellitus , bmi , sbp , tg , and hdl .
a total of 1755 participants , aged 45 to 65 years submitted to health check - ups at a medical center in taipei city from january 2012 to december 2013 .
we retrospectively retrieved participants profiles from the health check - up database and conducted a cross - sectional observational study .
this study was approved by the institutional review board of the tri - service general hospital , national defense medical center ( tsghirb103 - 05 - 106 ) .
all of the participants received health examinations , which included a physical examination , biochemical analysis , and hrv examination . among these participants ,
bowel preparation before colonoscopy included oral administration of 2 bottles ( 45 ml each ) of oral sodium phosphate , 12 hours apart , 1 day before the health examination .
this study analyzed the data on the participants demographic characteristics ( age and gender ) , weight ( kg ) and height ( cm ) , body mass index ( bmi ; kg / m ) , waist circumference ( cm ) , systolic blood pressure ( sbp ; mm hg ) and diastolic blood pressure ( dbp ; mm hg ) and biochemical values , including triglyceride ( tg ; mg / dl ) , high - density lipoprotein ( hdl ; mg / dl ) , low - density lipoprotein ( ldl ; mg / dl ) , fasting plasma glucose ( mg / dl ) , sodium ( mmol / l ) ,
potassium ( mmol / l ) , calcium ( mmol / l ) , phosphorus ( mmol / l ) , blood urea nitrogen ( mg / dl ) , and creatinine ( mg / dl ) . participants with a fasting blood glucose level above 126 mg / dl were considered to be diabetic .
hrv was assessed using a measuring system ( sa-3000p ; medicore co. , ltd , korea ) to acquire , store , and process electrocardiography ( ecg ) signals for 5 minutes .
the participants were examined in a seated position in a quiet and dark room with a regular temperature ( 2224c ) , and the ecg recording was performed in the morning ( 8:00 am12:00 noon ) to minimize the diurnal fluctuation . the time and frequency domains were calculated automatically . for the time domain , standard deviation of all normal to normal intervals ( sdnn , ms ) , and root mean square of successive differences ( rmssd , ms ) were measured .
for the frequency domains , low - frequency component of spectral hrv ( lf , 0.040.15 hz , ms ) , high - frequency component of spectral hrv ( hf , 0.150.4 hz , ms ) , and lf / hf ratio were measured using the fast fourier transform system .
hf reflects the vagal modulation of the sinus node , and lf represents the sympathetic modulation or a combination of the sympathetic and vagal influences .
the lf / hf ratio is considered to reflect the sympathovagal influence on the heart rate control . the time and frequency domain parameters
the statistical significance of the differences between the respective groups was evaluated using student t test for the continuous data and pearson test for the categorical variables .
the association between hrv and bowel preparation was derived by a multivariable linear regression with adjusted factors , including age , gender , diabetes mellitus , bmi , sbp , tg , and hdl .
the characteristics of the participants are listed in table 1 . in comparison to participants without bowel preparation ( control group ) ,
participants with bowel preparation showed few differences in clinical or demographic data but higher serum levels of tg and phosphorus as well as lower levels of hdl cholesterol and potassium .
demographic and clinical data of participants the hrv parameters of both groups are provided in table 2 .
the bowel preparation group showed significantly lower sdnn , rmssd , lf , and hf results ; however , there was no difference between groups in the lf / hf ratio . based on the lf / hf ratio
, the subjects were further classified into low ( < 0.5 ) , normal ( 0.52 ) , and high groups ( > 2 ) , although no differences were observed between participants with and without bowel preparation ( = 0.334 , p = 0.84 ) .
comparison of heart rate variability between participants with and without bowel preparation to determine the effect of bowel preparation on hrv parameters , we used a multiple linear regression model that included potentially confounding factors ( age , bmi , gender , diabetes mellitus , sbp , tg , and hdl ) .
as shown in table 3 , bowel preparation was significantly associated with lower sdnn , rmssd , lf , and hf values and no difference in the lf / hf ratio .
factor - adjusted associations between bowel preparation and heart rate variability pearson partial correlation coefficients between the hrv parameters and the electrolyte changes in the bowel preparation group are shown in table 4 .
the serum phosphorus and potassium levels were significantly correlated with the hrv , although this relationship was not noted in the control group ( data not shown ) .
the major finding of this study is that bowel preparation before colonoscopy was significantly associated with decreased hrv , including lower sdnn , rmssd , lf , and hf values .
the relationship between bowel preparation and hrv was verified using multiple linear regression analysis after adjusting for potentially confounding factors .
the association between hrv and changes in serum phosphorus and potassium levels was only demonstrated in the bowel preparation group .
one serious concern raised in our study is that hrv data acquired using the current health examination protocol might not reflect the basal status ; instead , these data might reflect an acute condition in people who undergo bowel preparation before colonoscopy .
the details of the mechanisms by which bowel preparation affects hrv remain unclear , although an electrolyte imbalance may play an important role . studies investigating the relationship between serum electrolytes and hrv are rare . most available research in this field has focused on the relationship between hrv and serum electrolytes in particular pathological conditions , such as chronic kidney disease ( ckd ) . using a multivariable linear regression model ,
drawz et al demonstrated that elevated serum phosphorus was associated with lower sdnn and rmssd in patients with ckd .
in addition , elevated serum phosphorus has been reported to be associated with a lower lf / hf ratio .
wen et al studied the relationship between a rapid change in the serum phosphorus level and hrv in 23 ckd patients before and after hemodialysis ( hd ) .
a significant decrease in serum phosphorus was noted after hd , with no significant correlation between the serum phosphorus level and hrv . in our study ,
rapid elevation of the serum phosphorus level was noted in the bowel preparation group , and an association between the serum phosphorus level and hrv was noted in the bowel preparation group ; this association was not observed in the control group .
the mechanism by which an acute change in the serum phosphorus level interacts with hrv remains unclear .
hill et al reported that patients developing hypokalemia after bowel preparation were predisposed to cellular potassium depletion . decreased serum potassium
is negatively correlated with the basal intracellular potassium concentration , and a loss of serum potassium lowers the threshold for cell depolariration , increases the qt variation , induces cardiac arrhythmia and , theoretically , affects the hrv .
several studies have examined the effects of rapidly altered levels of serum potassium on hrv in ckd patients before and after hd , and the results revealed that decreased serum potassium after hd showed no significant correlation with hrv .
lutfi evaluated the effects of electrolyte concentrations on hrv in apparently healthy subjects , and these data revealed no significant correlations between the potassium level and the hrv indices . in our study , the serum potassium level was decreased in the bowel preparation group , and the relationship between the serum potassium level and hrv was only noted in the bowel preparation group , implicating a possible role of serum potassium in mediating hrv .
probable explanations for the discrepancy in the results of the relationship between serum potassium and hrv include variability between study groups ( eg , ckd patients ) and different study designs ( eg , without a rapid change of serum potassium ) .
in addition , the level of serum potassium might not accurately reflect the intracellular potassium levels , although the measurement of intracellular potassium levels is technically difficult .
physical or psychiatric stress can result from bowel preparation - related adverse events , including abdominal pain , abdominal fullness , nausea , vomiting , perianal irritation , dizziness , weakness , fatigue , thirst , and headache .
individuals under stress theoretically precipitate activation of the sympathetic nervous system and depression of the parasympathetic nervous system , and different types of stress might exert different hrv changes .
previous reports have shown that physiological ( eg , exercise ) and pathological stress ( eg , sepsis ) results in decreased sdnn ; however , a diminished lf / hf ratio was only noted with exercise .
in addition , psychological stress is correlated with increased lf , decreased hf , and/or an increased lf / hf ratio .
work stress has been reported to correlate with decreased sdnn , lf , and hf .
it is supposed that stress might play an important role in mediating the effect of the bowel preparation on hrv .
further studies are needed to investigate the association between bowel preparation - related stress and hrv .
the important contribution of our study is the demonstration that bowel preparation might affect hrv . to our knowledge , this study is the first to investigate the role of bowel preparation and rapid changes of electrolytes in relation to hrv in a relatively healthy population .
first , we could not determine when bowel preparation begins to affect hrv , when bowel preparation exerts the greatest effect on hrv or when hrv returns to normal .
we did not investigate the heart rhythm behavior during colonoscopy and whether there are more ventricular or supraventricular arrhythmias in the bowel preparation group of participants compared to the control group .
we also had insufficient data to determine whether other dosages or regimens of bowel preparation exhibit similar hrv responses .
second , the stress effect of bowel preparation might affect hrv ; however , we did not perform an objective assessment of the quantification of stress .
third , there are many factors that possibly mediate hrv that were not analyzed in our study , including the past medical history and cardiovascular medication history of each participant .
fourth , this work is an observational study , which limits our ability to assess causality ; thus , longitudinal studies and repeated measurements are needed . in conclusion ,
bowel preparation before colonoscopy was associated with lower hrv , and the underlying mechanism for the alteration of the cardiovascular autonomic nervous system after bowel preparation might be related to an electrolyte imbalance .
hrv data derived using the current health examination protocol might not reflect the basal status ; rather , these data might reflect an acute condition in participants who undergo bowel preparation before colonoscopy .
additional studies to elucidate the role of bowel preparation in hrv are required , and caution may be necessary for interpreting hrv reports in people having undergone bowel preparation . | abstractin current health examination setting , people frequently undergo heart rate variability ( hrv ) analysis and colonoscopy on the same day .
however , it remains unclear whether the bowel preparation before colonoscopy affects hrv .
this study aimed to evaluate the association between hrv and bowel preparation.we conducted a cross - sectional observational study of 1755 people from january 2012 to december 2013 in taipei , taiwan .
the participants , aged 45 to 65 years , received health examinations that included a physical examination , blood tests , and an hrv analysis . among these people ,
1099 additionally received a colonoscopy on the same day and underwent bowel preparation 1 day before the colonoscopy .
the association between hrv and bowel preparation was derived by a multivariable linear regression with adjusted confounding factors.bowel preparation was associated with a lower standard deviation of the normal - to - normal intervals ( sdnn ) , the root mean square of the successive differences ( rmssd ) , low - frequency power ( lf ) , and high - frequency power ( hf ) ( all p < 0.0001 ) . after adjusting confounding factors , bowel preparation remained correlated with lower sdnn , rmssd , lf , and hf ( all p < 0.0001 ) .
higher serum phosphorus and lower serum potassium levels were noted in the bowel preparation group ( p < 0.0001 ) , and an association between lower hrv and higher serum phosphorus and lower serum potassium levels was only noted in the bowel preparation group.bowel preparation was significantly associated with lower hrv .
