This study aims to gather information either supporting or rejecting the hypothesis that acute stress may induce ovulation in women. The formulation of this hypothesis is based on 2 facts: 1) estrogen-primed postmenopausal or ovariectomized women display an adrenal-progesterone-induced ovulatory-like luteinizing hormone (LH) surge in response to exogenous adrenocorticotropic hormone (ACTH) administration; and 2) women display multiple follicular waves during an interovulatory interval, and likely during pregnancy and lactation. Thus, acute stress may induce ovulation in women displaying appropriate serum levels of estradiol and one or more follicles large enough to respond to a non-midcycle LH surge.A literature search using the PubMed database was performed to identify articles up to January 2010 focusing mainly on women as well as on rats and rhesus monkeys as animal models of interaction between the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes.Whereas the HPA axis exhibits positive responses in practically all phases of the ovarian cycle, acute-stress-induced release of LH is found under relatively high plasma levels of estradiol. However, there are studies suggesting that several types of acute stress may exert different effects on pituitary LH release and the steroid environment may modulate in a different way (inhibiting or stimulating) the pattern of response of the HPG axis elicited by acute stressors.Women may be induced to ovulate at any point of the menstrual cycle or even during periods of amenorrhea associated with pregnancy and lactation if exposed to an appropriate acute stressor under a right estradiol environment. It is known that the percentage of pregnancies resulting from single episodes of forced penile-vaginal intercourse (rape) is significantly higher  than the percentage of pregnancies resulting from single episodes of consensual, unprotected intercourse (3.1% in a sample of 221 women with no fertility problems planning to become pregnant in USA)  ; or (2) As far as we know, there are no studies reporting increases in HPA axis reactivity to acute stressors in estrogen-treated perimenopausal, menopausal or ovariectomized women. Only one study in young men has found increased peak ACTH and cortisol responses to a brief psychosocial stress  after 24-48 h of estradiol treatment compared to a placebo group .The studies focused on the effects of hormone replacement on HPA axis response suggest that estrogen treatment in ovariectomized females or perimenopausal and menopausal women may modify the HPA axis response to acute stressors. In rats, it appears that estradiol treatment increases the responsivity of the HPA axis to different acute stressors. In rhesus monkeys, the HPA axis response to a 30-min intracerebroventricular infusion of interleukin-1α depends on the plasma level of estradiol after estrogen replacement. In particular, low and high plasma concentrations increase the response whereas intermediate concentrations are inhibitory. In contrast, estrogen replacement in women does not change or even may decrease the typical response of the HPA axis to acute stressors. Although it is difficult to compare among studies the absolute plasma levels of estradiol exhibited by women before being exposed to acute stressors because different measurement methods were used, it is worth mentioning that, in all the studies analyzed in this section, independently of the replacement regimen followed, the HPA axis always displayed a positive response when plasma levels of estradiol were relatively low (≤ 73.4 pmol/L) or intermediate (115.0-315.7 pmol/L) whereas the response was decreased when plasma levels of estradiol were relatively high (587.4-1118.0 pmol/L). Thus, it can be hypothesized that the response of the HPA axis to inflammatory or psychological stressors evidenced in perimenopausal, menopausal or ovariectomized women may depend on the range of plasma estradiol concentration present in women when exposed to these stressors.Table Brown-Grant et al.  reportedIn cycling rhesus monkeys, Norman et al.  found thThe surgical stress of bilateral ovariectomy plus total hysterectomy performed under general anesthesia during the mid- to late-follicular phase of the menstrual cycle induces a small increase in plasma levels of LH 36 h after surgery (from ≈ 6 IU/L to 8 IU/L), preceded by an elevation of adrenal progesterone . LikewisAlthough some studies report that light, heavy or exhaustive exercise does not alter  or even The studies analyzed in this section show conflicting results on the effects of ovarian cycle on acute-stress-induced LH release in rats. Some studies seem to indicate that the adenohypophysis is much more responsive to acute stressors on the morning of proestrus than at diestrus. In contrast, other studies suggest the opposite. It can be hypothesized that the degree of adrenal-progesterone response depends on the strength of the stressor applied. Thus, strong stressors such as intracerebroventricular injection of interleukin-1α or β or rapid blood volume depletion may induce an adrenal-progesterone response higher than that elicited by more moderate stressors such as laparotomy. Such an adrenal-progesterone response would inhibit the release of LH if plasma levels of estradiol are elevated (proestrus morning) but it would stimulate the release of LH under low estradiol backgrounds (e.g. the diestrus stage). Likewise, a moderate rise in adrenal-progesterone concentrations equivalent to that observed during the preovulatory midcycle rise would be stimulatory if it takes place under high plasma levels of estradiol (proestrus morning) but it would be unable to elicit a release of LH under low estradiol backgrounds (e.g. the diestrus stage).In rhesus monkeys, females at the mid-follicular and mid-luteal phases, but not at the early-follicular stage, display appropriate plasma levels of estradiol for acute stressors to induce a release of LH.Although there are some conflicting reports in women, it seems that the mid-follicular phase gathers more favorable conditions than the mid-luteal phase for the release LH by the adenohypophysis in response to acute stressors.Table In contrast to ovariectomized non-estrogen-primed rats, ovariectomized estrogen-primed rats respond with a small but significant LH peak (≈ 0.017 IU/L) either 1 h after the onset of a prolonged (11 h) immobilization  or 10 miIn ovariectomized rhesus monkeys after 5 days of estrogen replacement therapy at doses simulating the late follicular phase, a 30-min intracerebroventricular infusion of interleukin-1α results in a small but significant increase in LH release during the first 5 h after treatment (from 0.014 IU/L to 0.019 IU/L) . HoweverEstrogen-treated menopausal or ovariectomized women respond to an inflammatory/immune-like stress, induced by single intravenous injection of endotoxin, with an ovulatory-like LH peak (50 IU/L) 7 h after injection. In contrast, non-estrogen treated women do not modify their basal LH levels after endotoxin injection .Studies in ovariectomized rats show paradoxical results as far as the response of the adenohypophysis to different acute stressors is concerned. Whereas 2 studies ,71 foundLiterature focused on rhesus monkeys shows a stimulatory effect of estradiol replacement on acute-stress-induced LH release at doses that result in plasma estradiol levels reproducing the late follicular phase. This stimulatory effect, however, is not found after estradiol treatment at doses that result in plasma estradiol levels simulating the early-mid follicular phase.Finally, estrogen-treated menopausal or ovariectomized women respond with a clear ovulatory-like LH peak after a single intravenous injection of endotoxin .The studies reviewed in this bioessay indicate that the ovarian cycle and, in particular, the female's estradiol background modulates the response of the HPA and HPG axes to acute stressors. The pattern of response, however, differs between the HPA and HPG axes. In fact, although the highest responses of the HPA axis within the ovarian cycle are observed when females display relatively high plasma levels of estradiol , the HPA axis exhibits positive responses in practically all phases of the ovarian cycle. In contrast, it seems that there is only one specific period of time within the ovarian cycle during which the HPG axis response is possible. In particular, positive responses of the HPG axis are found under relatively high plasma levels of estradiol on the morning of proestrus in rats, during the mid-follicular and mid-luteal phase in rhesus monkeys and during the mid-follicular phase in women. This conclusion is endorsed by the fact that the HPG axis of estrogen-treated ovariectomized females and perimenopausal or menopausal women also exhibit a positive response to acute stressors.Two studies ,72 in thIn women, the pattern of LH release elicited by acute stressors may vary from small non-ovulatory rises in plasma concentrations and/or slight changes in pulse characteristics to the typical ovulatory surge that spontaneously takes place at the middle of the menstrual cycle. As women present waves of follicular development during an interovulatory interval (and likely during pregnancy and lactation), they may be induced to ovulate at any point of the menstrual cycle or even during periods of amenorrhea associated with pregnancy and lactation if exposed to an appropriate acute stressor under a right estradiol environment. We should bear in mind that the maximum diameters (range of 10-17 mm) of the dominant follicles from anovulatory major waves, which account for 21% of the anovulatory follicular waves, are compatible with potential ovulations if follicles were correctly stimulated by an ovulatory-like LH surge. Follicles large enough (≥ 15 mm of diameter) to ovulate may be found in the ovaries of all the regular-cycling women at the late-mid follicular phase . In addition, they may be present in the ovaries of women exhibiting (1) a major-major 2-wave pattern at the mid-luteal phase ; (2) a minor-major-major 3-wave pattern at the early-mid-follicular phase ; and (3) a major-major-major 3-wave pattern at the late-mid-luteal and early-mid follicular phases  . We shouWe should bear in mind that an acute-stress-induced surge of LH is shortly preceded by an elevation of serum progesterone from the adrenal glands. This fact suggests that such an elevation of serum progesterone may advance the secretory transformation of the endometrium resulting in embryo-endometrium asynchrony and consequently reduced chances of implantation and pregnancy if ovulation and fertilization took place. However, a recent systematic review and meta-analysis  has not The authors declare that they have no competing interests.JJT has been involved in conception and design, acquisition, analysis and interpretation of data and drafting the article. TH and AC have been involved in analysis and interpretation of data, and revising the article critically for important intellectual content. All authors read and approved the final manuscript.