the underlying mechanism may be related to an electrolyte imbalance .
cautions may be needed when interpreting hrv reports for people receiving bowel preparations before colonoscopy . | INTRODUCTION
METHODS
Participants
HRV Evaluation
Statistical Analysis
RESULTS
DISCUSSION |
7 m acetone - fixed frozen sections from spleen or kidney were air - dried and stained with various antibodies for 30 min at room temperature in a moist chamber . for the identification of germinal centers , sections were labeled with pe conjugated anti - b220 antibodies ( bd , ra3 - 6b2 ) and fitc conjugated anti - gl-7 ( bd , clone gl7 ) antibodies . for assessment of igg deposition in the kidney ,
> 10 sections stained with fitc - conjugated anti - mouse igg ab ( sigma ) were examined for each experimental condition , verified for reproducibility and quantified as positively - stained using imagej software .
single - cell suspensions were prepared and maintained in the dark at 4c for immunofluorescent analysis , washed in ice - cold facs buffer ( 2% fatal calf serum , 0.1% nan3 in pbs ) and incubated with each antibody for 30 min and washed with facs buffer before analyzing .
anti - cd4 , anti - cd8 , anti - b220 , anti - cd44 , anti - cd62l , anti - fas , anti - qa-1b , anti - cd45.2 , anti - pd-1 , anti - cd11b , anti - cxcr5 , anti - ifn , anti - il10 , anti - il4 , anti - icos , anti - vb5.1/5.2 , anti - nkg2a , anti - igm ( bd bioscience ) or anti - cd8 , anti - ctla4 , anti - ccr7 , anti - gitr , anti - icosl , anti - cd127 , anti - cd25 , anti - klrg1 and anti - vla4 ( ebioscience ) were used followed by analysis of cells using a facscalibur ( bd biosciences ) and flowjo software ( tristar ) . for detection of np specific antibodies ,
elisa plates were coated with 0.5 g / ml np4-bsa or 1 g / ml np23-bsa ( biosearch technologies ) and serum harvested 14 days after immunization with np19-klh in cfa and reimmunization with np19-klh in ifa was used as a standard .
total igg , igg1 and igg2a were detected by incubating plates with biotinylated anti - mouse igg , igg1 or igg2a followed by streptavidin - peroxidase .
anti - dsdna and anti - nuclear antibodies in mouse sera were determined by elisa ( alpha diagnostic international ) and porcine thyroglobulin ( sigma ) and porcine insulin ( sigma ) were used to detect relevant ab .
ifn and il-4 were measured using opt elisa kit ( bd biosciences ) ; il-21 and il-17 were measured using elisa duo set ( r&d systems ) .
68 week old or 6 month old b6.qa-1(wt ) or b6.qa-1(d227k ) mice were infected i.p . with 110 pfu lcmv - armstrong .
at day 8 , mice were reinfected i.p . with 110 pfu lcmv - armstrong .
serum samples were prepared at day 8 ( prior to reinfection ) and day 13 and subjected to the elisa test ( biotech trading partners ) .
cells from spleen and lymph nodes were collected from wt ot - ii or b6.qa-1(d227k ) ot - ii mice .
210 cells / ml ot - ii cd4 cells were stimulated with 1 g / ml ot - ii peptides ( isqavhaahaeineagr ) in the presence of 210 irradiated total splenocytes .
for the differentiation of ot - ii cd4 cells to follicular helper t cell phenotype , 50 ng / ml il-21 , 10 g / ml anti - ifn ab , 10 g / ml anti - il-4 ab , 20 g / ml ril-6 , and 20 g / ml anti - tgf ( 1d11 ) abs were added into the culture . at day 5 ,
recipient mice were immunized i.p . with either 100 g np13-ova in cfa or 100 g np19-klh in cfa .
statistical analyses were performed using wilcoxon - mann - whitney rank sum test for comparison of two conditions and kruskal - wallis test for comparison of more than two conditions .
p value < 0.05 was considered statistically significant ( * = < 0.05 , * * = < 0.01 , * * * = < 0.001 ) .
7 m acetone - fixed frozen sections from spleen or kidney were air - dried and stained with various antibodies for 30 min at room temperature in a moist chamber . for the identification of germinal centers , sections were labeled with pe conjugated anti - b220 antibodies ( bd , ra3 - 6b2 ) and fitc conjugated anti - gl-7 ( bd , clone gl7 ) antibodies . for assessment of igg deposition in the kidney ,
> 10 sections stained with fitc - conjugated anti - mouse igg ab ( sigma ) were examined for each experimental condition , verified for reproducibility and quantified as positively - stained using imagej software .
single - cell suspensions were prepared and maintained in the dark at 4c for immunofluorescent analysis , washed in ice - cold facs buffer ( 2% fatal calf serum , 0.1% nan3 in pbs ) and incubated with each antibody for 30 min and washed with facs buffer before analyzing .
anti - cd4 , anti - cd8 , anti - b220 , anti - cd44 , anti - cd62l , anti - fas , anti - qa-1b , anti - cd45.2 , anti - pd-1 , anti - cd11b , anti - cxcr5 , anti - ifn , anti - il10 , anti - il4 , anti - icos , anti - vb5.1/5.2 , anti - nkg2a , anti - igm ( bd bioscience ) or anti - cd8 , anti - ctla4 , anti - ccr7 , anti - gitr , anti - icosl , anti - cd127 , anti - cd25 , anti - klrg1 and anti - vla4 ( ebioscience ) were used followed by analysis of cells using a facscalibur ( bd biosciences ) and flowjo software ( tristar ) .
for detection of np specific antibodies , elisa plates were coated with 0.5 g / ml np4-bsa or 1 g / ml np23-bsa ( biosearch technologies ) and serum harvested 14 days after immunization with np19-klh in cfa and reimmunization with np19-klh in ifa was used as a standard .
total igg , igg1 and igg2a were detected by incubating plates with biotinylated anti - mouse igg , igg1 or igg2a followed by streptavidin - peroxidase .
anti - dsdna and anti - nuclear antibodies in mouse sera were determined by elisa ( alpha diagnostic international ) and porcine thyroglobulin ( sigma ) and porcine insulin ( sigma ) were used to detect relevant ab .
ifn and il-4 were measured using opt elisa kit ( bd biosciences ) ; il-21 and il-17 were measured using elisa duo set ( r&d systems ) .
68 week old or 6 month old b6.qa-1(wt ) or b6.qa-1(d227k ) mice were infected i.p . with 110 pfu lcmv - armstrong . at day 8
serum samples were prepared at day 8 ( prior to reinfection ) and day 13 and subjected to the elisa test ( biotech trading partners ) .
cells from spleen and lymph nodes were collected from wt ot - ii or b6.qa-1(d227k ) ot - ii mice .
210 cells / ml ot - ii cd4 cells were stimulated with 1 g / ml ot - ii peptides ( isqavhaahaeineagr ) in the presence of 210 irradiated total splenocytes .
for the differentiation of ot - ii cd4 cells to follicular helper t cell phenotype , 50 ng / ml il-21 , 10 g / ml anti - ifn ab , 10 g / ml anti - il-4 ab , 20 g / ml ril-6 , and 20 g / ml anti - tgf ( 1d11 ) abs were added into the culture . at day 5 , live cd4 cells were harvested from the culture by percoll gradient centrifugation . ot -
recipient mice were immunized i.p . with either 100 g np13-ova in cfa or 100 g np19-klh in cfa .
statistical analyses were performed using wilcoxon - mann - whitney rank sum test for comparison of two conditions and kruskal - wallis test for comparison of more than two conditions .
p value < 0.05 was considered statistically significant ( * = < 0.05 , * * = < 0.01 , * * * = < 0.001 ) . | the ability to produce vigorous immune responses that spare self tissues and organs depends on elimination of autoreactive t and b cells .
however , purging of immature and mature self - reactive t and b cells is incomplete and may require additional censorship by cells programmed to suppress immune responses 1 .
regulatory t cells belonging to the cd4 + t cell subset may play a role in preventing untoward inflammatory responses , but t cell subsets programmed to inhibit the development of autoantibody formation and sle - like disease have not been defined 2 .
here we delineate a cd8 + regulatory t cell lineage that is essential for maintenance of self tolerance and prevention of autoimmune disease .
genetic disruption of the inhibitory interaction between these cd44 + icosl+ cd8 + t cells and their target qa-1 + follicular t helper cells results in the development of a lethal sle - like autoimmune disease .
these findings define a sublineage of cd8 t cells programmed to suppress rather than activate immunity that represents an essential regulatory element of the immune response and a guarantor of self tolerance . | Additional Methods
Immunohistochemistry
Reagents and flow cytometry
ELISA for antibodies
LCMV infection and determination of Ab titer
Generation of T
Statistical analyses
Supplementary Material |
skin grafting is a widely used method for the reconstruction of wound closures that can not be closed primarily .
skin grafts are indicated to accelerate the healing of burns , ulcers , and other wounds , reduce scar contractures , and enhance cosmesis .
a skin graft may be meshed to cover a greater surface area at the recipient site .
surgical staples are a common and expeditious method of affixing a mesh skin graft , particularly to large recipient areas .
the sewing of the graft using absorbable sutures may be more prudent in children or sensitive areas of adults . however , these affixing procedures have disadvantages , such as pain during staple removal and the time required for suturing .
the edges of a meshed skin graft are generally not mesh like but exhibit a dendritic structure .
these dendritics are sometimes trimmed or placed on normal skin , but they are not merely left to dry and drop off .
dendritics are too small to thread , but they can be attached using octyl-2-cyanoacrylate ( dermabond ; ethicon inc . ,
however , we do not affix the graft on the recipient 's defective skin area because of the danger of obstructing the epithelialization of the mesh .
alternatively , we use negative - pressure dressings ( vac ; kinetic concepts inc . ,
a nonadherent material must be placed as an interface between the skin graft and the vac sponge to prevent a disruption of the graft during removal of the dressing .
the initial dressing should remain in place for approximately 5 days ( 3 - 7 days ) unless pain , odor , discharge , or other signs of complication develop .
a total of 14 patients ( five males and nine females ) under went skin graft using this technique .
the ages ranged from 9 months to 86 years ( mean = 62.3 years ) .
the skin defects included two burn ulcers , two decubitus , and 10 chronic leg ulcers .
all grafts were accepted without complications . a 72-year - old man with a chronic leg ulcer after debridement for necrotizing fasciitis
the dendritics of the mesh skin graft were affixed using dermabond six - month postoperative result
dermabond was initially used to replace the suturing of superficial wounds , but it is currently used in a variety of techniques .
dermabond has been used recently as a skin bolster to assist the primary closure of wounds in patients with thin skin .
the fixation time is equivalent to surgical stapler fixation and the removal of sutures is not required because the graft is affixed with absorbable sutures .
local anesthesia may be necessary for pain relief during surgical stapling and sewing , but it is not necessary for the dermabond technique .
dermabond costs approximately $ 18 per tube , whereas skin staples are $ 16 and absorbable sutures are $ 6 per suture .
the dendritic bonding technique is a quick and easy method for the fixation of mesh skin grafts in any patient . | we describe a newly designed technique for the quick , easy , and cost - effective fixation of mesh skin grafts in a range of skin conditions and patients .
we fixed the skin graft using octyl-2-cyanoacrylate ( dermabond ; ethicon ) , which was termed
dendritic bonding .
this technique exhibits several advantages over surgical stapling and suturing with absorbable sutures . | INTRODUCTION
SURGICAL TECHNIQUE
RESULTS
DISCUSSION |
the prevalence of lower urinary tract symptoms ( luts ) in men has been clearly shown to increase with age .
although the etiologies of luts in aged men are well understood , relatively little research effort has been devoted to studying luts in adolescents and young men .
obtaining a clinical diagnosis on the basis of the history of symptoms and physical examination alone is often not possible for chronic luts in adolescents and young men .
however , detailed investigations are often not performed in the belief that they will be inconclusive .
therefore , diagnosis is often empiric , and most patients are diagnosed with nonbacterial prostatitis , simple overactive bladder syndrome , or psychogenic voiding dysfunction .
recent research has focused on bladder and urethral dysfunction as a cause of luts and pelvic pain in young men , and urodynamic investigation has enhanced awareness of possible etiologies of chronic luts in adolescents and young men . to date , some specific urodynamic etiologies have been described as a cause of chronic luts in adolescents and young men . of these ,
primary bladder neck dysfunction ( pbnd ) has been reported to be the most frequent , followed by dysfunctional voiding ( dv ) , detrusor overactivity ( do ) , and detrusor underactivity ( du)/acontractile detrusor ( ad ) . because the management strategy differs for each urodynamic diagnosis , accurate diagnosis with urodynamic testing may be helpful in counseling the patient on the most appropriate treatment .
previous studies concerning this issue considered only small populations and focused on young men who had previously been diagnosed with chronic prostatitis .
we investigated the etiologies of luts and compared the urodynamic characteristics between different diagnostic groups in young men with chronic luts who did not have symptoms suggestive of chronic prostatitis . to the best of our knowledge
, there have been no large studies of luts in young men without symptoms suggestive of chronic prostatitis .
we reviewed the medical records of male patients aged 18 to 50 years who had undergone a urodynamic study for work - up of luts at our institution between may 2003 and may 2013 .
all of the cases were reviewed , and patients who had experienced symptoms for less than 6 months and those with a neurogenic abnormality , diabetes mellitus , or interstitial cystitis that affected micturition function ; a history of surgery on the lower urinary tract ; anatomical deformation of the lower urinary tract such as urethral stricture ; or impairment of general health that affected voiding , such as having recently undergone surgery , were excluded .
in addition , patients with pelvic or inguinal pain or bacterial infection or more than 10 leukocytes in expressed prostatic secretions ( eps ) at any time before the urodynamic study were excluded from the study population .
the initial evaluation of patients presenting with luts at our institution consists of taking a history of the luts and performing a physical examination , followed by having the patient document an international prostate symptom score ( ipss ) , overactive bladder symptom score ( since 2008 ) , and a 3-day frequency - volume chart ( fvc ) , including the urinary sensation scale at every voiding .
episodes of urgency were defined as those with a urinary sensation scale rating of 3 .
free uroflowmetry ( daba , endo tech , seongnam , korea ) and measurement of the postvoid residual volume ( bladderscan bvi-3000 , verathon inc . , bothell , wa , usa ) were performed before urodynamic evaluation .
the result showing a higher maximum flow rate ( qmax ) was selected from two sets of uroflowmetry measures with a voided volume over 150 ml .
after discontinuation of all drugs that could possibly affect micturition function for at least 3 to 7 days , a multichannel urodynamic study ( ud-2000 , medical measures system b.v . , enschede , the netherlands ) , including a pressure - flow study ( pfs ) , was carried out in accordance with the guidelines of the international continence society . a 6-fr double - lumen catheter and a 9-fr balloon catheter
were used to measure the transurethral intravesical and abdominal pressures in all of the urodynamic studies .
pelvic floor electromyography was performed by using surface electrodes attached near the anus at the 3 and 9 o'clock positions .
intravesical pressure was recorded under conditions of room - temperature saline infusion at 50 ml / min .
however , the filling rate was decreased to 20 ml / min in patients with severe storage symptoms or a lower functional bladder capacity according to the results of the 3-day fvc .
bladder compliance was considered reduced when the v/pdet was 20 ml / cm h2o . during the pfs , the patient was instructed to void in a standing or sitting position under quiet and relaxed circumstances . if the first voiding trial failed , an additional trial was performed to allow for the possibility that the failure was due to cortical inhibition .
because the present study was conducted retrospectively , the treatment efficacy data retrieved had not been collected according to uniform criteria .
the clinical urinary symptoms were divided into 3 categories : storage luts ( 8 episodes of frequency per day , 2 episodes of nocturia per night , or 3 episodes of urgency or urgency urinary incontinence on a 3-day fvc ) , voiding luts ( intermittency score 3 , weak stream score 3 , or straining score 3 on the ipss ) , and postmicturitional luts ( incomplete emptying score 3 on the ipss ) .
pbnd , dv , du , and ad were considered to represent voiding phase dysfunction and do , small cystometric capacity , and reduced bladder sensation were considered to represent storage phase disorders .
the urodynamic diagnosis was made on the basis of the retrospective interpretation by the two investigators ( s.j.j and s.c.l . ) who were blinded to the patients ' clinical characteristics .
pbnd was diagnosed if bladder outlet obstruction , defined as an abrams - griffith ( ag ) number of 40 or greater or 20 - 39.9 with a slope of the linear passive urethral resistance ratio of > 2 cm h2o / ml / s , where the ag number was calculated as the detrusor pressure at qmax ( pdetqmax)-2qmax , was present concomitant with electromyography evidence of external sphincter relaxation , and neither urethral stricture nor prostatic enlargement was observed in urethrocystoscopy and transrectal ultrasonography ( trus ) , which were performed only in suspected cases in our young population .
dv was diagnosed on the basis of the electromyography activity of the external sphincter / pelvic floor during voiding in the absence of abdominal straining .
if dv was diagnosed during a pfs , a free uroflow measurement was performed in a private setting to identify undulating intermittent increases and decreases in flow .
if patients exhibited dv in a pfs and free uroflow , we repeated the same procedure in an effort to reduce false positive results caused by performance anxiety or bashful voiding during the urodynamic evaluation .
we performed an additional voiding cystourethrography ( vcug ) in cases with equivocal findings of pbnd or dv during pfs .
du was diagnosed when the ag number was less than 20 and the qmax was less than 12 ml / s during a pfs and no obstruction was recognized in urethrocystoscopy or trus . for the purpose of classification of cases with the absence of a detrusor contraction during 2 consecutive pfs ,
patients with a measureable uroflow during free uroflow were considered to have du and those who did not generate a measureable uroflow in free uroflow were regarded as having an ad .
patients were regarded as positive for idiopathic do if a spontaneous or provoked involuntary detrusor contraction was observed during the filling cystometry .
maximum cystometric capacity was defined as the volume at which the patient felt that he could no longer delay micturition and small bladder capacity was defined as a maximum cystometric capacity of < 350 ml . reduced bladder sensation was defined as a diminished bladder sensation during filling cystometry .
after assessment of the urodynamic etiologies of luts , comparisons among specific urodynamic etiologies were analyzed by a one - way analysis of variance with scheffe 's method for multiple comparisons or by linear by linear association depending on the type of variable .
the collected data were presented as the meanstandard deviation or as the number ( percentage ) . the ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) was used , and a 2-tailed p value of < 0.05 was determined to indicate statistical significance .
a total of 308 young men who did not meet any of the exclusion criteria were included for analysis .
mean age was 40.4 ( 10.1 ) years and mean symptom duration was 38.8 ( 49.2 ) months .
storage luts were present in 247 men ( 80.2% ) , voiding luts in 166 men ( 53.9% ) , and postmicturitional luts in 129 men ( 41.9% ) .
table 1 shows the urodynamic diagnoses categorized by voiding phase and storage phase dysfunctions in all patients .
seventy - nine patients ( 25.6% ) had normal urodynamic findings . among the total patient population , the incidence of pbnd was highest among those in their 30s ( p=0.04 ) and that of do was highest among those in their 40s ( p=0.03 ) ( fig .
small bladder capacity was more prevalent among men under 40 years of age ( p=0.03 ) .
dv was more frequent among those under 40 years of age , but the difference was not significant ( p=0.18 ) .
the clinicodemographic and urodynamic characteristics of men with urodynamically defined voiding phase and storage phase dysfunction are shown in tables 2 , 3 .
the demographics and types of clinical symptoms were not significantly different between the diagnostic groups except for storage luts in distinguishing the type of storage urodynamic dysfunctions . whereas 53.9% of patients ( 103/191 ) with voiding phase dysfunction had concomitant storage dysfunction ( table 2 ) , 69.6% ( 78/112 ) of those with storage dysfunction
sixty - five and 73.2% of patients with dv and do had concomitant other storage phase and voiding phase dysfunctions ( p<0.01 ) .
qmax did not differ significantly between men with dv and those with normal urodynamics , but was significantly lower in men with pbnd or du / ad than in those with normal urodynamics .
men with dv or du / ad exhibited lower maximum cystometric capacity than did those with normal urodynamics ( p<0.001 , respectively ) .
of the storage phase dysfunctions , only small bladder capacity was associated with a lower free qmax than in men with normal urodynamics ; however , the qmax during a pfs was not significantly different .
the free postvoid residual was higher in patients with do than in those with small bladder capacity .
men with do were shown to have higher detrusor pressure during voiding than that in men with other storage phase dysfunctions .
medications were administered in 83.1% of men and -blockers were the most frequently prescribed , followed by anticholinergics , muscle relaxants , and cholinergics .
interestingly , no treatment was chosen in 46.8% of men with normal urodynamics , as abnormal findings were not demonstrated in their urodynamic evaluation despite their symptoms . however , the other urodynamically normal patients received treatment for luts .
although chronic luts are not uncommon in young men , they have received little scientific attention . because the incidence and clinical implications of each urodynamic etiology for chronic luts of young men remain largely unknown , the proper diagnosis and management of each condition is challenging for physicians .
furthermore , these young men frequently experience the recurrence of symptoms despite various medications such as antibiotics , -blockers , and anticholinergics .
our study showed that most demographics and types of clinical symptoms did not differ among patients with specific urodynamic etiologies of luts .
insisted that in young men with pbnd , dv , do , or du , clinical features frequently overlap and are not as defining as they are often presumed to be .
therefore , treatment of young men on the basis of a presumed diagnosis from presenting luts is less likely to be successful , because the sensitivity of luts for the prediction of any specific urodynamically defined condition is not strong .
urodynamic investigation seems to be justified in adolescents and young men with chronic luts to confirm the correct diagnosis and avoid unnecessary and ineffective treatment .
the incidence of pbnd has been reported as 40% to 50% among young men with refractory luts .
table 4 compares the incidence of each urodynamic etiology between our study and previously published studies . in our study ,
incidences of pbnd and dv were 26% and 23.4% , showing a relatively lower rate among young men with chronic luts .
we believe that this was because our population differed from those in the previous studies .
unlike previous studies that focused on men who had been previously diagnosed with nonbacterial chronic prostatitis , we studied young men with chronic luts who did not have positive eps findings or pelvic or perineal pain responsible for nonbacterial chronic prostatitis .