HIV care and treatment services are primarily delivered in vertical antiretroviral (ART) clinics in sub-Saharan Africa but there have been concerns over the impact on existing primary health care services. This paper presents results from a feasibility study of a fully integrated model of HIV and non-HIV outpatient services in two urban Lusaka clinics.Integration involved three key modifications: i) amalgamation of space and patient flow; ii) standardization of medical records and iii) introduction of routine provider initiated testing and counseling (PITC). Assessment of feasibility included monitoring rates of HIV case-finding and referral to care, measuring median waiting and consultation times and assessing adherence to clinical care protocols for HIV and non-HIV outpatients. Qualitative data on patient/provider perceptions was also collected.Provider and patient interviews at both sites indicated broad acceptability of the model and highlighted a perceived reduction in stigma associated with integrated HIV services. Over six months in Clinic 1, PITC was provided to 2760 patients; 1485 (53%) accepted testing, 192 (13%) were HIV positive and 80 (42%) enrolled. Median OPD patient-provider contact time increased 55%  and decreased 1% for ART patients . Median waiting times increased by 36 (p<0.001) and 23 minutes (p<0.001) for ART and OPD patients respectively. In Clinic 2, PITC was offered to 1510 patients, with 882 (58%) accepting testing, 208 (24%) HIV positive and 121 (58%) enrolled. Median OPD patient-provider contact time increased 110%  and decreased for ART patients by 23% . Median waiting times increased by 47 (p<0.001) and 34 minutes (p<0.001) for ART and OPD patients, respectively.Integrating vertical ART and OPD services is feasible in the low-resource and high HIV-prevalence setting of Lusaka, Zambia. Integration enabled shared use of space and staffing that resulted in increased HIV case finding, a reduction in stigma associated with vertical ART services but resulted in an overall increase in patient waiting times. Further research is urgently required to assess long-term clinical outcomes and cost effectiveness in order to evaluate scalability and generalizability. In Zambia as elsewhere in sub-Saharan Africa, HIV care and treatment is primarily delivered in stand-alone or vertical antiretroviral (ART) clinics located next to primary health care clinics To develop a model of integrated service delivery, the Centre for Infectious Disease Research in Zambia (CIDRZ) working with LDHMT, conducted site assessments of the ART and OPDs of ten urban primary health care clinics, documenting and comparing clinical space, staff availability, referral protocols, patient flow and medical record keeping. Findings were analyzed and cross referenced to provide the basis for a model of integrated service delivery which focuses on three key modifications: i) amalgamation of physical space and patient flow; ii) standardization of medical records and screening forms and iii) introduction of routine provider initiated testing and counseling (PITC).Primary operational objectives of the pilot were to i) increase HIV case finding; ii) improve adherence to clinical care protocol for OPD patients iii) maintain adherence to clinical care protocol for ART patients; iii) reduce clinic-based HIV-associated stigma and iv) improved continuum of care between departments. To meet these objectives, the integrated patient flow adopted a modified first-come, first-serve approach with a fast-track mechanism . All patnot know their HIV status. Any patient who did not have a recent (<6 months) written record of a negative HIV test was offered counselling and opt-out (finger-prick) testing by one of two lay counsellors with results recorded in patient files. This is an innovation in Zambia, where routine PITC is not offered in OPDs, and patients must attend stand-alone VCT services in order to test. Patients testing positive were offered a chance to enrol in the ART program immediately or return at a later (pre-determined) date. Finally, Enrolment Track was for returning HIV positive patients referred for ART enrolment. After recording of vitals signs, patients in this track went through enrolment/registration procedures before being screened by a clinician. On subsequent visits, these patients were triaged to Fast Track , doctors (MOs) and lay personnel. Those who have ART training are eligible to work in the ART clinics and receive ‘over-time’ payment for this work Clinics 1 and 2 were purposively selected by District officials based on moderate catchment population , presumed high rates of undiagnosed HIV, and extreme space and staffing constraints that were being exacerbated by vertical departments. In the first phase of implementation, a series of information and planning meetings were held with clinic staff and community representatives. Initial meetings involved working with clinic leadership to map available infrastructure and plan re-allocation of space to accommodate the integrated patient flow. Essential small-scale renovations were planned such as installation of shelving to accommodate files. Meetings with the wider clinic staff were held to introduce the concept of integration and the specific model being implemented, providing a chance for staff to voice concerns and offer feedback. Meetings with neighbourhood health committees (NHC) also took place to inform members of the upcoming changes and provide them with information to disseminate to their respective catchment areas.During the second phase, community sensitization was scaled up to include drama performances by a trained theatre troupe in strategic locations. Performances were designed to use common themes and experiences to inform community members that clinic services would be integrated and to explain how the changes would affect them. Renovations were completed and a series of three didactic and interactive staff trainings were conducted with lay and professional health workers to ensure familiarity with the integrated model. In the third phase clinic space was re-arranged to accommodate the altered patient flow and the integrated service model initiated. For three weeks following initiation, intensive support and in-service mentoring for clinical and registry staff was provided to facilitate a smooth transition. Thereafter on-going monthly follow-ups were conducted by CIDRZ staff with LDHMT oversight.University of Alabama IRB: Protocol No: X080403013; Continuing Review received 28 April 2009University of Zambia REC: Protocol No: 003-02-08; Continuing Review received 28 April 2009This feasibility study collected data in six areas matching the pilot objectives. Rates of clinic-based HIV case-finding and referral; adherence to collection of vital signs for ART and OPD patients; adherence to 6 indicators of clinical protocol for ART patients; amount of with-provider time and per visit waiting times for OPD and ART patients and finally, patient and provider perceptions.HIV case-finding and referrals were measured by collating operational data from clinic registers on routine PITC services , S2 as wAdherence to collection of patient's vital signs was assessed via random file review of 100 OPD and ART files at each site. This provided an estimate of the number of patients with vital signs recorded at their last visit, pre- versus post-implementation. Adherence to 6 indicators of ART protocol was measured for all patients enrolled in the ART program at both sites, during the two quarters pre and two quarters post integration. Data for the two sites were extracted from the electronic medical record system on a quarterly basis starting from April 2008 through July 2009. Indicators were selected to investigate associations (not measure causation) and included: (1) percentage of newly enrolled patients with a baseline CD4 measurement, (2) percentage of delinquent patients per quarter (3) percentage of newly enrolled, eligible patients who are on antiretroviral drugs (4) percentage of newly enrolled, eligible patients who are on cotrimoxizole, (5) percentage of newly enrolled patients on zidovudine who had a hemoglobin measurement, (6) percentage of active patients who attended a scheduled follow-up visits in the last four months. A two-sided Wilcoxon Rank Sum Test was used to measure differences in performance between: i) the quarter immediately pre- and quarter immediately post-implementation and ii) the quarter immediately pre- and the second quarter post-implementation.A time-in-motion study was conducted to measure median waiting and with-provider times per patient visit pre- and post-implementation. With-provider and per-visit waiting times were intended to provide a rough proxy for quality of care and provide a standard indicator that could be used to compare the effect of integration across the two cadres. Data were collected over two seven day periods, pre- and post-implementation. Data was recorded using a study form , S4 attaPatient and provider perceptions were collected in one-on-one interviews using semi-structured questionnaires and free-listing techniques. Patient interviews investigated perceptions of their experience in either the vertical or the integrated services asking them to list positive and negative features of their visits. Patients were randomly selected and interviews were conducted in a private room by trained community interviewers in English, Nyanja or Bemba. Interviews with health care workers were conducted in English, and focused on positive and negative aspects of working in the clinic, pre- and post- integration. Interview questions were open ended and responses were manually recorded, entered into an electronic database, coded inductively and analyzed for common themes. All participants provided written informed consent.This study was approved by the University of Alabama at Birmingham Institutional Review Board  and the University of Zambia Research Ethics Committee .In Clinic 1 over six months, PITC was provided to 2760 OPD patients, with 1485 53%) accepting testing, 192 (13%) found HIV positive and 80 (42%) subsequently enrolled in ART care with a minimum follow up of 6 months. In six months of integration at Clinic 2, PITC was offered to 1510 OPD patients, with 882 (58%) accepting testing, 208 (24%) HIV positive and 121 (58%) enrolled in care. Although it is beyond the scope of this particular paper to investigate characteristics of those enrolled in ART, continued demand for clinics' stand-alone voluntary counseling and testing (VCT) service suggests that the population being reached by PITC is different from that accessing VCT ,3.