. the etiology of nonbacterial chronic prostatitis may be similar to the etiology of luts in young men .
abnormalities of pelvic floor muscle relaxation and poor relaxation of bladder neck during voiding have been suggested as the etiology of nonbacterial chronic prostatitis and chronic luts in adolescents and young men .
therefore , young men with nonbacterial chronic prostatitis appear to have more voiding phase dysfunctions than storage phase dysfunctions .
our results demonstrated that pbnd was the most frequent single specific dysfunction , followed by dv and du / ad .
as a whole , pbnd and dv accounted for about 50% of the specific diagnoses . while approximately half of patients with voiding phase dysfunction had concomitant storage dysfunction , storage phase dysfunction
similarly , nitti et al . reported that the majority of men with do ( 85% ) also had voiding phase abnormalities and stated that primary do without voiding phase dysfunction would appear to be unusual in young men .
men with dv or du / ad exhibited lower maximum cystometric capacity than that in men with normal urodynamics , and men with do displayed higher detrusor pressure during voiding than did those with other storage dysfunction .
although the plausible reasons for these findings need more investigations , similar results were demonstrated in our previous research with female overactive bladder patients .
the etiology of du in aged persons is degeneration of muscle cells and axons . however , the mechanism of du in young men is not fully understood .
therefore , it is somewhat interesting to find that 12.7% of our young men had du / ad .
while cholinergic agonists may be used to facilitate voiding efficacy in aged patients who have du without bladder outlet obstruction , the impact of such treatment on voiding efficacy has not been explored in young men with du without neurogenic abnormalities .
the reported prevalence of do among young men with refractory luts is found to be 6% to 18% .
although the diagnosis of overactive bladder syndrome is based on subjective symptoms rather than objective criteria , overactive bladder syndrome is frequently described clinically as a condition associated with urodynamic do , with the latter potentially responsible for the clinical symptoms . in general , urodynamic
do is detected in approximately 50% of women with symptoms of overactive bladder . in the present study ,
first , the present study was a retrospective study and therefore had several potential shortcomings , in particular being prone to several forms of selection bias .
second , we did not perform fluoroscopic assessment when diagnosing pbnd or dv . the robust method for the diagnosis of pbnd may be video - urodynamic .
however , pbnd can also be determined indirectly by the urodynamic findings of bladder outlet obstruction with obstructive symptoms in the absence of urethral stricture , prostatic enlargement , and striated sphincter dyssynergia .
our patients were aged 50 years or less and we performed an additional vcug in cases with equivocal findings of pbnd during a pfs ( 26 cases ) .
the diagnosis of pbnd was eventually made when vcug demonstrated narrowing only at the bladder neck .
similarly , for the cases with equivocal results for the diagnosis of dv in a pfs ( 13 cases ) , dv was finally diagnosed when vcug demonstrated brief and intermittent closing at the level of the membranous urethra during voiding .
third , for the purpose of classification of cases with the absence of a detrusor contraction during two consecutive pfs , patients with a measureable uroflow in free uroflow were arbitrarily considered to have du , and those who did not generate a measureable uroflow in free uroflow were regarded as having an ad . however , the number of these patients was small ( 6.0% ) .
we recommend that it is essential to perform urodynamic evaluation to investigate the possible etiologies of luts in young men with chronic luts , especially in those with refractory symptoms .
a high index of suspicion for possible etiologies for luts may be important for accurate and timely diagnosis of treatable luts in young men .
we need to learn more about the natural history of pbnd , dv , and other possible etiologies of luts in young men .
chronic luts among young men have a variety of underlying etiologies . as a single specific dysfunction ,
urodynamic investigation in this population is helpful in making an accurate diagnosis and may guide adequate treatment , because clinical symptoms are not useful in predicting a specific urodynamic etiology . | purposewe investigated the etiologies of lower urinary tract symptoms ( luts ) and compared urodynamic characteristics between different diagnostic groups in young men with chronic luts.materials and methodswe reviewed the medical records of 308 men aged 18 to 50 years who had undergone a urodynamic study for chronic luts ( 6 months ) without symptoms suggestive of chronic prostatitis.resultsthe men 's mean age was 40.4 ( 10.1 ) years and their mean duration of symptoms was 38.8 ( 49.2 ) months .
urodynamic evaluation demonstrated voiding phase dysfunction in 62.1% of cases ( primary bladder neck dysfunction [ pbnd ] in 26.0% , dysfunctional voiding [ dv ] in 23.4% , and detrusor underactivity [ du]/acontractile detrusor [ ad ] in 12.7% ) and a single storage phase dysfunction in 36.4% of cases ( detrusor overactivity [ do ] in 13.3% , small cystometric capacity in 17.9% , and reduced bladder sensation in 5.2% ) .
most of the demographic characteristics and clinical symptoms did not differ between these diagnostic groups .
whereas 53.9% of patients with voiding dysfunction had concomitant storage dysfunction , 69.6% of those with storage dysfunction had concomitant voiding dysfunction .
men with dv or du / ad exhibited lower maximum cystometric capacity than did those with normal urodynamics .
low bladder compliance was most frequent among patients with pbnd ( 10.0% , p=0.025 ) . in storage dysfunctions , men with
do exhibited higher detrusor pressure during voiding than did those with other storage dysfunctions ( p<0.01).conclusionsbecause clinical symptoms are not useful for predicting the specific urodynamic etiology of luts in this population , urodynamic investigation can help to make an accurate diagnosis and , potentially , to guide appropriate treatment . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
urinary tract infection ( uti ) is considered the most prevalent infectious disease insofar as it has reached the overall incidence of 18/1000 persons per year in the general population ( 1 , 2 ) .
uti affects both sexes , irrespective of their age , although it is more common in females ( 3 , 4 ) due to anatomical predisposition or large bacterial load in urothelial mucosa or other host factors ( 5 ) including obstruction in the urinary tract , sexual activity , and pregnancy ( 6 ) .
one out of every two females contracts uti at least once in her life ( 7 ) .
diverse groups of uropathogens ( gram - positive and negative ) are involved in the etiology of uti , but the gram - negative facultative anaerobe and uropathogenic escherichia coli is responsible for the majority of the infections in the general population ( 8 , 9 ) .
it is estimated that 80% of uti cases in healthy women aged 18 - 39 years are caused by e. coli , followed by staphylococcus saprophyticus ( 15 - 20% ) ( 10 ) .
other probably less common uropathogens include klebsiella , enterobacter , serratia , proteus , pseudomonas , and enterococcus ( 11 ) . in the treatment of uti
, mostly empirical antibiotic therapy is used by clinicians ; thus , frequent misuse of antibiotics may increase resistance in uropathogens ( 12 , 13 ) .
the gradual increasing antimicrobial resistance among uropathogens has become a challenge worldwide ( 14 ) .
the antibiotic susceptibility of bacterial strains isolated from urine cultures decides the best choice of antimicrobial drugs for the treatment and prophylaxis of uti .
prompt diagnosis and timely antimicrobial treatment help to minimize renal scarring and progressive kidney damage ( 15 , 16 ) .
the present study was designed to investigate antibiotic susceptibility and resistant pattern in isolates obtained from uti patients .
this would be helpful to clinicians to make new choices of antibiotic therapy for the management of uti .
during the period of december 2012 to january 2014 , a total of 1429 urine samples were collected from uti patients in chughtais lahore lab , lahore , pakistan .
sterile plastic containers were used to collect mid - stream urine samples , and the samples were immediately processed for different procedures such as gram staining , culture , and antibiotic susceptibility testing .
the urine samples were streaked on cysteine lactose electrolyte deficient ( cled ) media ( oxoid , england ) for the isolation of uropathogens and incubated at 37c for 24 hours .
urine volume ( 0.01 ml ) ( loopful ) was inoculated with a sterile calibrated wire loop and was used for the colony count of the isolates .
bacterial growth was reflected significant as per kass count ( single species count > 10 organisms / ml of urine ) ( 17 ) . the colonies were biochemically characterized according to the guidelines ( 18 ) .
the isolates were subcultured on the macconkey agar ( oxoid , england ) and incubated at 37c for 24 hours in order to obtain pure growth .
standardized identification ( api 20 e system ) ( biomrieux , france ) was used to identify and confirm the groups of different isolates .
the antibiotic susceptibility pattern of the isolates was determined by the kirby - bauer disk diffusion method .
plates of mller - hinton agar were used to find the sensitivity pattern and incubated at 37c for 24 hours .
the zone of the inhibition of the bacterial growth was measured after incubation and compared with the clinical and laboratory standards institute ( clsi , 2013 ) guidelines .
e. coli ( atcc 25922 ) , pseudomonas
aeruginosa ( atcc 27853 ) , e. faecalis ( atcc2 9212 ) , and s. aureus ( atcc 29213 ) were used as reference strains according to the clsi protocol .
a total of 392 different gram - negative and gram - positive isolates were subjected to sensitivity testing for 38 different antibiotics , viz .
ampicillin ( 30 g ) , amoxicillin ( 25 g ) , amoxicillin / clavulanic acid ( 30 g ) , ampicillin / sulbactam ( 30 g ) , cefepime ( 30 g ) , cefotaxime ( 30 g ) , cefoxitin ( 30 g ) , cefuroxime ( 30 g ) , ceftazidime ( 30 g ) , ceftriaxone ( 30 g ) , cephalexin ( 30 g ) , cefradine ( 30 g ) , cefaclor ( 30 g ) , cefixime ( 30 g ) , imipenem ( 10 g ) , meropenem ( 10 g ) , vancomycin ( 30 g ) , amikacin ( 30 g ) , gentamycin ( 30 g ) , tobramycin ( 30 g ) , azithromycin ( 30 g ) , erythromycin ( 30 g ) , doxycycline ( 30 g ) , ciprofloxacin ( 10 g ) , levofloxacin ( 10 g ) , norfloxacin ( 10 g ) , nitrofurantoin ( 300 g ) , fosfomycin ( 50 g ) , clindamycin ( 10 g ) , fusidic acid ( 50 g ) , linezolid ( 30 g ) , cefoperazone ( 30 g ) , aztreonam ( 30 g ) , nalidixic acid ( 30 g ) , ofloxacin ( 5 g ) , moxifloxacin ( 5 g ) , trimethoprim / sulfamethoxazole ( 25 g ) , pipemidic acid ( 20 g ) , cefoperazone / sulbactam ( 30 g ) , and piperacillin / tazobactam ( 110 g ) . these antibiotics are routinely prescribed for uti patients by the clinicians in the punjab region .
the detection of extended - spectrum beta - lactamases ( esbl ) production was carried out using the cephalosporin / clavulanic acid combination discs method .
this test was performed on mller - hinton agar via the disc diffusion method as suggested by the clsi .
the organism was considered as esbl producing if the zone diameter was 5 mm for ceftazidime ( 30 g ) tested in combination with clavulanic acid versus its zone diameter when tested alone .
during the period of december 2012 to january 2014 , a total of 1429 urine samples were collected from uti patients in chughtais lahore lab , lahore , pakistan .
sterile plastic containers were used to collect mid - stream urine samples , and the samples were immediately processed for different procedures such as gram staining , culture , and antibiotic susceptibility testing .
the urine samples were streaked on cysteine lactose electrolyte deficient ( cled ) media ( oxoid , england ) for the isolation of uropathogens and incubated at 37c for 24 hours .
urine volume ( 0.01 ml ) ( loopful ) was inoculated with a sterile calibrated wire loop and was used for the colony count of the isolates .
bacterial growth was reflected significant as per kass count ( single species count > 10 organisms / ml of urine ) ( 17 ) . the colonies were biochemically characterized according to the guidelines ( 18 ) .
the isolates were subcultured on the macconkey agar ( oxoid , england ) and incubated at 37c for 24 hours in order to obtain pure growth .
standardized identification ( api 20 e system ) ( biomrieux , france ) was used to identify and confirm the groups of different isolates .
the antibiotic susceptibility pattern of the isolates was determined by the kirby - bauer disk diffusion method .
plates of mller - hinton agar were used to find the sensitivity pattern and incubated at 37c for 24 hours .
the zone of the inhibition of the bacterial growth was measured after incubation and compared with the clinical and laboratory standards institute ( clsi , 2013 ) guidelines .
e. coli ( atcc 25922 ) , pseudomonas
aeruginosa ( atcc 27853 ) , e. faecalis ( atcc2 9212 ) , and s. aureus ( atcc 29213 ) were used as reference strains according to the clsi protocol .
a total of 392 different gram - negative and gram - positive isolates were subjected to sensitivity testing for 38 different antibiotics , viz .
ampicillin ( 30 g ) , amoxicillin ( 25 g ) , amoxicillin / clavulanic acid ( 30 g ) , ampicillin / sulbactam ( 30 g ) , cefepime ( 30 g ) , cefotaxime ( 30 g ) , cefoxitin ( 30 g ) , cefuroxime ( 30 g ) , ceftazidime ( 30 g ) , ceftriaxone ( 30 g ) , cephalexin ( 30 g ) , cefradine ( 30 g ) , cefaclor ( 30 g ) , cefixime ( 30 g ) , imipenem ( 10 g ) , meropenem ( 10 g ) , vancomycin ( 30 g ) , amikacin ( 30 g ) , gentamycin ( 30 g ) , tobramycin ( 30 g ) , azithromycin ( 30 g ) , erythromycin ( 30 g ) , doxycycline ( 30 g ) , ciprofloxacin ( 10 g ) , levofloxacin ( 10 g ) , norfloxacin ( 10 g ) , nitrofurantoin ( 300 g ) , fosfomycin ( 50 g ) , clindamycin ( 10 g ) , fusidic acid ( 50 g ) , linezolid ( 30 g ) , cefoperazone ( 30 g ) , aztreonam ( 30 g ) , nalidixic acid ( 30 g ) , ofloxacin ( 5 g ) , moxifloxacin ( 5 g ) , trimethoprim / sulfamethoxazole ( 25 g ) , pipemidic acid ( 20 g ) , cefoperazone / sulbactam ( 30 g ) , and piperacillin / tazobactam ( 110 g ) . these antibiotics are routinely prescribed for uti patients by the clinicians in the punjab region .
the detection of extended - spectrum beta - lactamases ( esbl ) production was carried out using the cephalosporin / clavulanic acid combination discs method .
this test was performed on mller - hinton agar via the disc diffusion method as suggested by the clsi .
the organism was considered as esbl producing if the zone diameter was 5 mm for ceftazidime ( 30 g ) tested in combination with clavulanic acid versus its zone diameter when tested alone .
significant bacteriuria was observed in 392 ( 27.4% ) samples , while the remaining 1037 samples demonstrated either nonsignificant bacteriuria , very low bacterial count , or were sterile . among all the patients , 62.6% were female and the remaining 37.4% were male . out of the 392 uti patients with significant bacteriuria , 263 ( 67% ) were female and 129 ( 33% ) male .
overall , 244/392 ( 64% ) samples were positive for the presence of e. coli : 163 ( 67% ) samples in the females and 81 ( 33% ) in the males .
the presence of the gram - negative and gram - positive uropathogens among the uti patients is depicted in table 1 .
, which was isolated from 3 ( 60% ) females and 2 ( 40% ) males ; proteus spp . in 3 ( 100% )
females only ; pseudomonas in 15 ( 65% ) females and 8 ( 35% ) males ; s. aureus in 2 ( 67% ) females and 1 ( 33% ) male ; e. faecalis in 42 ( 72% ) females and 16 ( 28% ) males ; and candida in 35 ( 63% ) females and 21 ( 38% ) males .
the frequency of esbl positivity within the individual organism group was the highest amongst e coli ( 66.8% ) , followed by klebsiella spp .
( 40% ) and proteus
mirabilis ( 33.3% ) , as is shown in figure 1 .
the e. coli isolates were resistant to cephalexin ( 95% ) , cephradine ( 95% ) , pipemidic acid ( 92% ) , amikacin ( 91% ) , and nalidixic acid ( 91% ) and had the highest sensitivities for imipenem ( 97% ) , meropenem ( 97% ) , cefoperazone ( 97% ) , and tazobactam ( 94% ) .
cephalexin ( 100% ) and cephradine ( 100% ) , with high sensitivity ( 100% ) for nitrofurantoin .
the most sensitive antibiotics were imipenem , meropenem , cefoperazone , and tazobactam , as is illustrated in table 2 .
the pseudomonas isolates exhibited the least susceptibility ( 43% ) to ciprofloxacin , levofloxacin , norfloxacin , ofloxacin , and moxifloxacin and the highest susceptibility to tazobactam , imipenem , meropenem , amikacin and aztreonam ( 87% , 78% , 78% , 78% , and 70% , respectively ) .
abbreviations : amc , amoxicillin / clavulanic acid ; ak , amikacin ; atm , aztreonam ; caz , ceftazidime ; ce , cephradine ; cec , cefaclor ; cfp , cefoperazone ; cfm , cefixime ; cl , cephalexin ; cn , gentamicin ; cip , ciprofloxacin ; cro , ceftriaxone ; ctx , cefotaxime ; cxm , cefuroxime ; do , doxycycline ; fep , cefepime ; fos , fosfomycin ; ipm , imipenem ; lev , levofloxacin ; mem , meropenem ; mox , moxifloxacin ; na , nalidixic acid ; nor , norfloxacin ; nt , not tested ; ofx , ofloxacin ; pip , pipemidic acid ; sam , ampicillin / sulbactam ; scf , cefoperazone / sulbactam ; sxt , trimethoprim / sulfamethoxazole f nitrofurantoin ; tob , tobramycin ; tzp , piperacillin / tazobactam . among the gram - positive isolates , s. aureus exhibited utmost resistance ( 67% ) against ampicillin , amoxicillin , tobramycin , ciprofloxacin , levofloxacin , norfloxacin , and nitrofurantoin and had the highest ( 100% ) susceptibility to vancomycin , amikacin , fosfomycin , clindamycin , fusidic acid , and linezolid .
s. faecalis presented maximum sensitivity rates ( 97% , 93% , and 86% ) against vancomycin , linezolid , and fosfomycin , respectively , and maximum antimicrobial resistance rates ( 88% , 83% , 83% , and 83% ) against gentamicin , ciprofloxacin , levofloxacin , and norfloxacin , correspondingly . the corresponding resistance patterns of the 31 antimicrobial agents on the gram - negative and gram - positive uropathogens are illustrated in tables 2 and 3 .
abbreviations : ak , amikacin ; amp , ampicillin ; aml , amoxicillin ; amc , amoxicillin / clavulanic acid ; azm , azithromycin ; caz , ceftazidime ; ce , cephradine ; cec , cefaclor ; cfp , cefoperazone ; cfm , cefixime ; cl , cephalexin ; cn , gentamicin ; cip , ciprofloxacin ; cro , ceftriaxone ; ctx , cefotaxime ; cxm , cefuroxime ; da , clindamycin ; do , doxycycline ; e , erythromycin ; f , nitrofurantoin ; fd , fusidic acid ; fep , cefepime ; fos , fosfomycin ; ipm , imipenem ; mem , meropenem ; lev , levofloxacin ; nor , norfloxacin ; lzd , linezolid ; nt , not tested ; sam , ampicillin / sulbactam ; tob , tobramycin ; va , vancomycin .
the present study focused on the local status of antimicrobial susceptibility pattern in uropathogens with a view to offering assistance in monitoring the continuous changing environment of bacterial resistance and further improvements in uti treatment .