% acceptiFrom a pre-implementation baseline of nil, vital signs were collected during 76% (n = 76) and 73% (n = 73) of OPD patients' latest visits at Clinic 1 and 2 respectively. No change was observed in collection of vitals for ART patients with over 96% recorded pre- and post-integration.In Clinic 1, data on 151 ART and 357 OPD patients were captured in the pre-implementation time-in-motion study, and data on 129 ART and 385 OPD patients post-implementation. Six month follow-up at Clinic 1 showed 55% increase  in median patient-provider contact time per OPD visit and 1% decrease  for ART patients . Median In Clinic 2, data on 201 ART and 585 OPD patients were captured pre-implementation, and on 221 ART and 248 OPD patients post implementation. In both clinics, seasonal fluxes in weekly and monthly clinic attendances are common and may account for the uneven OPD attendance pre- and post-implementation . Median Semi-structured interviews were carried out with ART and OPD patients in Clinic 1 (Post: n = 16) and Clinic 2 . Semi-structured interviews were conducted with ART and OPD staff in Clinic 1  and Clinic 2  summarized in In this report, we demonstrate the feasibility of complete integration of HIV care and treatment with non-HIV outpatient services in a high-HIV prevalence, low-resource setting. Over the first six months, positive outcomes matching the pilot objectives included: i) an additional 4270 patients being counseled of whom 2367 (55%) accepted testing, 400 (17%) identified as HIV-positive, and 201 (50%) enrolled into HIV care; ii) A 70% increase (from a baseline of zero) on OPD patients receiving pre-screening collection of vital signs; iii) a reduction in both patient and staff perceptions of stigma associated with HIV care and treatment; iv) improved staff communication and teamwork. Negative or unintended outcomes of the pilot included an increase in the waiting times for both OPD and ART patients and a drop in some indicators for delivery of care for ART patients; a further concerning outcome was some ART patients' perception that the integrated service provides a less secure environment in which to share experiences with fellow patients.Although small in scale, this intervention constitutes perhaps the first documented attempt to fully integrate ART and OPD services in a setting where HIV care and treatment was established vertically. This pilot stands apart from other documented integration models in that it constitutes a complete harmonization of point-of-care HIV and non-HIV services versus strengthened referral between still vertical services recommended for testing by a clinician are required to visit stand-alone services before returning to the clinician with their result. The stigma associated with being seen at VCT, fear of the test itself and lack of accompaniment by clinic staff have been shown to affect testing uptake and reduce the likelihood of HIV-positive patients returning to seek care Over time, the stand-alone nature of both HIV testing and HIV treatment services has contributed to a culture of HIV/AIDS exceptionalism in the Zambian health care system. Patients independently seeking testing or treatment may be deterred by the stigma associated with being seen at that service. Patients With a national HIV prevalence of 14.3% and 50% of HIV- infected individuals not yet in careall health servicesVertical ART and OPD services have exacerbated already limited space and human resources in the Zambian health care setting. WHO recommends a minimum of 20 physicians and 100 nurses per 100,000 population while in 2007, Zambia had with 7 physicians, 9.2 clinical officers and 113 nurses per 100,000 spread across Pre-implementation interviews demonstrated that vertical services contributed to ART and OPD staff working in relative isolation hindering communication, transfer of medical records and referrals. Continuum and quality of care was undermined by logistical breakdowns that diminished clinicians' ability to make accurate diagnoses, prescribe appropriate medications, and track potential drug interactions. The well documented difficulties involved in referring patients between vertical OPD, ART, antenatal care and other services were similarly experienced in this setting beginning of the patient flow  resulted in exponentially larger delays further along the process. For example, a 15–20 minute delay in transferring the first files from registry to vitals can result in the final ‘station’  not seeing a patient for several hours.Results from this study indicate a number of operational challenges. First, this model of integrated HIV and primary health services resulted in increased patient waiting times. Disaggregated results from the time-in-motion study (not shown) suggest that the increase is attributable to two factors. First, the re-introduction of collection of OPD vital signs and the addition of PITC automatically increased patient waiting time by creating additional stops in the patient flow. Second, by combining two groups of patients and increasing the number patients moving through a single system, small delays at the Although visit times remained within manageable limits in the pilot sites literature documenting the effects of waiting times clearly indicate that longer wait times can be a barrier to retention in care Routinely collected data on HIV case-finding and referral demonstrated significant increases in the uptake of testing but only a minimal increase in ART enrollment rates with six month follow-up. Since delay between the time-of-testing to time-of-enrolment is common, a longer follow-up period may demonstrate overall higher rates of enrolment. However, these results highlight the continued difficulty in enrolling HIV patients into care and we posit that an integrated service may facilitate increased HIV case-finding but not improve the rates of successful enrolment into care. Further work to systematize this process and promote duty-based responsibilities is necessary to minimize reliance on individuals.less able to discuss their problems with fellow patients due to the mixing of OPD and ART queues. Careful consideration of these two factors is necessary when planning integration. Extensive community sensitization in advance of integration minimizes the shock experienced by patients attending a newly integrated clinic, while ongoing, within-clinic patient sensitization and counseling is essential to strengthen understanding and a sense of security among both cadres of patients.Post-integration patient interviews indicated that many OPD and ART patients experienced a reduction in perceived stigma associated with HIV care and treatment in the integrated service. A contradictory finding was that some ART patients felt Although this study did not formally evaluate infrastructure, we note that appropriate infrastructure is an essential pre-requisite to effective integration. Inclusion of provider-initiated counselling and testing and harmonized registry and pharmacy services all require re-allocation of space. In this setting, innovative and experimental approaches were necessary to make use of limited space while ensuring appropriate infection control measures were in place. Nonetheless, in some sites integration of this type would not be possible without significant renovations or new infrastructure altogether.This pilot was undertaken in Lusaka urban clinics and our findings may not be generalizable to other settings. We note that six months follow-up time is a relatively short period and precludes a rigorous cost-effectiveness evaluation or assessment of the impact of integration on retention-in-care and long-term clinical outcomes for ART-enrolled patients. A cost effectiveness study will be necessary to provide information on the scalability of this model.The model presented here is one of the first case studies documenting the feasibility of formal integration of ART into general outpatient services at the primary health care level. Integration of care allowed shared use of space and staffing that resulted in increased HIV case finding, improved collection vital signs for OPD patients, a reduction in stigma associated with ART services but an overall increase in patient waiting times. This paper demonstrates ways in which resources for ART scale-up may be directed towards harmonizing service delivery systems in a high HIV-prevalence, low-resource setting. A rigorous evaluation is urgently required to assess true scalability, generalizability, long-term clinical outcomes and cost effectiveness.Appendix S1PITC Register, page 1.(0.01 MB TIF)Click here for additional data file.Appendix S2PITC Register, page 2.(0.00 MB TIF)Click here for additional data file.Appendix S3Pre-Implementation Time-in-Motion Study Form.(0.01 MB TIF)Click here for additional data file.Appendix S4Post-Implementation Time-in-Motion Study Form.(0.22 MB TIF)Click here for additional data file.Appendix S5Monthly OPD Attendances Clinics 1 & 2, 2006–2008.(0.44 MB TIF)Click here for additional data file.


Low serotonergic (5-HT) activity correlates with increased impulsive-aggressive behavior, while the opposite association may apply to cognitive impulsiveness. Both types of impulsivity are associated with attention-deficit/hyperactivity disorder (ADHD), and genes of functional significance for the 5-HT system are implicated in this disorder. Here we demonstrate the separation of aggressive and cognitive components of impulsivity from symptom ratings and test their association with 5-HT and functionally related genes using a family-based association test (FBAT-PC).Our sample consisted of 1180 offspring from 607 families from the International Multicenter ADHD Genetics (IMAGE) study. Impulsive symptoms were assessed using the long forms of the Conners and the Strengths and Difficulties parent and teacher questionnaires. Factor analysis showed that the symptoms aggregated into parent- and teacher-rated behavioral and cognitive impulsivity. We then selected 582 single nucleotide polymorphisms (SNPs) from 14 genes directly or indirectly related to 5-HT function. Associations between these SNPs and the behavioral/cognitive groupings of impulsive symptoms were evaluated using the FBAT-PC approach.In the FBAT-PC analysis for cognitive impulsivity 2 SNPs from the gene encoding phenylethanolamine N-methyltransferase  attained corrected gene-wide significance. Nominal significance was shown for 12 SNPs from BDNF, DRD1, HTR1E, HTR2A, HTR3B, DAT1/SLC6A3, and TPH2 genes replicating reported associations with ADHD. For overt aggressive impulsivity nominal significance was shown for 6 SNPs from BDNF, DRD4, HTR1E, PNMT, and TPH2 genes that have also been reported to be associated with ADHD. Associations for cognitive impulsivity with a SERT/SLC6A4 variant (STin2: 12 repeats) and aggressive behavioral impulsivity with a DRD4 variant (exon 3: 3 repeats) are also described.A genetic influence on monoaminergic involvement in impulsivity shown by children with ADHD was found. There were trends for separate and overlapping influences on impulsive-aggressive behavior and cognitive impulsivity, where an association with PNMT  was more clearly involved in the latter. Serotonergic and dopaminergic mechanisms were implicated in both forms of impulsivity with a wider range of serotonergic mechanisms  potentially influencing cognitive impulsivity. These preliminary results should be followed up with an examination of environmental influences and associations with performance on tests of impulsivity in the laboratory. Impulsive actions usually damage not only the victim but often their perpetrator. In fights, suicide or fire-setting the aggressive component is immediately apparent. But the risky and overly rapid decisions behind these actions demonstrate a cognitive component that is often revealed in gambling and seeking immediate gratification. Impulsivity is a core construct of many mental disorders . In partAcross a group of youngsters with ADHD-ct there are many who show abrupt bursts of impulsive aggression (related to conduct disorder), but many others who (merely) make rapid and wrong decisions in everyday life, or abhor delayed gratification to their cost . Here, wImpulsivity has been briefly and broadly defined as "action without foresight" . More geMore differentiated views of impulsiveness  suggest The goal of the current study is to disentangle the relationship between aggressive impulsivity of a more behavioral nature and cognitive impulsivity in ADHD by exploring the differential patterns of association of these two forms of impulsivity with genes that have significance for 5-HT function, a putative neurobiological marker of impulsivity. There is mounting evidence that polymorphisms for genes influencing several features of the 5-HT system are associated with ADHD , and a cWe therefore explored whether there would be commonalities and differences between the two forms of impulsivity and 5-HT genes controlling the activation or suppression of activity within 5-HT systems, along with other genetic markers of neuronal activity (e.g. dopamine) which influence or are influenced by the activity in 5-HT systems .The initial data-set contained 614 families with 776 European Caucasian cases of DSM-IV ADHD-ct and 706 siblings without the disorder. These families had been recruited as part of the International Multicenter ADHD Genetics Project (IMAGE: details in ). They hExclusion criteria were genetic or medical disorders associated with behavior mimicking ADHD, autism, epilepsy, IQ<70, and the absence of a medication-free period permitting evaluation in the previous 2 years. Approval for the study was obtained from ethical review boards at each clinic, which were registered with and monitored by the National Institutes of Health, and were in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).Clinical assessments were made during medication free periods or based on recall of such periods in the last 2 years. Diagnoses of ADHD-ct were based on the semi-structured Parental Account of Children's Symptoms . IntervFor both the cases and the siblings, parents completed the long form of the revised CPRS (80 items) and teachers the long form of the revised CTRS (59 items ). Both aWe selected 14 genes with 582 single nucleotide polymorphisms (SNPs) from the data available from the IMAGE study for the core analysis reported here. For the 5-HT system, markers for pre- and post-synaptic receptors , and synaptic availability controlled by synthesis (TPH2) and re-uptake (SERT/SLC6A4) were chosen. We reasoned that some dopaminergic (DA) biomarkers should be included in view of widespread interactions between 5-HT and DA systems in the brain . TheThe DNA sample used here is a part of a larger genetic sample from the IMAGE study. Details of the DNA isolation and SNP selection can be found elsewhere . BrieflyTo determine that the 47 symptoms selected for association analysis would indeed group into major factors of cognitive and aggressive behavioral impulsivity we ran a preliminary factor analysis (Promax) with an oblique rotation in the SAS statistical package. This rotation is in line with clinical experience that subjects present concurrently with symptoms from different dimensions . Four faWe then performed separate association analyses at each SNP for each of the two groupings while applying the FBAT-PC approach. The FBAT-PC approach generates a composite phenotype by using a weighted sum of the individual phenotypes  the conditional mean model is used to calculate the power to detect an association using the composite phenotype; 2) SNP/genetic model combinations are selected based on the power to detect an association and then ranked; 3) for the top ten power rankings in the FBAT-PC statistics are calculated for the selected SNP/genetic model combinations and their associated phenotypes; 4) the results are adjusted for multiple comparisons using the False Discovery Rate  with cognitive impulsivity. Other tests for association  proved not to be significant.Genotype information on repeat polymorphisms (microsatellites) was available for the DRD4 , the DAT1/SLC6A3 (3' untranslated region VNTR) and the SERT/SLC6A4 (a VNTR in intron 2: STin2) and an in/del promoter polymorphism (5-HTTLPR) from an analysis based on 1116 ADHD families. There were nominally significant associations on an additive model for the 3-repeat DRD4 allele (p = 0.014) with impulsive aggressive behavior, and the SERT VNTR  and receptor-mediated mechanisms (HTR1E) may be relevant to both forms of impulsivity. Indeed the same SNP for HTR1E rs1406946) was nominally associated with both types of impulsiveness in the dominant transmission model. It was over-expressed in the cognitive and under-expressed in the behavioral or aggressive form of impulsivity , and the efficacy of 5-HT action via the 5-HT2 and 5-HT1 receptor families . GeneralCorrected significant associations for the 5-HT2A gene have also been reported recently for children and adults with ADHD  and corrThe presence of 3 SNPs for TPH2 with near gene-wide significant associations with cognitive impulsiveness along with the significant relationship for a SERT allele  would sLastly, we studied SNPs representing 3 other genes  on the grounds that their activity could influence neurotransmission including that based on 5-HT, and that some associations with ADHD have been reported . Brain dThe design of the study involved recruiting children and adolescents who showed to a greater or lesser extent the features of impulsivity we sought. Alternative approaches could contrast children with and without these features, and compare pre-, post-pubertal and young adult subjects to investigate putative developmental changes in gene expression. A comparison of groups with high comorbidity for externalizing or internalizing symptoms would be instructive both for the integrity of our concepts of impulsivity in affect and cognition, as well as for identifying the grounds for separating subgroups of ADHD with 'comorbid features'. We would also point out the severe limitations of interpreting symptoms based on lay responses to questionnaires. It is important that the preliminary work described here be extended to neuropsychological testing that would contrast not only provoked aggression with impulsive decision making, but incorporate the role of reinforcement (e.g. delayed gratification).There are also several limitations with regard to the sample and genotyping. Although this sample is reasonably sized for moderate genetic effects, the number of individuals could be larger to detect the modest genetic effects that one would expect to observe here. In addition, individuals enrolled in the IMAGE study are from several different places throughout Europe, including Spain and Israel. Although the FBAT statistic is robust to population stratification, the PBAT screening algorithm can be affected by this. Finally, current genome-wide scans are suggesting many genes that one could not have suspected of being of interest a short time ago. Therefore some candidate genes that may now seem important were not included in this analysis.ADHD is a highly heterogeneous disorder: it has been argued that there are potentially multiple variants of the condition with different etiological bases, pathophysiological signatures and neuropsychological and behavioral markers . ConsequIn particular, this study has taken an exploratory look at whether some SNPs from genes related to 5-HT function are associated with symptom ratings that reflect aggressive behavioral and cognitive impulsivity. Clearly different results could emerge from the study of other genes/SNPs as they become available. These SNPs cover genes for only four of the 22 types 5-HT receptor identified. There are indications that other receptors could be involved ; DAT1 (SLC6A3): dopamine transporter; FADS2: Fatty-acid desaturase gene-2; FBAT-PC: family-based association test with principle components; IMAGE: International Multicenter ADHD Genetics study; NURR1/NR4A2: orphan nuclear receptor/transcription factor; PACS: Parental account of children's symptoms; PNMT: phenylethanolamine-N-methyl transferase; SDQ: Strengths and difficulties questionnaire; 5-HT: Serotonin ; SERT: serotonin transporter; SNP: single nucleotide polymorphism; STin2: Serotonin transporter [VNTR] in intron 2; TPH: tryptophan hydroxylase; VNTR: variable number tandem repeat.The authors declare that they have no competing interests.The first two authors  conceived and executed the analysis, and drafted the manuscript. They were supported and advised by the working group  assisted by TB, WC, and EJSS-B. Recruitment and initial data processing was performed by the CIs under the supervision and organization of the PIs who set up the IMAGE investigation  under the direction of SVF.


Periprosthetic fractures associated with total knee arthroplasty are rare but present a challenging problem particularly when associated with revision arthroplasty. Fractures around tibial stems are particularly difficult with no accepted technique in their management. This case describes a tibial periprosthetic fracture following a revision knee arthroplasty which was successfully managed with a Pulsed ElectroMagnetic Field bone stimulation device. We believe this to be first reported use of a bone stimulation device in this clinical environment. A 75 year old Caucasian lady suffering from osteoarthritis of her right knee with 20 degrees of valgus deformity proceeded to a total knee replacement . Twelve months later the valgus deformity reoccurred with loss of function of the medial collateral ligament (MCL). The MCL was reconstructed using Leeds-Keio connective tissue prosthesis and biotenodesis screw  & 1B. HoA radiograph taken immediately post-operatively demonstrated a periprosthetic fracture at the level of the tibial stem on the medial side  & 1D. ThTwo months later the patient described increasing pain in the operated limb, with radiographs demonstrating extension of the fracture to the lateral cortex but with no displacement. Therefore, a non weight-bearing long-leg light cast was applied, unfortunately a further fall two weeks later fractured the fibula producing a valgus deformity  & 2B.The tibial prosthesis appeared well fixed proximally and therefore non-operative management was initiated with Manipulation under Anaesthesia (MUA) and long-leg plaster of paris (POP). The fracture remained mal-aligned therefore a further MUA and POP was performed and a Pulsed Electromagnetic Field Device (PEMFD)  was applied. The device was used for at least 3 hours per day until the fracture healed.At 4 weeks from application the fracture was assessed under anaesthesia and found to have some stability and converted from a long leg POP to a hinged knee brace. The patient was allowed to increase weight-bearing to 50% of body-weight converting to full weight-bearing 4 months from the injury. The fracture was monitored with serial radiographs, clinical examination in the outpatient department and theatre to assess fracture healing and ensure acceptable limb alignment.Eight months from sustaining the periprosthetic fracture and 7 months from the application of the PEMFD complete bony union was achieved clinically and radiologically  & 2D. AtTotal knee arthroplasty (TKA) surgery is performed in ever increasing numbers. One potential complication is of periprosthetic fractures during primary and revision surgery or a result of trauma . PeriproThe management of tibial periprosthetic fractures is a challenging problem, attributed to its rarity and patient population with poor bone stock and healing. Tibial periprosthetic fractures have been classified by Felix et al. based upon anatomical location (Types I-IV), fixation of implants (A: well-fixed or B: loose) or timing of the fracture (C: intra-operative) . PossiblType-II fractures occur adjacent to the tibial stem of which Type-A represent a well fixed implant as in this case (given it was noted postoperatively and the lack of cement extrusion in the immediate post-operative films). However, in-keeping with the literature the patient sustained this fracture following a mild traumatic event, which is commonly seen with modern condylar knee designs -3 and waDisplaced Type-IIA fractures present a particularly challenging management problem. Those that remain undisplaced can be treated non-operatively. However, if displaced open reduction internal fixation (ORIF) techniques are employed to achieve correct alignment and stability ,3,6. In Current bone stimulating devices fall into two broad technologies that of ultrasound and electric/electromagnetic . Low-intThe mechanism of action remains under investigation but evidence suggests PEMFD regulate proteoglycan and collagen synthesis and increase bone formation in models of endochondral ossification, encouraging mineralisation and angiogenesis, increasing DNA synthesis and altering the cellular calcium content in osteoblasts to produce results equivalent to bone grafts in clinical studies with union in approximately 80% of cases achieved between 14 and 21 weeks -13. The In this case complete bony union required 7 months from application which is 1 month longer than that expected by the manufacturers. However, given the complexity of the fracture this still represents a successful outcome. There is only one other case report in the literature describing the use of capacitively coupled electromagnetic field in the management of a Type-IIIA  periprosthetic fracture. However, our case represents a much more complex fracture pattern involving the tibial metaphysic and fibula in which there was a fracture gap greater than 10 mm and the PEMFD was applied immediately.This is the first report in the literature of the successful use of a PEMFD for Type-IIA tibial periprosthetic fractures. Although there are limitations of a single case, the successful application of the PEMFD in this environment could suggest broadening the scope of indications of such devices and increase the armamentarium available to deal with such injuries, in an increasingly frail population.