the present data belong to patients bearing the cost of medical check - up in a private laboratory ; therefore , they may not imitate the true prevalence of uti as most of the patients were treated empirically for this infection . it has been reported previously that in 80% of acute and recurrent uti cases in women , e. coli is involved as the primary organism , followed by s. saprophyticus ( 10% - 15% ) .
other less common uropathogens with the potential to cause uti include klebsiella , enterobacter , serratia , proteus , pseudomonas , and enterococcus ( 11 ) .
our findings indicate that the frequency of e. coli was 62% in uti from a total of 392 culture - positive urine samples .
in some previous reports , the e. coli prevalence was 68% ( 19 , 20 ) , followed by pseudomonas ( 6% ) , klebsiella spp .
akram et al . reported that in the majority of their uti patients , e. coli was the predominant organism ( 21 ) : e. coli was found in 67% of the females and 33% of the males from a total of 62% of the uti cases , in comparison with pseudomonas , which was detected in 65% of the females and 35% of the males from only 6% of the uti cases .
our results are relatively similar to a previous study from lahore , pakistan , which reported that the prevalence rate of e. coli was 73% as opposed to only 27% in the other uti - causing organisms ( 22 ) . among the gram - positive pathogens ,
e. faecalis was found as the most frequent organism ( 15% ) , followed by s. aureus ( 1% ) .
the prevalence rate of e. candida was 14% , which is quite similar to the finding of a previous study from nepal , stating that the prevalence rate of e. faecalis was 18% ( 23 ) .
a previous study conducted in 2008 found resistant patterns in e. coli against ampicillin ( 92% ) , co - trimoxazole ( 80% ) , ciprofloxacin ( 62% ) , gentamicin ( 47% ) , nitrofurantoin ( 20% ) , and amikacin ( 4% ) ( 24 ) .
in contrast , our study revealed significant changes in the e. coli resistant patterns , especially against amikacin , where e. coli exhibited high resistance ( 91% ) .
our results also demonstrated that 23 out of the 31 different drugs showed greater than 70% antimicrobial resistance against e. coli , the most common pathogen . -lactam
drugs like amoxicillin / clavulanic acid , ampicillin , and aztreonam were also ineffective against e. coli as it had high antimicrobial resistance ( 84% , 84% , and 72% , respectively ) .
a study conducted in the lahore region indicated high resistance of e. coli to -lactam antibiotics such as amoxicillin/ clavulanic acid , ampicillin , and aztreonam ( 22 ) .
e. coli showed the maximum susceptibility ( 97% ) against drugs like imipenem , meropenem , and cefoperazone / sulbactam .
tazobactam / piperacillin and fosfomycin also provided significant activity against e. coli ( 96% and 90% , respectively ) . in our study , pseudomonas was found as the second most frequent gram - negative isolate and had maximum resistance against ciprofloxacin , levofloxacin , norfloxacin , ofloxacin , and moxifloxacin , while it was highly susceptible to tazobactam / piperacillin .
klebsiella has a resistance pattern comparatively similar to e. coli with high resistance ( 100% ) to cephalexin and cephradine and less resistance against nalidixic acid ( 20% ) . a previous study from pakistan reported high sensitivity ( 80% ) to cefepime versus a greater resistance rate ( 87% ) against ciprofloxacin ( 25 ) .
nitrofurantoin was the most efficient drug against this pathogen as it had peak sensitivity ( 100% ) .
proteus was the least common pathogen and had maximum resistance ( 100% ) against 12 antibacterial drugs but was found to have 100% sensitivity against meropenem , cefoperazone / sulbactam , and piperacillin / tazobactam .
our results indicated that only 16% of the uti cases were caused by gram - positive microorganisms .
moreover , e. faecalis showed a high rate of resistance to gentamicin ( 88% ) , ciprofloxacin ( 83% ) , levofloxacin ( 83% ) , and norfloxacin ( 83% ) but linezolid was the most effective antimicrobial drug .
vancomycin , amikacin , fosfomycin , clindamycin , fusidic acid , and linezolid had strong antimicrobial activity against the gram - positive isolates .
the s. aureus isolates from uti were susceptible to amikacin , augmentin , and oxacillin in a previous study by bano et al . while marked resistance to amoxicillin , ampicillin , tobramycin , ciprofloxacin , levofloxacin , norfloxacin , and fusidic acid was found in another study ( 25 ) .
( 2013 ) also reported that nitrofurantoin was the most effective drug against gram - positive uropathogens ( 13 ) .
our study demonstrated the highest frequency for esbl production in e. coli ( 66.8% ) , followed by klebsiella spp .
our results are similar to a previous study from karachi , pakistan , which reported that the esbl frequencies among e. coli and proteus mirabilis isolates were 68.55% and 28.57% , respectively .
in contrast , the frequency of klebsiella was different ( 84.61% ) from our finding , which may be due to the lower number of klebsiella isolates in our study ( 26 ) .
the antibiotic resistance of uropathogens to trimethoprim / sulfamethoxazole , ampicillin , and cephalothin is increasing worldwide ( 27 ) . in developing countries , the frequent prescription of antibiotics for the treatment of
uti and other infections , self - medication , suboptimal concentration and quality of antimicrobial agents , and community level poor hygiene are reasons for the ever - growing antimicrobial resistance in uropathogens . in conclusion
, cefoperazone / sulbactam and vancomycin would be the first - line drug and most effective for the empirical treatment of uti . in conclusion ,
the present study demonstrated increased antibiotic resistance in uti isolates , which necessitates the careful selection of antimicrobials and their conservative use . | background : urinary tract infection ( uti ) is deemed the most prevalent infectious disease in that it has now touched the overall incidence of 18/1000 persons per year in the general population.objectives:this study sought to determine the characteristics of isolates from patients with uti and their susceptibility to commonly used antibiotics in punjab , pakistan.patients and methods : totally , 1429 urine samples were analyzed from uti patients for the isolation of uropathogens at chughtai s lahore lab , lahore , pakistan , during a period of 14 months .
the antimicrobial susceptibility test was performed via the disc diffusion method for the isolates obtained from 392 ( 26% ) positive cultures.results:the highest percentage ( 67% ) of isolates was from females in comparison to males ( 33% ) .
the frequency of escherichia coli was the highest ( 62% ) in culture - positive urine samples , followed by e. faecalis ( 15% ) , candida ( 14% ) , pseudomonas ( 6% ) , klebsiella spp .
( 1% ) , proteus ( 1% ) , and staphylococcus aureus ( 1% ) .
e. coli was highly resistant to antimicrobial drugs , viz .
cephalexin ( 95% ) , cephradine ( 95% ) , pipemidic acid ( 92% ) , amikacin ( 91% ) , and nalidixic acid ( 91% ) .
most of the routine -lactam antibiotics like amoxicillin / clavulanic acid , ampicillin , and aztreonam were also ineffective against e. coli , with resistance rates of 84% , 84% , and 72% , correspondingly .
this pathogen showed maximum susceptibility ( 97% ) against three drugs , namely imipenem , meropenem , and cefoperazone .
piperacillin and fosfomycin also provided significant results against e. coli with respective susceptibility rates of 96% and 90%.conclusions : our results showed that broad - spectrum antibiotics such as imipenem , meropenem , fosfomycin , cefoperazone / sulbactam , and vancomycin would be the first line and the most effective drugs for the empirical treatment of urinary tract pathogens due to their higher resistance rates against other drugs like cephalexin , cephradine , ciprofloxacin , levofloxacin , and norfloxacin . | 1. Background
2. Objectives
3. Patients and Methods
3.1. Isolation
3.2. Identification of Microorganisms
3.3. Antibiotic Susceptibility
4. Results
5. Discussion |
surgery is the major therapeutic method in soft tissue sarcomas of the extremity ( e - sts ) .
treatment of large high - grade tumours , which resection can not be performed with a wide safe margin , should include complementary radiation and/or chemo - therapy .
hopefully , the use of adjuvant brachytherapy will improve the prognosis of e - sts .
after a long process of diagnosing a tumour in the medial compartment of the thigh , a 65-year - old woman with diagnosed synovial sarcoma underwent a surgery .
compartment resection was performed and the tumour was removed with a 10 mm safety margin of healthy tissue .
adjuvant brachytherapy was delivered with ir ( microselectron , nucletron electa group , stockholm , sweden ) with 10 ci of nominal activity to a dose of 55 gy in 16 days because of large tumour size ( 99 78 73 mm ) and its proximity to the neurovascular bundle .
the wound healed without any complications and the outpatient follow - up is being continued .
adjuvant brachytherapy is rarely used after surgical treatment due to its limited accessibility in hospitals with surgical and orthopaedic departments .
there are numerous publications proving positive influence of brachytherapy on local control and decreased number of recurrences .
the recurrence - free survival time also increased significantly , however no direct impact on the number of distant metastases was found .
the most common adverse effects include : peripheral neuropathy , skin necrosis and osteonecrosis of the long bones .
treatment of large soft tissue sarcomas of the extremity ( e - sts ) should include combination of surgical intervention and external beam radiotherapy or brachytherapy .
adjuvant brachytherapy improves local control rate up to 78% , is well tolerated and rarely causes complications .
the extremities are the most common anatomic site where soft tissue sarcomas develop i.e. almost 50% of all cases [ 1 , 2 ] .
symptoms of these malignancies are not characteristic . usually , tumours do n't cause any pain .
knowledge about this type of neoplasms presented by surgeons and orthopaedists is incomplete , which often results with delays and even errors concerning diagnostic and therapeutic interventions .
one should be aware that every tumour , especially fast growing ones , located beneath the fascia requires special attention and is always an indication for a diagnostic biopsy prior final surgical intervention .
diagnostic procedures should include mri scans of the extremity with the tumour and ct scans of the lungs in order to exclude pulmonary metastases [ 37 ] .
surgical resection of the tumour with a safety margin of healthy tissue is the fundamental method of treatment of soft tissue sarcomas . at present , an amputation is avoided and the number of these procedures performed in soft tissue sarcomas has decreased by 20% . however , an amputation is recommended in sarcomas infiltrating the neurovascular bundle and tumours causing large changes extending outside compartments where limb salvage surgery can not preserve extremity function .
the application of brachytherapy and chemotherapy seems to be associated with improved local control and the lower rate of recurrences [ 35 ] . at present
, brachytherapy is not a standard method used for complementary treatment of soft tissue sarcoma of the extremity ( e - sts ) despite reports about its effectiveness .
the biggest problem seems to be its limited accessibility and technical limitations due to the fact that brachytherapy facilities are rarely located near surgical or oncologic orthopaedic departments with the possibility of shared surgical path .