Mycobacterium tuberculosis (MTB), as well as its contribution to the dissemination of multi-drug resistant TB (MDR-TB) in rural areas of eastern China.The aim of this study was to investigate the molecular characteristics of isoniazid resistant 6110 restriction fragment length polymorphism (RFLP) and spoligotyping.A population-based epidemiological study was conducted in two rural counties of eastern China from 2004 to 2005. In total, 131 isoniazid resistant MTB isolates were molecularly characterized by DNA sequencing and genotyped by ISkatG315Thr mutation was observed in 74 of 131 isoniazid resistant isolates and more likely to be MDR-TB (48.6%) and have mutations in rpoB gene (47.3%). Spoligotyping identified 80.2% of isoniazid resistant MTB isolates as belonging to the Beijing family. Cluster analysis by genotyping based on IS6110 RFLP, showed that 48.1% isoniazid resistant isolates were grouped into 26 clusters and katG315Thr mutants had a significantly higher clustering proportion compared to those with katG wild type . Thirty-one of the 53 MDR-TB isolates were observed in 19 clusters. Of these clusters, isoniazid resistance in MDR-TB isolates was all due to the katG315Thr mutation; 18 clusters also contained mono-isoniazid resistant and other isoniazid resistant isolates.The katG315Thr is likely to be clustered in a community, develop extra resistance to rifampicin and become MDR-TB in Chinese rural settings.These results highlighted that isoniazid resistant MTB especially with  Mycobacterium tuberculosis (MTB). It is a cornerstone of the modern short-course chemotherapy for tuberculosis, and also widely used to treat the latent MTB infection (LTBI) to prevent the active disease and the subsequent TB transmission.Isoniazid (INH) is one of the most effective and specific agents for the treatment of the disease caused by However, recent increases both in INH-resistant and multidrug-resistant (MDR) tuberculosis have been jeopardizing the efforts of global TB control through the implementation of the Directly Observed Treatment, Short course (DOTS) ,2. In ChThe development of INH resistance is a common first step in the evolution to MDR . Thus, tkatG [inhA [katG gene is a major mechanism of INH resistance in MTB [katG gene, which is present in approximately 50-90% of all INH-resistant isolates and is associated with relatively high-level resistance to INH [inhA or its promoter region can cause 21-24% of INH resistance, with the promoter mutation (mainly in the inhA-15 position) being more common than the mutation in the structural gene [katG315Thr mutation had a strong association with the development of MDR, and the successful transmission of MDR MTB as well [Resistance to INH is mediated by mutations in different genes in MTB, with katG  and inhAtG [inhA  being the in MTB -9. The mral gene . A study as well .Based on the above knowledge and findings, we hypothesized that specific INH resistant MTB strains could perpetuate the epidemic of MDR-TB in high TB burden countries like China.katG and inhA genes/promoters as well as the IS6110 restriction fragment length polymorphism (RFLP) genotyping technique to analyze the level of clustering or recent transmission of INH-resistant MTB. Combined with epidemiological evidence, the molecular information obtained from this study could give us a better understanding of the possible mechanism behind the high prevalence of MDR-TB in rural China.Here we presented an in-depth study to determine the molecular basis of INH-resistant MTB and the transmission pattern of INH resistant MTB circulating in two rural counties of eastern China. We applied direct DNA sequencing on hotspots of The epidemiological methods and study population of this study were described previously . It is aAll MTB isolates were tested for drug susceptibility using the proportion method on egg-based LJ medium . The crirpoB, katG genes and the inhA promoter region, corresponding to the drug resistance to RIF and INH respectively. The Primers pairs were CCCATGGCCGCGGCGGTCGACATT and CGCCGTCCTTGGCGGTGTATTGCC for katG gene (GeneBank: X68081), and CCTCGCTGCCCAGAAAGGGA and ATCCCCCGGTTTCCTCCGGT for inhA promoter region (GeneBank: U41388), and GGGAGCGGATGACCACCCA and GCGGTACGGCGTTTCGATGAAC for rpoB gene (GeneBank: L27989). Mutations in these genes were determined by amplification of the corresponding hotspot DNA region by PCR followed by direct DNA sequencing with ABI 3770 DNA sequencer . The new alleles were confirmed by further PCR and re-sequencing from the original DNA. Sequencing data was independently analyzed by two biologists for quality control purposes.All isolates available were investigated for the presence of the hotspot mutations related to drug resistance by direct DNA sequencing. The hotspot genes included 6110 RFLP was done on the INH resistant isolates according to the standard protocol of van Embden et al, with data analyzed by the Gel Compar software  [6110 RFLP pattern. Unique strains denote unparallelness of the IS6110 RFLP profiles in the study collection. Typically, clustered strains indicate recent transmission while unique strains indicate reactive disease from a remote infection.ISBelgium) . A clusthttp://cgi2.cs.rpi.edu/~bennek/SPOTCLUST.html) and assigned with the corresponding spoligotype international type (SIT). The Beijing family MTB was defined as the strain that hybridized only to the last nine spacer oligonucleotides (spacers 35 to 43).Spoligotyping was carried out by using the commercial kit from Isogen Bioscience BV  . The INHkatG315Thr alleles and other INH resistant isolates using the Mantel-Haenszel chi-square test. Binary logistic regression model was used for univariate and multivariate analysis to qualify and quantify the difference in clustering proportion between groups of subjects with different socio-demographic and clinical characteristics. The adjusted Odds Ratio (OR) and 95% confidence interval (CI) were calculated by adjusting for the possible confounders .SPSS software  was utilized for the statistics analysis. Clinical and bacteriological characteristics were compared between The study was approved by the Institutional Review Board of Fudan School of Public Health. Written informed consent was obtained from all the participants.st line anti-TB drugs. Resistance to INH was the most common form of drug resistance (140/251) with a majority of cases mono-resistant to INH (55/140). Resistance to RIF was observed in 65 isolates, only 2 of which were mono-resistant. A total of 58 isolates were resistant to INH and RIF simultaneously. The details of the resistance have been reported previously [In total, drug resistance profile and baseline information were successfully obtained from 399 pulmonary TB patients, 182 in Deqing and 217 in Guanyun during the study period. Of the 399 MTB isolates from these patients, 251 (62.9%) were resistant to at least one of 1eviously .DNA samples were successfully extracted from 131 of the 140 INH resistant isolates. Of the 131 isolates, 80 were from patients who were previously diagnosed with TB and 51 were from the patients newly diagnosed. Regarding the drug resistance profile, apart from the 55 INH mono-resistant isolates, 53 were also resistant to RIF and referred to as MDR-TB; 23 were poly-resistant (INH + STR and/or EMB)  INH resistant isolates contained the katG Ser315Thr nucleotide substitution. katG315Arg and katG315Asn mutations were detected in 3 and 4 isolates respectively. In addition, 28 of 131 INH resistant isolates (21.4%) presented the mutation in the inhA promoter region, all of which possessed the inhA-15 C→T nucleotide substitution. Two INH resistant isolates shared the mutation in katG and inhA gene simultaneously: one with katG 315Thr and inhA-15T mutations and the other with katG315Arg and inhA-15T mutations. No nucleotide substitutions were identified in 220 INH sensitive isolates either at the katG315 position or the inhA-15 position. The frequency of the katG315Thr mutation was significantly higher in MDR-TB compared to the other drug resistant form of MTB isolates , while inhA-15T mutations did not differ significantly between these two major drug susceptibility groups .DNA sequencing demonstrated that 107 of 131 INH resistant isolates had mutations either in the er Table : 61.8% , 516Val (2/53), 526Arg (4/53), 526Tyr (11/53) and 531Leu (31/53). Double-spot mutations were presented in two MDR isolates, one with 516Tyr/531Leu mutations and the other with 516Val/531Leu mutations. No rpoB mutations were observed in isolates susceptible to RIF.The resistance to RIF was due to mutations in the MTB clades designations were available for 122 of 131 isolates, with the following distribution of the genotypic lineages , 28 unique patterns/isolates in Deqing, 15 cluster patterns (36 isolates) and 40 unique patterns/isolates in Guanyun. The genotype and phenotype patterns of drug resistant TB were further investigated among the clustered isolates. Ten clusters (25 isolates) from Deqing and 12 clusters (32 isolates) from Guanyun contained isolates with different phenotype and/or genotype of drug resistance. In these clusters, INH resistant isolates with the katG315Thr mutation had the highest occurrence (51/57 or 89.5%). Additionally, 31 of 53 MDR-TB isolates (58.5%) were observed in 19 clusters, with all carrying the katG315Thr mutation as well as the mutations either in position 516, 526 or 531 of the rpoB gene. In these clusters containing MDR-TB isolates, 18 clusters had INH-mono resistant and/or polydrug-resistant TB isolates simultaneously.In the clustering analysis of INH resistant isolates specific to counties Figure , IS6110 p, 0.032; 95%CI,1.075-4.863), as well as the previously diagnosed patients when compared to newly diagnosed patients . A significant higher proportion of MDR-TB isolates was clustered compared to INH mono resistant isolates . Compared with the wild type (wt) isolates, the isolates with the katG315Thr mutation were more likely to be clustered . The Beijing family isolates was mainly observed in clusters . The INH resistant inhA-15T mutant isolates were more likely to be "unique" compared to those inhA-15 wild type isolates .Binary logistic regression was applied to analyze the "clustering" of INH resistant isolates in association with patients' demographics, clinic profiles and the bacteriologic features Table . Smear-pkatG315Thr mutant, the host and bacteriological variable were compared between the INH resistant isolates with the katG315Thr mutation group and those with other mutation/wild type group , the relevant mutation in rpoB gene  compared to other mutation/wild type group. And meanwhile, this allele was more likely to be clustered  and belonged to the Beijing family .To further investigate the characteristics of This study attempted to gain further insight into the molecular basis of the INH resistant MTB circulating in the rural area of eastern China. Transmission of INH resistant MTB and its possible contribution to the epidemic of MDR-TB were also a concern. The majority of INH strains from TB dispensaries in the rural Chinese area, exhibited INH mono-resistance and the double-drug-resistance profile especially including extra resistance to RIF 53/131) and STR (37/131). These resistance profiles corroborated the pattern of acquisition of resistance to extra drug resistance especially for RIF and STR in INH resistant isolates [31 and STkatG315Thr mutation in MTB strains around the world varies, especially with regard to the prevalence of TB. High TB burden regions [katG315Thr mutation in INH resistant strain compared to low TB burden areas [katG 315Thr mutant allele in eastern rural China in around 50% of INH resistant isolates, which is consistent with the other report from China [katG315Thr mutation in INH resistant strains and the presence of resistance to other drugs, especially for RIF. These observations might suggest that the INH resistant strain with the katG315Thr mutation may be more likely to develop the resistance to other 1st line drugs especially for RIF [katG 315Thr mutant allele bore the mutation in rpoB gene, which was related to RIF resistance reported in China [The prevalence of the  regions  always oen areas -20. The om China . Further for RIF -24 than in China  and coulkatG mutations were in clusters compared to wild type INH resistant isolates, suggesting its strong transmissibility, full virulence and survival advantage under the drug pressure in the host. This could be explained by the maintenance of the 30%~40% catalase-peroxidase activity in the INH resistant strain with the katG 315 Thr mutation [katG 315 mutation in the index person was more likely to experience a series of subsequent mutations, leading to the accumulation of extra drug resistance and the development of MDR- and/or extreme drug resistant(XDR)-TB [The extensive transmission of the INH resistant MTB strain was observed in rural areas of eastern China, with 48.1% of the INH resistant MTB isolates in clusters. Most of INH resistant isolates with mutation  comparedmutation -28 of IN(XDR)-TB . The acckatG315Thr mutation, the transmissibility of INH resistant strain might be related to endemic characteristics of the Beijing family in rural China. The Beijing genotype is apparently the most prevalent genotype in China, representing of 80.2% INH resistant and 83% MDR-TB isolates in the current study. Our study also found that the MTB strain from the Beijing family was also the main source of recent transmission causing INH resistant TB. The Beijing genotype has been reported to be associated with treatment failure and relapse. Although the reasons for this are not known, it appears that the infections with the Beijing genotype strain may be more persistent, probably leading to longer duration of infectiousness and creating a better opportunity for transmission under the anti-TB drug pressure [Apart from the role of the pressure .Additionally in the current study, INH resistant MTB was more likely to be clustered in smear-positive and previously diagnosed patients. The smear-positive status might extend the transmission period of the pathogen between hosts and allow MTB to attack more people in the surroundings of the index case. Although the previous TB could be cured by direct observed chemotherapy, those formerly treated cases could still be at risk for the TB re-infection from recent transmissions since the risk factors for TB  are still present. In combination with these conditions, the risk of INH resistant MTB transmission between hosts increases.katG315Thr mutation, to acquire extra drug resistance and become MDR-TB. It deserves further investigation to determine which mechanism may play the critical role in the epidemic of MDR-TB, since the implication behind it could be meaningful to evaluate the performance of local TB control as well as to determine the MDR-TB control strategies suitable for rural areas of China and as well as other similar high burden settings.The epidemic of MDR-TB can occur as a result of the spontaneous mutations in MTB and selection under suboptimal drug therapy or extensive transmission of the drug resistant MTB, or both combined. In the current study, a significant high proportion of MDR-TB isolates were observed in clusters compared to the other form of INH mono-resistant isolates. Most clusters with MDR-TB isolates also contained isolates with other drug resistance profiles including INH mono-resistance and poly drug-resistant tuberculosis simultaneously. Based on these observations, the epidemic of MDR-TB in rural China could be explained by two possibilities: 1) the epidemic of MDR-TB might result from the recent transmission of MDR-TB strains in an area ridden by INH resistant MTB. 2) Recent transmission of the drug resistant MTB and the selection of MTB under drug pressure might exert the symbiotic interaction in an epidemic of MDR-TB. The combination of INH resistance and maintained virulence might make it possible for some INH resistant strains, especially those with the katG315Thr mutation and MDR-TB was confirmed. Therefore, it is important to recognize the katG315Thr mutants among INH-resistant strains, which could be seen as a risk factor for subsequent development of MDR-TB. Early detection of the patients with INH resistant strains would facilitate the modification of treatment regimens and appropriate infection control measures can be taken in time to reduce the risk of further development and transmission of MDR-TB.INH resistant MTB was transmitted widely in eastern rural areas of China. Also the correlation of prevalence and transmission between INH resistant isolates especially with the Mycobacterium tuberculosis; MDR: multidrug-resistant; INH: isoniazid; RIF: rifampicin; STR: streptomycin; EMB: ethambutol; RFLP: restriction fragment length polymorphism; SIT: spoligotype international type.MTB: The authors declare that they have no competing interests.YH carried out the data collection and molecular genotyping studies, participated in the PCR and sequence alignment and drafted the manuscript. SH has revised it critically for important intellectual content. WJ was involved in all the microbiological research. WW participated in the conception and design, acquisition of data, its analysis and interpretation. BX conceived the study, developed the design, coordinated the implementation, and helped to revise the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/43/prepub


To form myelin oligodendrocytes expand and wrap their plasma membrane multiple times around an axon. How is this expansion controlled?Here we show that cell surface area depends on actomyosin contractility and is regulated by physical properties of the supporting matrix. Moreover, we find that chondroitin sulfate proteoglycans (CSPG), molecules associated with non-permissive growth properties within the central nervous system (CNS), block cell surface spreading. Most importantly, the inhibitory effects of CSPG on plasma membrane extension were completely prevented by treatment with inhibitors of actomyosin contractility and by RNAi mediated knockdown of myosin II. In addition, we found that reductions of plasma membrane area were accompanied by changes in the rate of fluid-phase endocytosis.In summary, our results establish a novel connection between endocytosis, cell surface extension and actomyosin contractility. These findings open up new possibilities of how to promote the morphological differentiation of oligodendrocytes in a non-permissive growth environment.See related minireview by Bauer and ffrench-Constant:  During their life cycle many cells undergo dramatic changes in their cell morphology including rapid alterations in shape and surface area. One way by which cells change their surface area is to alter membrane traffic between surface and interior. For example, loss of integrin-mediated adhesion during tumor cell growth triggers the rapid caveolin-mediated endocytosis that leads to cell rounding -3. Anoth3 μm2 of myelin surface membrane per day [A striking example of cell-shape change is the morphological differentiation of oligodendrocytes during the development of the central nervous system. First, small and bipolar oligodendrocyte precursor cells (OPCs) migrate into the brain, where they differentiate into oligodendrocytes and expand their plasma membrane many folds in order to wrap it several times around an axon ,6. In th per day .Since the generation of myelin occurs at the appropriate time during neuronal development, it is assumed that neuron-glia communication coordinates this process. However, in cell culture oligodendrocytes differentiate and extend large myelin membrane sheets in the absence of neurons  indicatiIn this study, we tested how the extracellular environment controls cell shape and membrane traffic in oligodendroglial cells.The following primary antibodies were used: actin , O1 , MBP , nonmuscle myosin II heavy chain A and B  and betaIII-Tubulin . Secondary antibodies were purchased from Dianova .To prepare acrylamid gels on glass coverslips and crosslink adhesion proteins to the gels, we adapted the protocol by . BrieflyTo prevent polyacrylamid gels from drying, they were covered with 150 μl 1 mg/ml sulfo-SANPAH  immediately after polymerisation. The gels were then places under an ultraviolet lamp at approximately 10 cm distance and irradiated for 10 min. The polyacrylamid gels were immersed in 1 ml sterile 200 mM HEPES pH 8.6 in multi well plates . The HEPES solution was aspirated and 1 ml 70% Ethanol was added for 1 min to sterilize