currently three types of brachytherapy are used : neoadjuvant , adjuvant and a separate treatment method in tumours which can not be removed surgically .
the application of brachytherapy as a complementary therapy in soft tissue sarcomas has a history of 30-years . for many years , due to a small number of patients , published reports were fragmentary and unequivocal as far as recommendations were concerned . at present , publications include comparisons of different brachytherapy techniques i.e. hdr ( high - dose - rate ) , ldr ( low - dose - rate ) , and a recent one ultra - low - does - rate meaning the application of radioactive seeds .
effectiveness of brachytherapy is a result of its conformality as well as imaging methods used in order to spare critical organs .
the major advantage of brachytherapy is its clear influence on the increased recurrence - free five - year survival rate and better local control .
these adverse effects concern peripheral nerves , less frequently skin , and very rarely radiation - induced muscle damage and bone loss .
few randomized trials comparing the effectiveness of brachytherapy and external beam radiation therapy did n't unambiguously indicate which method was better [ 9 , 10 ] .
a 65-year - old woman was diagnosed outside the oncology centre because of a fast growing tumour localised in the medial compartment of the left thigh . at the beginning
the patient was treated in an outpatient surgical clinic where a puncture was performed as the surgeon suspected a synovial cyst .
the tumour was growing between and infiltrating three muscles : adductor magnus , sartorius and gracilis .
the large size of the malignancy ( 99 78 73 mm ) , its fast growth , increasing pain and numbness in the foot forced the need of faster diagnostics and treatment ( fig .
mri images of patient 's left thigh revealing a large soft tissue tumour located in the medial compartment .
sagittal plane through the largest dimension of the tumour ( a ) and a cross section ( b ) histopathological examination of tissue taken during the excision biopsy confirmed the suspected malignant change .
the patient had a second mri scan and the decision about limb salvage surgery was made .
ct scans of the lungs and abdominal cavity did n't reveal any metastases ( fig .
2 ) . a picture of a soft tissue tumour of the left thigh before the surgery .
a scar after the prior biopsy is visible on october 22 , 2012 , compartment resection with intraoperative histopathological assessment of safety margins was performed .
3 ) . the pictures of a tumour bed after resection of the neoplasm and muscle compartment ( a ) and the tumour with a tunnel after the biopsy ( b ) the surgery was performed in the presence of brachytherapy specialists ( radiation oncologists ) . after resection was finished applicators ( nucletron elekta group )
the distance between adjacent applicators was 1 cm whereas the proximal and distal margins vs. the tumour location in the longitudinal axis of the limb was 2 cm .
4 ) . the pictures presents the tumour bed in the thigh ( a ) and secured applicators for brachytherapy ( b ) brachytherapy was performed using hdr technique with an ir radioactive source .
treatment planning ( reconstruction ) was based on ct images ( ge bright speed 8) ( figs . 5 and 6 ) .
the total dose was 55 gy ( fraction dose of 2.5 gy ) delivered twice a day with a minimum 6-hour inter - fraction interval .
3d reconstruction of target volume ( green ) , bone structures ( white ) , nerve structures ( blue ) , muscles ( red ) with inserted applicators dose distribution for the target volume and organs at risk with corresponding dose volume histograms ( dvh ) no complications were reported during the surgery as well as postoperative period and brachytherapy .
the patient was discharged from the hospital on the 28 day after the limb salvage surgery and is in outpatient follow - up care .
we reviewed literature and found that doses delivered in similar localizations and application of hdr technique ranged between 32 and 55 gy regardless of treatment sequence i.e. perioperative or postoperative . in our case fraction dose was limited while the total dose remained unchanged mainly due to the close proximity of femoral nerve .
it seems that r0 resection is the optimal solution in small tumours graded as g1 and g2 .
large malignancies and those graded as g3 require resection with an adequate safety margin of soft tissue , which usually is impossible . therefore , a complementary therapy should be delivered .
chemotherapy in soft tissue sarcoma is usually delivered within the frames of clinical trials rather than standard treatment protocols .
brachytherapy in sts is rarely administered as a therapy complementary to surgical interventions due to its technical requirements and limited accessibility .
the advantage of brachytherapy is the fact that applicators are inserted under visual control therefore the process is very precise and decreases the number of complications .
however , recent publications report that new techniques with j seeds were used to deliver the dose ranging from 40 to 70 gy .
the complications rate is estimated to be around 10% and peripheral nerve damage is the major adverse effect comprising 5% [ 8 , 10 ] . according to published reports , the 5-year recurrence free survival rate was 78% [ 1215 ] .
brachytherapy in sts significantly decreases the number of local recurrences , however it has no direct effect on the number of distant metastases . at present
it is impossible to decide which type of radiation therapy is more effective , but shorter time of treatment seems to be relevant .
postoperative brachytherapy usually takes from 5 to 10 days whereas external beam radiation therapy starts from 4 to 6 weeks after the surgery .
there are no clear reports indicating that teletherapy is necessary in extremity soft tissue sarcomas .
surgical treatment is the basic therapeutic method in soft tissue sarcomas of the extremity ( e - sts ) , however delays in the diagnostic process often force the use of complementary oncologic therapy in order to maintain local control .
brachytherapy seems to be a good method of adjuvant therapy in e - sts , however its accessibility is limited .
further multicentre randomized studies should decide which method of adjuvant treatment ( external beam radiation therapy vs. brachytherapy ) is better and what is the optimal sequence of therapies improving local control . | purposesurgery is the major therapeutic method in soft tissue sarcomas of the extremity ( e - sts ) .
treatment of large high - grade tumours , which resection can not be performed with a wide safe margin , should include complementary radiation and/or chemo - therapy .
hopefully , the use of adjuvant brachytherapy will improve the prognosis of e-sts.case descriptionafter a long process of diagnosing a tumour in the medial compartment of the thigh , a 65-year - old woman with diagnosed synovial sarcoma underwent a surgery .
compartment resection was performed and the tumour was removed with a 10 mm safety margin of healthy tissue .
adjuvant brachytherapy was delivered with 192ir ( microselectron , nucletron electa group , stockholm , sweden ) with 10 ci of nominal activity to a dose of 55 gy in 16 days because of large tumour size ( 99 78 73 mm ) and its proximity to the neurovascular bundle .
no complications were reported .
the patient was discharged from the hospital on the 28th day after the surgery .
the wound healed without any complications and the outpatient follow - up is being continued.discussionadjuvant brachytherapy is rarely used after surgical treatment due to its limited accessibility in hospitals with surgical and orthopaedic departments .
there are numerous publications proving positive influence of brachytherapy on local control and decreased number of recurrences .
the recurrence - free survival time also increased significantly , however no direct impact on the number of distant metastases was found .
treatment is well tolerated and short .
the complication rate varies between centres from 5 to 30% .
the most common adverse effects include : peripheral neuropathy , skin necrosis and osteonecrosis of the long bones.conclusionstreatment of large soft tissue sarcomas of the extremity ( e - sts ) should include combination of surgical intervention and external beam radiotherapy or brachytherapy .
adjuvant brachytherapy improves local control rate up to 78% , is well tolerated and rarely causes complications .
we could n't determine which type of adjuvant radiation therapy is more effective . | Purpose
Case description
Discussion
Conclusions
Purpose
Case description
Discussion
Conclusions |
empyema thoracis is defined as the presence of pus in the pleural space or a purulent pleural effusion .
empyema is divided into three stages ( phases ) : stage i ( acute exudative phase ) , stage ii ( subacute fibrinopurulent phase ) , and stage iii ( chronic organizing phase ) . in stage
i , the visceral pleura remains elastic , and dimensions of the thoracic cavity are maintained .
stage ii is characterized by a turbid , infected fluid with fibrin deposits constructing bridges that septate the effusions , creating multiple loculations . in stage iii , this construct is replaced by formal granulation tissue .
the acute and sub - acute forms of empyema are easily managed with chest tube insertion and antibiotics with intrapleural fibrinolytics , and may occasionally require surgery .
chronic empyema is characterized by thickened visceral and parietal peels which hamper the ability of the affected lung to re - expand and requires definitive surgical intervention , i.e. , decortication with or without lung resection and/or pleural obliteration procedures like thoracoplasty and/or myoplasty .
these procedures , however , require a fit patient with a healthy ipsilateral lung for a successful outcome . frequently , these patients are so debilitated that they are eligible for neither decortication nor pleural obliteration surgeries , but still require drainage for uncontrolled empyema
. such patients may have a thick pleural rind and entrapment of the ipsilateral lung by the thick empyema membrane , which stabilizes the mediastinum .
in addition , there are often areas of pleural symphysis along the area of lung that is not entrapped .
these processes allow the pleural space to be opened to the atmosphere without fear of lung collapse .
thoracostomy ( opw ) , i.e. eloesser flap , or some modification of this surgery .
a prosthesis for open pleurostomy ( pop ) device has been described as a management option .
there are a few reports of successful treatment of an infected pleural space by drainage via tunneled indwelling pleural catheter ( pleurx ) , but placement is usually contraindicated in active intrapleural infection . although better tolerated than definitive surgical intervention , open pleural drainage strategies are not devoid of surgical morbidity and poor cosmetic results . thus , there is a need to devise a pleural drainage procedure that is simple , effective , and minimally invasive in nature .
such an intervention would be suitable for sick patients ineligible for most surgical drainage options . in the current series
, we describe our experience managing such patients by converting tube thoracostomy ( chest tube ) into an open pleural drain , which we have termed an empyema tube , as an alternative to the strategies previously described .
this observational study includes patients treated at the university of florida ( uf ) hospitals between january 2010 , and august 2014 .
we collected data on all patients who had received intrapleural fibrinolytics for the management of empyema but had nonresolution or inadequate treatment response .
patients considered unfit for surgical intervention after consultation with thoracic surgeons and with underlying unexpandable lung based on imaging findings were discharged with the empyema tube and were included for this study .
the study was approved by the institutional review board of uf , and a standardized protocol was followed throughout every stage of the study .
demographic information , age , sex , comorbidity , and procedure - related complications were recorded .
other data recorded included the length of hospital stay , drainage volume from the chest tube , duration of chest tube drainage , number and cumulative duration of antibiotics used , need for thoracic surgical intervention , blood and pleural fluid culture results , need for additional chest tubes , and functional status of the patient .
all patients underwent an initial 3-day course of once - daily sequential intrapleural tissue plasminogen activator ( 10 mg in 50 ml of sodium chloride ) , and deoxyribonuclease ( dnase , 5 mg in 30 ml of sodium chloride ) administration as per protocol followed at our center .