the polyacrylamid gels. To remove residual ethanol and sulfo-SANPAH the gels were washed overnight in 1 ml sterile 200 mM HEPES pH 8.6. Coating with poly-L-lysine was done for 1 h at RT. The gels were rinsed once with de-ionized water and dried for 10 min before plating with cells.The oligodendroglial cell line, Oli-neu, was cultured on poly-L-lysine as described previously . Alterna2).The effects of RGD-binding integrins were assessed by treating Oli-neu cells for 8 h with 100 nM RGD-peptide  or with the inactive control RAD-peptide . Poly-L-lysine pre-coated glass coverslips were incubated overnight at RT in 10 ng/ml to 500 ng/ml Chondroitin sulfate proteoglycans (CSPGs) , dried and rinsed with PBS before plating with cells. Cells were plated in a density of 50 000 per 12 well (3.8 cm6) were harvested, resuspended in 100 μl resuspension buffer and mixed with 160 pmol siRNA per transfection. The transfection was repeated after 72 h. For electroporation of primary oligodendrocytes, 1.5 × 106 cells were collected, nucleofected with both myosin II-A and II-B or control siRNA and subsequently cultured for 3 d.Smart pool siRNA targeting myosin II-A, myosin II-B  and of control non-targeting siRNA pool were purchased from Dharmacon . Electroporation was performed using the basic neuron transfection Kit  and Amaxa programm O-05  according to the manufacturer's protocol. Briefly, per transfection Oli-neu cells . For the quantification of fluorescence intensities of internalized dextran a fixed threshold was applied for all images. Immunofluorescence was performed as described previously [Endocytosis was assessed by incubating Oli-neu cells for 15 or 30 min at 37°C with rhodamin-labelled Dextran 10K (Molecular Probes) in culture medium. Cells were subsequently washed and fixed, followed by microscopic analysis. Image processing and analysis was performed using Meta Imaging Series 6.1 software  DMRXA microscope and a confocal laser scanning microscope  with 40× and 63× oil plan-apochromat objective (Leica), respecively. Image processing and analysis was performed using Meta Imaging Series 6.1 software . Quantification of fluorescence intensities was performed as described previously . The celAn atomic force microscope , mounted on a Zeiss Axiovert 200 M (Carl Zeiss) was used to perform elasticity measurement. 5 micron diameter silica beads (Kisker) were attached to tip-less cantilevers  using epoxy glue . Cantilever spring constants were determined using the equipartition theorem. Polyacrylamide gels were probed with a maximum indentation force of 1 nN at no less than 5 locations. Each indentation curve was fitted  according to the Hertz model  to obtaiWe started assessing the influence of integrins on cell surface area by treating the oligodendroglial cell line, Oli-neu, with a monovalent integrin ligand (RGD-peptide) or a non-binding control (RAD-peptide). Morphological analysis of Oli-neu cells grown on fibronectin or poly-L-lysine coated coverslips revealed a significant reduction in cell surface area after treatment with RGD-peptide compared to the inactive RAD-peptide Figure . PreviouSignals from integrins to the Rho family GTPases regulates morphological differentiation of oligodendrocytes . We specThere are number of recent studies demonstrating that cells sense and response to the stiffness of their substrates .For example, matrix elasticity directs mesenchymal stem cell lineage specification . In addiSince integrins in conjunction with actomyosin contractility are implicated in the sensing of these physical matrix properties , we examNext, we tested whether the inhibition of actomyosin contractility influences the effects of matrix rigidity on cell surface area. We observed that the inhibition of either Rho/ROCK signaling by C3 transferase and Y27632 or myosin II by blebbistatin restored cell surface expansion on soft matrices Figure . When OlBy measuring the elastic modules of acrylamide gels with atomic force microscopy, we ruled out that the RGD-peptide affected matrix rigidity (the elastic module of 0.75% gels was 7761 +/- 2200 in the presence of the RGD-peptide).Next, we investigated the effects of matrix rigidity on membrane sheet expansion in primary oligodendrocytes. Again, a considerable decrease in sheet size accompanied decreasing matrix rigidity Figure .One group of molecules that contribute to the non-permissive growth properties in the CNS environment are chondroitin sulfate proteoglycans (CSPG) . StudiesWe, therefore, determined the effects of CSPG on the surface area of Oli-neu cells by plating the cells on different concentrations of CSPG. We observed that the cell surface area decreased with increasing concentration of CSPG. The highest concentration of 500 ng CSPG resulted round cells with diameters half that of control cells Figure . QuantitBlebbistatin is a small molecule inhibitor with high affinity and selectivity towards myosin II . To confTo analyze whether the changes in cell surface area again correlate with changes in fluid-phase endocytosis, we analyzed the rate of dextran uptake in Oli-neu cells cultured on CSPG. We found that dextran was taken up more efficiently with increasing concentrations of CSPG Figure . When ceNext, we used primary cultures of oligodendrocytes for our experiments. These cells differentiate within a few days from cells with multiple branches to myelin-membrane sheet producing cells. In cell culture the differentiation of oligodendrocytes follows a default pathway and occurs in the absence of extrinsic signals from neuronal cells.When primary cells were treated with blebbistatin or Y27632 cell surface area did not change significantly. We analyzed whether growth on a non-permissive substrate suppresses myelin-membrane extension and if so, whether inhibition of actomyosin contractility could overcome the block.2) there was a significant inhibition of membrane sheet extension , molecules associated with non-permissive growth properties within the central nervous system (CNS), block cell surface spreading. In addition, we observed that the reductions of plasma membrane area were accompanied by changes in the rate of fluid-phase endocytosis. We found that this form of endocytosis is dependent on actomyosin contractility and possibly also on integrins. A possible explanation is that actomyosin contractility pulls membrane into the cell and thereby accelerates vesicle budding. Loss of integrin-mediated anchoring to the extracellular matrix may increase these pulling forces of the actin cytoskeleton. We show that this mechanism is important for cell surface spreading of Oli-neu cells, but it is likely to be involved in the control of cell surface area in other systems. It may be particularly relevant in processes where rapid changes of cell surface area occur, for example in a blood vessel or a bladder .Previous work shows that extracellular matrix proteins, like laminin, promote the formation of myelin membrane sheets in oligodendrocytes by its interaction with integrin α6β1 ,29. OligIn addition to signals from the extracellular matrix, paracrine signals from oligodendrocytes may also regulate myelin-membrane extension. Interestingly, the effects of the CSPG peptide on oligodendrocyte cell surface area was not observed when cells were grown at a high density, indicating oligodendrocyte present factors to neighboring cells that are able to overcome the inhibitory cues.Integrin-mediated cell attachment is known to regulate endocytosis and cell surface shape. A striking example of the importance of this mechanism is the loss of integrin-mediated adhesion during tumor growth, which triggers rapid endocytosis and cell rounding . The groThe finding that actomyosin contractility acts downstream of integrins is consistent with a report describing a signal transduction pathway from integrins to Fyn that facilitates RhoA GTPase inactivation and the morphological differentiation of oligodendrocytes . Our resIn the adult brain OPCs are kept in an undifferentiated state. This may in part be due to the presence of myelin from mature oligodendrocytes that blocks their differentiation . WhetherWe do not know by what means CSPGs affects cell surface size, but the finding that knocking down myosin II overcomes CSPGs inhibitory effects points to a role of actomyosin contractility in this process. Earlier work examining the role of CSPGs on axon outgrowth suggested that CSPGs interfere with extracellular matrix/integrin interactions .Indeed, increased integrin expression is sufficient to eliminate the inhibitory effects of CSPGs on neuronal growth cone function . A down-Our results demonstrating that inhibition of Rho/ROCK/myosin II activity is able to overcome a non-permissive growth property of the extracellular environment on oligodendrocytes extends the role of this system in cellular regeneration.These findings not only show that the actomyosin system plays a role in cell surface spreading of oligodendrocytes, but also point to potential target for therapeutic interventions. However, we do not yet know whether the inhibition of actomyosin contractility affects the ability of oligodendrocytes to wrap their plasma membrane around an axon to form compact myelin. Clearly, in vivo studies are now required to determine whether inhibition of actomyosin contractility promotes myelin repair in the CNS.In summary, we find that cell surface area depends on actomyosin contractility and is regulated by physical properties of the supporting matrix. CSPG blocks cell surface spreading, but the inhibitory effect of CSPG on plasma membrane extension is prevented by treatment with inhibitors of actomyosin contractility and by RNAi mediated knockdown of myosin II. These results establish a novel connection between cell surface extension and actomyosin contractility in oligodendrocytes and open up new possibilities of how to promote the morphological differentiation of oligodendrocytes in a non-permissive growth environment.AK performed the experiments, analyzed the data, drafted the manuscript; JH performed the atomic force microscopy measurements; DF performed experiments, analyzed the data and approved the final manuscript; MS designed and coordinated the work and wrote the manuscript. All authors read and approved the final manuscript.