those with chronic empyema with unexpandable lung , deemed to be poor surgical candidates , were given the option to convert their chest tubes to empyema tubes .
unexpandable lung was confirmed based on visceral pleural thickening on computed tomographic scan , and lack of expansion of the ipsilateral lung with continued negative suction ( 20 cm of h2o ) applied to the chest tube .
the interventional pulmonary and thoracic surgery teams assessed the suitability of each patient before finalizing the decision to implement an empyema tube . for each patient ,
a narrow - bore pigtail chest tube was replaced by a standard chest tube under imaging guidance into the infected space .
the tube was then cut in close proximity to the skin , and sterile metal clips were used to secure the tube and prevent dislodgement into the pleural space [ figure 1 ] .
the end of the tube was covered with sterile gauze or an ostomy bag which was changed as needed , based on the amount of drainage .
the tube was withdrawn 12 cm at each visit until the pleural space became progressively obliterated and the exudate dried up , or the empyema tube fell out without intervention .
approximately 4 cm of the empyema - tube extends out of the chest wall with the rest still inside the pleural space .
the tube is covered with a sterile gauze or with a colostomy bag based on the amount of drainage discharge instructions included avoidance of swimming , submerging , or exposing the end of the tube to water , and the patient was instructed to lie preferentially on the side of the empyema tube to aid gravitational drainage .
posteroanterior and lateral chest x - ray was obtained during each clinic visit to assess radiological improvement , and the patient was evaluated for worsening infection .
clinic visits were scheduled every 2 weeks for the first 6 months and every 4 weeks thereafter .
all patients underwent an initial 3-day course of once - daily sequential intrapleural tissue plasminogen activator ( 10 mg in 50 ml of sodium chloride ) , and deoxyribonuclease ( dnase , 5 mg in 30 ml of sodium chloride ) administration as per protocol followed at our center .
those with chronic empyema with unexpandable lung , deemed to be poor surgical candidates , were given the option to convert their chest tubes to empyema tubes .
unexpandable lung was confirmed based on visceral pleural thickening on computed tomographic scan , and lack of expansion of the ipsilateral lung with continued negative suction ( 20 cm of h2o ) applied to the chest tube .
the interventional pulmonary and thoracic surgery teams assessed the suitability of each patient before finalizing the decision to implement an empyema tube . for each patient ,
a narrow - bore pigtail chest tube was replaced by a standard chest tube under imaging guidance into the infected space .
the tube was then cut in close proximity to the skin , and sterile metal clips were used to secure the tube and prevent dislodgement into the pleural space [ figure 1 ] .
the end of the tube was covered with sterile gauze or an ostomy bag which was changed as needed , based on the amount of drainage .
the tube was withdrawn 12 cm at each visit until the pleural space became progressively obliterated and the exudate dried up , or the empyema tube fell out without intervention .
approximately 4 cm of the empyema - tube extends out of the chest wall with the rest still inside the pleural space .
the tube is covered with a sterile gauze or with a colostomy bag based on the amount of drainage discharge instructions included avoidance of swimming , submerging , or exposing the end of the tube to water , and the patient was instructed to lie preferentially on the side of the empyema tube to aid gravitational drainage .
posteroanterior and lateral chest x - ray was obtained during each clinic visit to assess radiological improvement , and the patient was evaluated for worsening infection .
clinic visits were scheduled every 2 weeks for the first 6 months and every 4 weeks thereafter .
eight patients ( six males , two females ; four right , and four left ) , mean age 60.38 6.07 ( 95% confidence interval [ ci ] ) years , had a chest tube converted to an empyema tube [ figure 1 and table 1 ] .
two had primary metastatic lung cancer involving the ipsilateral lung , causing endobronchial obstruction and lung collapse .
the average length of hospital stay was 15.50 3.91 days ( 95% ci ) .
the mean cumulative pleural fluid drainage was 3343.75 662.72 ml ( 95% ci ) .
pleural fluid cultures were positive in seven of eight , with streptococci spp . and staphylococci spp . being the most prevalent species [ table 1 ] .
the mean duration of antibiotic use was 37 1.04 days ( 95% ci ) .
course and type of antibiotics were determined based on culture results after consultation with the infectious disease specialist .
no patient developed secondary infection related to thoracostomy duration , nor did any require premature removal of the tube .
one patient required the tube for 240 days ; excluding him , the average duration of drainage was 49.85 20.11 ( 95% ci ) days .
characteristics of the eight patients included in the analysis one patient [ no . 7 , table 1 ] had a slightly different clinical course .
a repeat surgical procedure was considered very high risk ; therefore , the chest tube was converted to an empyema tube .
all of our patients had severe illness ; functional class varied from eastern cooperative oncology group performance status 34 .
the american society of anesthesiology ( asa ) class varied from asa iii to asa iv . despite their tenuous conditions , all of our patients experienced empyema resolution .
figures 2 and 3 show sequential radiographic follow - up on two separate patients with chronic empyema who initially had pigtail catheters placed for drainage . once underlying unexpandable lung
was identified , the pigtail catheters were exchanged for surgical chest tubes , which were then converted to empyema tubes and gradually withdrawn as described in the protocol above .
we noted the subsequent progressive reduction in the size of the pleural cavity , which is eventually completely replaced with scarring and some degree of lung re - expansion , as seen in images posttube removal .
the pigtail has been replaced by a surgical chest tube in anticipation of discharge with an empyema tube ( b ) .
the chest tube has been converted into an empyema tube ( c ) and a clip has been placed over the chest tube to prevent the accidental retraction of the tube into the pleural space . the empyema cavity has resolved and the empyema tube has been removed ( d ) .
a small pleural scar has replaced the empyema cavity a pigtail has been placed to drain the right - sided empyema ( a ) .
the pigtail has been replaced with a surgical chest tube ; the pus has been mostly drained and a hydropneumothorax remains ( b ) .
the chest tube is cut close to the skin surface and a clip applied to it to prevent accidental retraction of the tube into the pleural space ( c ) .
a linear vertical pleural scar remains as a result of the empyema , but the infection has been controlled along with expansion of the ipsilateral lung
despite recent advances , management of chronic empyema remains a challenge for modern medicine . the overall mortality is still high , and the best treatment remains to be defined .
when there is a healthy lung that can re - expand and the patient can tolerate a major operation , pulmonary decortication is the treatment of choice , with or without the use of video - assisted thoracoscopic surgery .
however , the management of those patients deemed too unstable to undergo this procedure remains controversial .
most often , an opw ( eloesser flap ) procedure is performed . approximately , 620 cm of two to four ribs is usually resected during this procedure , creating a large defect in the chest wall .
although effective for the treatment of chronic empyema , major resection of the chest wall induced by an opw should , in our opinion , be avoided .
we feel especially strongly about this considering the current global trend toward minimally invasive operations .
another proposed management option , the pop , involves a surgical procedure under general anesthesia where the prosthesis is introduced in the intercostal space with about 2 cm of the tube protruding from the chest wall .
these patients may need thoracoscopic guidance and adhesiolysis before the prosthesis can be correctly placed .
the pop procedure is not routinely performed and the prosthesis is not ubiquitously available , nor is it currently available in the united states . due to significant morbidity , mortality , and nonavailability of open pleural drainage options mentioned above , many patients experience prolonged hospital stays with a draining chest tube , where they are susceptible to health - care associated infections , increased medical costs , and reduced quality of life .
we , in the form of this current case series , report our experience in management of these complex patients by converting the chest tube localized in the infected space with image guidance , into an
prior to executing this management option , we confirm the presence of chronic empyema , unexpandable lung , and high surgical risk .
our study demonstrates that empyema tube drainage is simple , safe , well - tolerated , and effective .
results from our study are comparable to those of a pop procedure and opw surgery in terms of treatment efficacy .
additionally , use of an empyema tube offers advantages over conventional approaches in that it is placed under local anesthesia , does not require a prolonged surgery , and avoids postoperative pain and other potential complications .
a significant advantage for the patient is that empyema tube drainage precludes major chest wall resection , thus avoiding gross esthetic compromise . among all patients in our series ,
the residual pleural space was replenished by fibrous tissue with some degree of lung re - expansion , and the empyema - tube was removed in 16 months .
although none of our patients underwent pleural space obliteration procedures , empyema - tubes can potentially be used as a bridge to thoracoplasty and muscle or omental transposition surgeries . some limitations of our study worth highlighting are its retrospective nature and small sample size .
we also did not employ any quality of life questionnaires to assess superiority of empyema tube to opw drainage .
our experience , however , demonstrated that empyema tubes allowed drainage of the infected space and pleural healing with complete success . in this clinical scenario where treatment options are limited and often dismal
we , therefore , believe that chronic empyema can now be treated with a much less invasive and nondeforming operation , with the same result as conventional surgical techniques .
empyema tube drainage provides a practical solution for managing extremely debilitated and marginal surgical candidates because of the simplicity of its design and effectiveness in helping with infection control , and could be considered a first - line management option for patients with chronic empyema , unexpandable lung , and poor surgical candidacy .
this study identifies a nonsurgical , minimally invasive method to treat chronic empyema in patients who are not candidates for decortication or other forms of definitive surgery .
the procedure is safe , obviates the need for surgical intervention , and can be performed without extensive training .
it shortens hospital stays , expedites the patient 's return to a normal lifestyle , and is free of major side effects . | objectives : high preoperative risk precludes decortication and other surgical interventions in some patients with chronic empyema . we manage such patients by converting the chest tube into an
empyema tube , cutting the tube near the skin and securing the end with a sterile clip to allow for open pleural drainage . the patient is followed serially , and the tube gradually withdrawn based on radiological resolution and amount of drainage.methods:between 2010 and 2014 , patients with chronic empyema and unexpandable lung , deemed high - risk surgical candidates , had staged chest tube removal , and were included for the study .
the volume of fluid drained , culture results , duration of drainage , functional status , and comorbidities were recorded.measurements and results : eight patients qualified .
all had resolution of infection .
the tube was removed after an average of 73.6 49.73 ( 95% confidence interval [ ci ] ) days .
the mean duration of antibiotic treatment was 5.37 1.04 ( 95% ci ) weeks .
none required surgery or experienced complications from an empyema tube.conclusion:a strategy of empyema tube drainage with staged removal is an option in appropriately selected patients with chronic empyema , unexpandable lung , and poor surgical candidacy . | INTRODUCTION
METHODS
Protocol
RESULTS
DISCUSSION
Financial support and sponsorship
Conflicts of interest
CONCLUSION |