Although traditional Chinese medicine has benefitted one fifth of the world's population in treating a plethora of diseases, its acceptance as a real therapeutic option by the West is only now emerging. In light of a new wave of recognition being given to traditional Chinese medicine by health professionals and regulatory bodies in the West, an understanding of their molecular basis and highlighting potential future applications of a proven group of traditional Chinese medicine in the treatment of a variety of cancers is crucial – this is where their calling holds much hope and promise in both animal and human trials. Furthermore, the rationale for combining conventional agents and modern biotechnological approaches to the delivery of traditional Chinese medicine is an avenue set to revolutionize the future practice of cancer medicine – and this may well bring on a new dawn of therapeutic strategies where East truly meets West. Reports of therapeutic success with traditional Chinese medicine (TCM) have until very recently been met with much scepticism and pessimism by the West, due in-part to the sheer lack of available credible and rigorous clinical data and at claims that a given TCM can remedy common ailments and be just as efficacious in eliminating life threatening diseases, such as cancer. The tide is now beginning to turn on this negative outlook, aided by the ever-increasing migration of people and along with them knowledge  from two of the world's fasting growing populations, China and India, to the West . This trThe age-old holistic approach employed by Chinese practitioners proposes that a multitude of events are key to returning a patient to a healthy state; where cancer therapy is concerned these primarily include an interplay between the induction of apoptosis/cell-cycle arrest, inhibition of angiogenesis, overcoming multidrug resistance (MDR), and boosting the immune system Figure . Followiprogrammed cell death' can be directly linked to prolonged tumour cell survival and resistance to therapy [via death receptors) or intrinsic  pathways and although their paths are not mutually exclusive, evidence suggests the latter certainly predominates where TCM is concerned [apoptosis' and 'angiogenesis' in the context of TCM, proposed to be key avenues responsible for imparting therapeutic efficacy against a wide range of cancers.The many physiological growth control mechanisms that regulate cell proliferation and tissue homeostasis are linked to apoptosis and it follows that resistance developed to this ' therapy ,3. The poncerned . Here weApoptosis is guided by a range of complex multi-step, multi-pathway programs that eventuate in the breakdown of cellular DNA leading to cell death . And by caspases' are key players with their role and function extensively reviewed elsewhere [Of the pro-apoptotic enzymes implicated in TCM activity the family of cysteine proteases, commonly termed 'lsewhere ,3,10. Brlsewhere . Pro-caslsewhere .Cellular stress is invoked by an imbalance in a cell's redox state. Nuclear factor-κB (NF-κB) is a key regulatory molecule, activated when a cell experiences oxidative stress . It is wNumerous studies have been aimed at deciphering the precise molecular basis for a variety of TCM and those most noteworthy are discussed below.Scutellaria Baicalensis , namely of the prostate, breast, lung, liver and ovaries [baicalin, although it is converted to baicalein by intestinal gut flora with this latter bioactive firmly considered to be the primary active constituent responsible for its pro-apoptotic and anti-proliferative effects [Scutellaria Baicalensi is via the inhibition of eicosanoid synthesis – being important mediators of pro-inflammatory (cyclooxygenase-2 (Cox-2)) and tumour cell proliferatory (lipoxygenase) markers [baicalein against cell proliferation in PC-3 and DU145 prostate cancer cell lines is that of cell cycle arrest (at G0-G1) while also inducing apoptosis – confirmed via detection of caspase-3, at concentrations typical for administration to humans [It is also one of the most widely studied by researchers, being used either alone or more often in combination with other TCM for a range of cancers  – comprising 12 herbs in various proportions [et al on promyelocytic leukeamic cells (HL-60) cells pre-treated with TRSDY revealed that apoptotic cell death was indeed caspase-3 induced [via selective members of the caspase family, namely caspase-3 [Further evidence of TCM acting portions . Caspase induced . Its act induced ,30. Intr induced ,32. Whetaspase-3 -36.Tripterygium wilfordii has been studied extensively for anti-inflammatory and immunosuppressive activity, being shown to sensitize various types of tumour cells to apoptosis induced by TRAIL, TNF-α and chemotherapy [et al evaluating PG490 suggests that it sensitizes previously resistant Calu-1 lung cancer cell lines to TRAIL-induced apoptosis while sparing normal human bronchial epithelial cells [via death receptors (TRAIL-R1 & R2) forms a death-inducing signalling complex (DISC); this then recruits an 'initiator', caspase-8, which begins a cascade of protease activation enlisting the 'executioner' – caspase-3, promoting cleavage of death substrates with the end-point being cell death. [et al confirmed the role of PG490 in apoptosis and went further to highlight that mitochondria, and not the death receptor, predominate in PG490 activity in mouse embryonic fibroblasts, due to caspase-9 activation [et al where caspase-8 was found to be the key pro-enzyme in triggering apoptosis highlighting that mechanistic variability indeed exists with TCM as would be expected, thus caution must be taken when making any broad claims relating to their precise mechanism of action [TRAIL has received considerable attention by researchers since the discovery that most cancer cells are sensitive to its apoptotic effect but that normal cells confer resistance to it . The pototherapy -44. A stal cells . PG490-ml death. -47. Carttivation . This cof action . Synergif action .All cancer cells possess an elevated apoptotic threshold and although the therapeutic interventions of chemotherapy and γ-irradiation are crucial they are commonly plagued with resistance, resulting in a cycle of remission and relapse. The ever-rising incidence of resistance to chemotherapy suggests an increase in this apoptotic threshold and so the challenge is whether it can be reduced sufficiently to break the cycle at the point of remission. Advances made in recognizing and activating the key molecules involved in apoptotic pathways are certainly very encouraging from the perspective of eradicating a tumour although resistance to these interventions are also emerging . The groArtemisia absinthium – Figure Curcuma longa – Figure Scutellaria Baicalensis are commonly employed by traditional practitioners and studies demonstrate that their actions are at least in-part achieved by blocking the critical process of tumour vascularisation [ras, HER-2, p53 and C-jun) and growth factors  have been identified as up-regulating VEGF-mRNA and so TCM that inhibits their expression and production, respectively, are also considered invaluable tools in cancer therapy.Angiogenesis – the creation of a healthy vascularised network by a tumour is a key underlying process in the induction and establishment of cancer . It, likrisation . In orderisation ,53. Starrisation . VEGF prrisation . A wide Artemisia absinthium – Figure Artemisinin, an active constituent of Chinese wormwood (in vivo. Artesunate (ART) and dihydro-ART are semi-synthetic derivatives of artemisinin with demonstrable activity against a wide range of cancer cell lines including KML-562 (chronic myeloid leukaemia), HeLa  and HO-8910 (ovarian cancer) [in vitro [Phytochemicals have long been used as 'lead compounds' to generate drugs with better pharmacokinetic profiles and reduced toxicity  cancer) -62. Humain vitro . Along wCurcuma longa – Figure via downregulation of matrix metalloproteinases (MMP), most notably MMP-2 & MMP-9 – responsible for the invasive growth property of tumours [Curcumin  and its derivatives suppress microtubule depolymerization, thus terminating cell mitosis [Cephalotaxus hainanensis) and camptothecin (Camptotheca acuminata) with similar mechanisms of action are already commonly used in the clinic as anticancer agents for a variety of cancers [Camptotheca acuminata) being to inhibit DNA topoisomerase I, consequently affecting DNA replication; paclitaxel is a mitotic spindle inhibitor (spindle poison), which can also bind with tubulin and prevent the normal physiological process of microtubule depolymerization.TCM often possess quite distinct and specialised modes of action, and consequently tumours normally resistant to conventional chemotherapy are reported to be more susceptible to TCM therapy . They ha mitosis . As a re cancers . The mecRhizoma Zedoariae) and oridonin (Rabdosia rubescens) lend themselves to being co-administered with conventional chemotherapeutic agents (e.g. doxorubicin and 5-fluorouracil (5-FU)) to impart a synergistic anti-tumour effect. Combining elemene (Rhizoma Zedoariae) with the pyrimidine base analogue 5-FU, resulted in significantly higher tumour growth inhibitive effects [Pteris semipinnata L. – Figure Anticancer properties of the TCM elemene ( effects . The antBerberis amurensis), namely EBB (O-(4-ethoxyl-butyl)-berbamine) when combined with cyclophosphamide and mitomycin-C respectively enhanced the antitumor capacity, while also significantly improving patients' quality of life [et al., reported that ginsenoside-Rg3 (Panax ginsenoside Rg3) promotes Rhodamine-123 accumulation in vincristine-resistant KBV20C human fibroblast cancer cells, reversing vincristine resistance acquired by a variety of cells [Similarly, derivatives of the herbal compound berbamine ( of life . Zhang a of life ,87. Kim of cells .The discovery that single components within TCM have the potential to overcome multidrug resistance developed by tumour cells opens the door to new avenues of multi-drug/TCM therapy. These findings justify and moreover pave the way for them to be used alongside conventional drugs, where significant resistance to therapy has already developed.Although only one clinical trial using TCM was reported up until 2001 it was poorly controlled and any conclusions drawn were deemed unreliable, it is noteworthy however that given the very recent attention being received by TCM a re-analysis study has since been conducted ,90. Thisin vitro/in vivo testing now common place within research facilities where drug discovery and delivery is a focus, the drive to engineer well-defined, targeted drug delivery systems offers a new dawn for this very traditional practice of medicine [In treating diseases/illnesses of a systemic nature the typical route of TCM administration is oral. This is deemed a far from optimal approach in cancer patients given patient-to-patient variability when formulating TCM, the unpredictable absorption profile of the various bioactives across the gastrointestinal tract together with compliance issues. Furthermore, the challenge of ensuring batch-to-batch reproducibility of any given formulation remains, although the adoption of cutting-edge genetic/chemical fingerprinting in conjunction with micro-array-based cell line testing offers unique solutions to this. With modern techniques of isolation, characterisation and functionalisation of compounds along with medicine -93. By iThe authors declare that they have no competing interests.HSP drafted and wrote the manuscript. MQW revised the manuscript critically for important and intellectual content. GL provided translation of articles written in Chinese and revised the manuscript for intellectual content. All authors read and approved the final manuscript.


Eluted natural peptides from major histocompatibility molecules show patterns of conserved residues. Crystallographic structures show that the bound peptide in class II major histocompatibility complex adopts a near uniform polyproline II-like conformation. This way allele-specific favoured residues are able to anchor into pockets in the binding groove leaving other peptide side chains exposed for recognition by T cells. The anchor residues form a motif. This sequence pattern can be used to screen large sequences for potential epitopes. Quantitative matrices extend the motif idea to include the contribution of non-anchor peptide residues. This report examines two new matrices that extend the binding register to incorporate the polymorphic p10 pocket of human leukocyte antigen DR1. Their performance is quantified against experimental binding measurements and against the canonical nine-residue register matrix.One new matrix shows significant improvement over the base matrix; the other does not. The new matrices differ in the sequence of the peptide library.One of the extended quantitative matrices showed significant improvement in prediction over the original nine residue matrix and over the other extended matrix. Proline in the sequence of the peptide library of the better performing matrix presumably stabilizes the peptide conformation through neighbour interactions. Such interactions may influence epitope prediction in this test of quantitative matrices. This calls into question the assumption of the independent contribution of individual binding pockets. HLA-A, -B, -C and class II HLA-D loci  peptide. IC50 values are deduced for each peptide from fluorescence values with respect to a range of concentration of the competitor peptide  peptide and unlabelled competitor peptide; the amount of the competitor peptide used is varied for each assay. The assay mixture is incubated in 50 mM NaCl, 100 mM Na8 peptideThe performance of the extended matrices was evaluated in prediction against experimental binding measurements of peptides derived from 65 kDa glutamic acid decarboxylase (GAD65) , 69 kDa 50 values through a logarithmic transform. This is correlated with predicted peptide scores through a fitted function by regression. Linear and nonlinear functions are used and evaluated based on the residual. The predicted scores are further evaluated through the r2 (R-square) function. Regression analysis was carried out using MATLAB . Predictions were carried out within the in-house MHC epitope prediction tool PREDICT .For each data set, a dependent variable was derived from experimental ICCSP wrote the prediction platform, tested and installed it. He programmed further for the extended matrices project; carried out the analysis; wrote and edited the manuscript.Supplementary tableClick here for file


Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms – a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte–macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12. © 1999 Cancer Research CampaignIntact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood.