Primary drug resistance in extra-pulmonary tuberculosis: a hospital-based prospective study from Pakistan.

SETTING: Tertiary level specialised tuberculosis (TB) hospital.OBJECTIVE: To determine the prevalence of antimicrobial resistance in new extra-pulmonary tuberculosis (EPTB) cases.DESIGN: Prospective cross-sectional study. Presumptive EPTB patients with enlarged lymph nodes or pleural effusion having no history of TB treatment were enrolled. Specimens were tested for smear, Xpert® MTB/RIF and culture. Indirect drug susceptibility testing (DST) was performed using MGIT 960 and line-probe assays (LPA).RESULTS: Among 671 cases, 255 were bacteriologically confirmed and 185 DSTs were performed. Multidrug resistance (MDR-TB) was reported in 2.2% (95%CI 0.6-5.4), any resistance to rifampicin (RMP) in 2.7% (95%CI 0.9-6.2), isoniazid (INH) in 7.6% (95%CI 4.1-12.4), ethambutol in 1.1% (95%CI 0.1-3.9), pyrazinamide in 2.2% (95%CI 0.9-5.5) and fluoroquinolones (FQ) in 6.0% (95%CI 3.0-10.4). The sensitivity and specificity of LPA-DST was 100% and >98.8% respectively for RMP, INH and FQ. Among 82 cases with RMP of the results available for all three methods used, five were reported to be resistant on Xpert but all five were susceptible on MGIT 960 and four on MTBDRplus.CONCLUSION: Prevalence of RMP resistance in new EPTB cases is 2.7% (95%CI 0.9-6.2). Caution is warranted for RMP resistance detected using Xpert in EPTB samples with a very low bacterial load.

Peritumoral dermis of squamous cell carcinomas in renal transplant recipients contains less CD11c+ myeloid dendritic cells and FoxP3+ T cells compared to immunocompetent controls.

BACKGROUND: Renal transplant recipients (RTR) have an increased risk of developing cutaneous squamous cell carcinomas (SCC). These SCC are often more aggressive than SCC in immunocompetent individuals. OBJECTIVES: In this comparative study, we analysed the cell composition in the tissue immediately surrounding invasive SCC in immunosuppressed RTR and immunocompetent controls in an effort to further elucidate the role of the local immune system. METHODS: Morphology and quantity of various dendritic cell (DC) subsets, macrophages and FoxP3+ T cells were analysed by immunohistochemical staining. RESULTS: The number of CD11c+ myeloid DC and FoxP3+ T cells was significantly reduced in RTR, whereas the number of plasmacytoid DC, Langerhans cells and macrophages was similar in RTR and controls. CONCLUSIONS: A reduction in CD11c+ mDC in peritumoral dermis in RTR might contribute to impaired immunosurveillance thus giving rise to an increased risk to develop aggressive SCC in these patients.

Long-term persistence of BCG Pasteur in lungs of C57BL/6 mice following intranasal infection.

Different Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine substrains may vary in their efficacy. Here, we describe differences in disease progression and pathology in the lungs of female C57BL/6 mice infected intranasally with BCG Russia or BCG Pasteur and followed for 17 months. The lungs were investigated for bacillary load, histopathology and expression of cytosolic and secreted proteins by immunohistochemistry. BCG Russia was cleared from the lungs by 8 months. BCG Pasteur reached a low-level persistence at 8 months and remained at this level until the end of the experiment. BCG Pasteur induced greater pathology than BCG Russia, and there were more macrophage and lymphocyte infiltrates in animals infected with BCG Pasteur (P < 0.05). Bacterial growth correlated with cellular infiltration. All selected mycobacterial proteins were found to be expressed in the lesions by both BCG strains, and there were only minor variations between the strains. Furthermore, we identified isolated cells containing a high mycobacterial protein load in the normal-looking lung parenchyma. The infected cells in the healthy areas of the lung may represent the ability of mycobacteria to evade immune activation and thereby persist in the host. Clearance of BCG Russia indicates that a more effective and sterilizing immune response is established by this strain. On the other hand, the ability of BCG Pasteur to persist could be important for long-term protection against challenge with Mycobacterium tuberculosis.

Identification and Genotyping of the Etiological Agent of Tuberculous Lymphadenitis in Ethiopia

Background: In Ethiopia; little has been done to assess how Mycobacterium bovis has contributed to human tuberculosis; though the population routinely consumes unpasteurized milk and raw meat. The aim of this study was to determine the proportion of M. tuberculosis and M. bovis as etiological agents of tuberculous lymphadenitis (TBLN). Methods: Patients with lymphadenopathy (n = 171) were included in a cross-sectional study at Butajira Hospital; Southern Ethiopia. Lymph node biopsies were cultured. Patients' HIV status was identified. DNA from positive cultures was tested by PCR to identify M. bovis and M. tuberculosis. Isolates were genotyped by multiplex ligation-dependent probe amplification (MLPA) assay. Results: Among 171 patients; 156 had culture results. Of these; 107 (69) were positive for M. tuberculosis complex (MTC). Six of the 10 HIV-positive patients were culture positive. M. tuberculosis specific sequences were identified in the DNA of each of 100 samples as assessed by RD10 targeted PCR; and each of the 95 isolates exhibited the M. tuberculosis specific TbD1 deletion by MLPA analysis. No M. bovis was identified. These results indicate that all the isolates were modern M. tuberculosis strains. Furthermore; MLPA studies confirmed that 42of the isolates showed the Haarlem genotype and 12displayed sequences compatible with INH resistance. No mutations conferring resistance to ethambutol or rifampicin were detected. Conclusions: Our data showed that M. tuberculosis strains had common characteristics with strains causing pulmonary TB; which appears to be the main etiological agent of TBLN

Diagnosis of tuberculous lymphadenitis in Ethiopia: correlation with culture, histology and HIV status.

SETTING: Butajira, Southern Ethiopia. OBJECTIVE: To compare the diagnostic capacity of the clinical criteria for tuberculous lymphadenitis (TBLN) with histological and/or culture results and to assess the association of human immunodeficiency virus (HIV) with tuberculosis (TB) lymphadenitis. DESIGN: Patients (n=171) were included in the study from October 2005 until July 2006 at Butajira Hospital. Laboratory tests were performed to confirm TBLN. HIV status was identified in TBLN patients and retrospectively in 1608 healthy individuals. RESULT: A total of 136/161 (84.5%) patients were diagnosed with TBLN by histology. TBLN was culture-confirmed in 107/156 (68.6%) patients. The sensitivity, specificity, positive and negative predictive values of histology were respectively 92.5%, 49%, 79.8% and 75% when compared to culture as gold standard. Patients positive for TBLN by cytology and Ziehl-Neelsen (ZN) were also positive by histology and culture. Among the 143 confirmed TBLN patients, nine (6.3%) were HIV-positive. Of the 1608 healthy individuals, 77 (4.8%) were HIV-positive. Younger age (P=0.0001), female sex (P=0.016), not being married (P=0.0001) and illiteracy (P=0.016) showed a strong association with HIV in healthy individuals. CONCLUSION: Clinical criteria alone over-diagnosed TBLN by 15.4% compared to histological and/or bacteriological results. The HIV prevalence in TBLN patients and healthy individuals was the same.

How does clinical diagnosis of mycobacterial adenitis correlate with histological findings?

OBJECTIVE: To describe and compare histopathological findings with clinical criteria in diagnosis of TB adenitis. METHODS: Lymph node biopsies were obtained from 213 patients. Specimens were processed for culture and histopathologic examination, using standard methods. One hundred blocks with good preservation of tissue morphology were selected for detailed histological examination. RESULTS: About 75% of 213 patients had granulomas. In the remaining 25%, neither histopathological nor microbiological evidence of mycobacterial disease was found. Of 100 blocks selected for detailed examination, 79 blocks had granulomatous changes. The granulomas were well organised in 24%, mixed in 33%, and poorly organised in 43%. Langhans giant cells and acid-fast bacilli were observed in 88.6% and 21.5% of the 79 blocks, respectively. Cultured specimens were positive in about 10% of 79 biopsy specimens. CONCLUSIONS: Histological evidence of mycobacterial disease was only found in three quarters of patients that were clinically diagnosed and started on empirical treatment for tuberculous adenitis. Neither histological nor mycobacteriological evidence was found in a quarter of the patients who were already on treatment for TB, basing on clinical criteria. These findings call for new research on simple diagnostic tools for patients who seek care for s ymptoms of extra-pulmonary TB.

Reduced apoptosis and increased inflammatory cytokines in granulomas caused by tuberculous compared to non-tuberculous mycobacteria: role of MPT64 antigen in apoptosis and immune response.

Inhibition of apoptosis of infected macrophages by pathogenic mycobacteria is suggested to be an important virulence mechanism, but little is known about the mycobacterial proteins involved in the inhibition of apoptosis. In this study we investigated differences in apoptosis and immune response and their correlation with the expression of Mycobacterium tuberculosis complex-specific secretory protein MPT64 in lesions caused by tuberculous or non-tuberculous mycobacteria by analysing the in situ expression of apoptosis-related proteins (FasL, Fas, Bax, Bcl-2), apoptotic cells, inflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-10, transforming growth factor (TGF)-beta, interferon (IFN)-gamma] and MPT64 antigen. The discrimination of mycobacteria was made by nested polymerase chain reaction (PCR) amplification of IS6110, which is specific for M. tuberculosis complex organisms. Forty-seven cases of lymphadenitis with necrotic granulomas were evaluated. With nested PCR, 30/47 cases were positive for M. tuberculosis. MPT64 antigen was detected specifically in the PCR-positive cases. Granulomas caused by tuberculous mycobacteria had fewer apoptotic cells, higher numbers of cells expressing TNF-alpha and TGF-beta and less extensive necrosis than granulomas caused by non-tuberculous mycobacteria. There was a significant negative correlation between apoptotic cells and the number of cells expressing MPT64 antigens, suggesting a role for MPT64 protein in the inhibition of apoptosis. Granulomas with higher amounts of MPT64 also showed a greater number of cells expressing TGF-beta than those with lower amounts of MPT64. In conclusion, this study supports the hypothesis that inhibition of apoptosis is a virulence mechanism for tuberculous mycobacteria. Correlation of MPT64 antigen with expression of macrophage deactivating cytokines and reduced apoptosis suggests its role in pathogenesis and bacillary persistence.

Patient knowledge, practices and challenges to health care system in early diagnosis of mycobacterial adenitis.

OBJECTIVE: To assess diagnostic delay, knowledge and practices related to tuberculosis among patients with mycobacterial adenitis. DESIGN: A cross sectional study involving comparison analysis of high-risk groups. SETTING: Seven hospitals in rural and semi-rural districts of Arusha. SUBJECTS: Four hundred and twenty six clinically diagnosed adenitis patients. INTERVENTIONS: Biopsy specimens were processed for culture, histology, and sera for HIV testing. A questionnaire was used to assess knowledge, practice, and diagnostic time. MAIN OUTCOME MEASURES: Tribal comparisons were made using proportions and means. RESULTS: About 90% (387/423) of patients first visited medical facilities within a mean time of 10.1(SD, 15.7) weeks after becoming aware of their illness, and a diagnosis was made at a mean of 27 (SD, 25) weeks. Non-Iraqw patients, especially the Datoga, practised drinking raw milk (35.2% 43/122), eating raw animal products (18.8% 24/128) and living in houses with poor ventilation (33.6% 44/131), more than Iraqw patients. Of the investigations done, 14.5% (60/415) were culture positive, 11.3% (16/142) were HIV positive, and 73.6% (128/174) had histological features consistent with tuberculosis. The knowledge of TB spread by air droplets was poorer in Iraqw (74.1%, 203/274) than in non-lraqw (61.1%, 77/126) patients. About 35.0% (45/129) of non-lraqw and 27.3% (79/289) of Iraqw patients were not aware that TB could be transmitted from animals to humans. CONCLUSIONS: The health system diagnostic delay is about twice the patient delay. The knowledge and practices related to both human and bovine TB transmission were poor in all patients, especially in the patients from nomadic tribes.

Skin biopsies for early diagnosis and prognosis of graft-versus-host disease in recipients of allogeneic stem cells from blood or bone marrow.

A total of 61 patients with haematological malignancies were randomised either to allogeneic transplantation with blood stem cells (BSC) or bone marrow (BM), of whom 37 patients gave their consent to participate in a skin biopsy trial. Skin biopsies were performed before and after transplantation. The main objective was to assess whether biopsies of normal and affected skin from patients allografted with BSC showed a different histopathological and immunohistochemical pattern as compared to biopsies taken from patients allografted with BM. In addition, we wished to clarify whether sequential skin biopsies could be of prognostic value with regard to graft-versus-host disease (GVHD). Biopsies from normal or affected skin in BSC allografted did not disclose a different pattern as compared to BM transplants. Biopsies taken before the outbreak of acute and chronic GVHD showed no substantial differences between the groups. Irrespective of the type of allograft, the immunohistochemical picture of affected skin consistent with acute GVHD was dominated by a significantly higher number of T-lymphocytes (CD8+). Biopsies from normal skin before the outbreak of GVHD had no predictive value with regard to the development of acute or chronic GVHD. Immunohistochemistry is of supplementary help in distinguishing changes caused by cytotoxic agents from those caused by acute GVHD.

In situ expression of CD40, CD40L (CD154), IL-12, TNF-alpha, IFN-gamma and TGF-beta1 in murine lungs during slowly progressive primary tuberculosis.

The distribution and expression of CD40, its ligand CD40L (154) and related cytokines interleukin-12 (IL-12), tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) were studied in the lungs of B6D2F1 hybrid mice during slowly progressive primary tuberculosis (TB) by immunohistochemistry. CD40 and CD40L are implicated in cell-mediated immunity (CMI) causing activation or apoptosis of infected cells. The phenomenon of apoptosis is associated with Mycobacterium tuberculosis survival. In this study, using frozen lung sections (n = 33), our results showed increased CD40, IL-12 and TGF-beta1 expression in macrophages with progression of disease. High percentages of mycobacterial antigens (M.Ags), CD40L and IFN-gamma expression were maintained throughout infection, and TNF-alpha-expressing cells were decreased. In lymphocytes, the percentage of IFN-gamma-positive cells was increased, but CD40L and IL-12 were maintained with the progression of disease. M.Ags, CD40 and CD40L were expressed in the same areas of the lesions. We conclude that changes in the expression of CD40-CD40L and cytokines associated with M. tuberculosis infection favour the hypothesis that M. tuberculosis causes resistance of host cells to apoptosis causing perpetuation of infection.

Heat shock protein 70: correlation of expression with degree of graft-versus-host response and clinical graft-versus-host disease.

BACKGROUND: The heat shock proteins are increasingly becoming associated with immunopathologic phenomena, being induced in response to inflammation. They are highly immunogenic and are postulated as playing a role in both innate and adaptive immunity. Their proposed role in peptide binding and antigen presentation could suggest a potential role in the alloreactive process that leads to graft-versus-host disease (GVHD) after bone marrow transplantation. METHODS: In this study we examined the expression of the widely studied heat shock protein 70 (hsp70) in an in vitro-generated graft-versus-host reaction in human skin, using streptavidin biotin immunohistochemistry and laser scanning confocal microscopy. RESULTS: Hsp70 expression was correlated with high graft-versus-host responses (P<0.001) and was confirmed using laser scanning confocal microscopy. Increased expression of hsp70 was further defined due to increases in the inducible form of hsp70. Expression of inducible hsp70 was predictive of both clinical acute GVHD (P=0.001) and incidence of chronic GVHD (P<0.001). CONCLUSIONS: This investigation has demonstrated for the first time the expression of hsp70 in a human model of GVHD, suggesting involvement in the pathogenesis of the disease and providing the basis for further investigation. Increased expression of inducible hsp70 in the model could provide a biologic marker for the prediction of clinical acute and chronic GVHD.

Increased Bcl-2 and reduced Bax expression in infected macrophages in slowly progressive primary murine Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (MTB) persists in host macrophages (Mphis) because it has developed mechanisms to escape Mphi killing. In vitro studies have shown that MTB can induce and inhibit apoptosis by causing the expression of Bax and Bcl-2, respectively, suggesting that the infected cells' fate depends on pro- and antiapoptotic signals. In the present study, we investigated the role of Bcl-2 in MTB infection in situ. The aim was to study the pattern and distribution of Bcl-2 and Bax in cellular infiltrates of MTB-infected B6D2F1 hybrid mice and correlate the expression with the presence of MTB antigens (MAgs). Using formalin-fixed lung tissues (n = 45), our results showed a significant difference in the percentage of Mphis stained for Bcl-2 or MAgs and Bax (P < 0.0001). Bcl-2 expression was increased in a population of Mphis and corresponded in intensity, colocalization and percentage with that of MAgs on the same cells, while Bax expression was reduced. In lymphocyte aggregates, Bcl-2 and Bax did not show any differences. We conclude that overexpression of Bcl-2 in Mphis containing MTB may be associated with intracellular survival of the bacilli, thus demonstrating one way by which MTB can escape the host's cellular response and killing.

The detection of apoptosis in a human in vitro skin explant assay for graft versus host reactions.

AIMS: Keratinocyte apoptosis is a major pathogenic mechanism in dermal complications, such as graft versus host disease (GVHD), after allogeneic bone marrow transplantation. However, the mechanisms by which recipient target cells undergo apoptosis in GVHD are still unclear, but may result from DNA damage caused by chemotherapeutic agents and/or by direct cytokine action. The basis of this investigation was to correlate keratinocyte apoptosis with (1) the severity of graft versus host reactions (GVHR) in vitro and (2) the clinical grade (0--III) of GVHD. METHODS: Skin sections generated from an in vitro skin explant model for detecting experimental or clinically relevant GVHR were investigated for the detection of apoptotic nuclei using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique. This investigation also aimed to establish whether the TUNEL assay could be used as an additional, predictive method for the severity of GVHD before transplantation in potential patient/donor pairs given standard GVHD prophylaxis (cyclosporin A and methotrexate). RESULTS: By comparing mean values of apoptosis for each GVHR grade in a cohort of 83 retrospective skin sections it was shown that as the severity of GVHR increased there was a parallel increase in the percentage of apoptotic cells (p < 0.0001). However, the correlation between clinical GVHD grade II--III and overall keratinocyte apoptosis (> 2.6%) did not reach this degree of significance (chi(2): 4.2; degrees of freedom, 1; p = 0.04; Fisher's exact test: p = 0.06). CONCLUSIONS: The detection of apoptosis correlated with degree of GVHR using an in vitro assay and a higher degree of apoptosis tended to correlate with more severe GVHD. Further studies in a larger cohort of patients, using other methods to detect apoptosis in conjunction with the TUNEL assay, may give additional insight into the complex immunopathophysiology of GVHD.

GvHD risk assessment in hematopoietic stem cell transplantation: role of cytokine gene polymorphisms and an in vitro human skin explant model.

This present review concentrates on the recent results investigating the role of certain cytokine gene polymorphisms, including tumor necrosis factor alpha, interferon gamma, interleukin-6 (IL-6), IL-10, and IL-1 receptor antagonist, in allogeneic stem cell transplantation. The review discusses their potential role in predicting outcome and the development of a genetic risk index for graft-versus-host disease in human leukocyte antigen matched sibling transplants. By the comparative use of an in vitro human skin explant model, initial results suggest that certain polymorphisms may be associated with more severe disease.

Histological correlation between different centers using the skin explant model to predict graft-versus-host disease following bone marrow transplantation.

Acute graft-versus-host disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT) with an incidence of 40-60% and a mortality of up to 50%. Several assays have been developed to try to predict the development of GVHD including the mixed lymphocyte culture reaction, cytotoxic and helper T lymphocyte precursor frequency assays. In the Northern region of England we have used an in vitro skin explant model for predicting GVHD in MHC compatible bone marrow transplant recipients since 1988. The aims of the present study was to test the reproducibility of the model in two other bone marrow transplant centers in Europe. The assay consists of incubating patient skin explants with effector cells from mixed donor versus recipient lymphocyte cultures and the subsequent detection of graft-versus-host reactions by histopathological grading (0-IV) of the skin explants. 503 slides from 134 patients were evaluated. All were graded for negative GVHR grade 0-I or positive grade II-IV. Results from control and test slides significantly correlated between centers to the p value of 0.0001 by Fisher's exact probability test. These results show that the skin explant assay is reproducible between centers and supports the continued use of the assay to predict GVHD in allogeneic stem cell transplant recipients.

An in vitro skin explant assay as a predictive assay for graft-versus-host disease in a cohort of pediatric transplants.

Severe acute graft-versus-host disease (GvHD) remains a serious complication of allogeneic stem cell transplantation. An in vitro skin explant assay was used to predict the occurrence and severity of acute GvHD in a cohort of 30 pediatric patients undergoing human leucocyte antigen (HLA)-matched sibling transplants (20 patients) and matched or one antigen mismatched unrelated donor transplants (10 patients). In the cohort of HLA-matched sibling transplants, the result appeared to reflect the degree of GvHD prophylaxis. The skin explant assay correlated with GvHD outcome in 12 of 20 children, but this did not reach statistical significance (chi-square 0.95, d.f.=1, p=0.32). These results support previous observations. In this present cohort, patients were treated either with cyclosporin A (CsA) monotherapy (n=7) or with CsA plus additional methotrexate (MTX) (n=13). We have previously demonstrated that the skin explant assay was not as predictive in patients receiving CsA plus additional MTX compared to cohorts treated with CsA alone. In the group of patients treated with CsA alone, four of five patients (80%) predicted to develop GvHD developed acute GvHD of grade II or above; of two patients predicted to develop only grade 0-I GvHD, one patient developed no GvHD and the other grade II GvHD. In the CsA plus MTX group, nine patients were predicted to develop GvHD. Five of nine (55%) developed acute GvHD of grade II or above, while three of four with grade 0 or I skin explant assay results developed only grade 0-I GvHD. In a cohort of 10 patients who received unrelated donor transplants, the skin explant assay correlated with GvHD outcome in all 10 patients (Fisher's exact test p=0.008). Hence, the skin explant assay is a pretransplant in vitro GvHD predictive test that predicts the occurrence and severity of acute GvHD in pediatric unrelated donor transplants and to varying degrees, depending on the GvHD prophylaxis protocols, in HLA-matched sibling cohorts.

[A method for modeling skin explants--an in vitro predictive test for graft vs host disease in allogenic hematopoietic cell transplantation]. / Metoda modelu kozního explantátu--in vitro graft versus host disease prediktivní test pro alogenní transplantace hematopoetických bunek.

BACKGROUND: Acute graft versus host disease (GvHD) remains a severe complication of allogeneic haematopoietic stem cell transplantation (HSCT). Our study summaries results of skin explant assay (SEA) as a pretransplant GvHD predictive test in a cohort of paediatric (n = 33) and adult (a = 8) patients receiving grafts from their HLA identical siblings (n = 28), haploidentical relatives (n = 3) and unrelated donors (n = 10). Results GvHD prediction are correlated with the occurrence and severity of acute GvHD posttransplant and effect of GvHD prophylaxis on GvHD clinical outcome is evaluated. METHODS AND RESULTS: SEA utilises responding lymphocytes of the donor, which are sensitized firstly in vitro by mononuclears cells of patient in allogeneic mixed lymphocyte culture (MLC) and subsequently co-cultured with recipient's skin. Histopathological changes found in patients' skin explants are evaluated according to standard Lerner classification for acute GvHD. In general, GvHD predictive results in SEA correlated with GvHd clinical outcome in 28 out of 41 tested patients (68%, p = 0.015). In a cohort of HLA identical sibling transplants GvHD predictive results correlated with clinical manifestation of acute GvHD only in 15 out of 28 patients on individual GvHD prophylaxis. GvHD prophylaxis in the form of cyclosporine A (CsA) combined with short-term methotrexate (MTX) reduced the risk of acute GvHD in 10 out of 14 transplanted patients (71%) meanwhile CsA alone prophylaxis only in 1 out of 5 patients (20%). In a cohort of unrelated pairs on CsA/MTX prophylaxis combined with horse anti-lymphocyte globuline (ALG) correlated the GvHD prediction with GvHD clinical outcome (100%, p = 0.003). In all patients transplanted with the grafts from their haploidentical relatives the occurrence of severe GvHD was predicted. CONCLUSION: Skin explant assay helps identify pretransplant patients at higher risk of severe acute GvHD. GvHD predictive results enable the transplantation team to individualise GvHD prophylaxis and to optimise selection of the donor.

Mitochondrial enteropathy: the primary pathology may not be within the gastrointestinal tract.

BACKGROUND: Mitochondrial DNA (mtDNA) defects are an important cause of disease. Although gastrointestinal symptoms are common in these patients, their pathogenesis remains uncertain. AIM: To investigate the role of the mtDNA defect in the production of gastrointestinal dysfunction. PATIENT: A 20 year old woman who presented at 15 years of age with recurrent vomiting and pseudo-obstruction, who did not respond to conservative management and ultimately had subtotal gastrectomy and Roux-en-y reconstruction. She subsequently presented with status epilepticus and was found to have a mitochondrial respiratory chain disorder due to a pathogenic mtDNA point mutation (A3243G). METHODS: Resected bowel was studied using light and electron microscopy and mtDNA analysed from both mucosal and muscular layers using polymerase chain reaction generated RFLP analysis. RESULTS: Histological and electron microscopic studies revealed no morphological abnormalities in the resected stomach, and molecular genetic analysis failed to identify the genetic defect in either the mucosal or muscle layers. CONCLUSION: This study suggests that in some individuals with gastrointestinal symptoms associated with established mitochondrial DNA disease, the primary pathology of the mitochondrial enteropathy lies outside the gastrointestinal tract.

Loss of nuclear p16 protein expression correlates with increased tumor cell proliferation (Ki-67) and poor prognosis in patients with vertical growth phase melanoma.

The CDKN2A (p16INK4alpha) cell cycle-inhibitory gene has been associated with development of familial melanoma. Additionally, recent studies indicate that p16 alterations occur frequently in sporadic melanomas. To investigate whether differences in p16 expression are associated with tumor cell proliferation, tumor progression, and patient survival, we examined the immunohistochemical staining of p16 protein in a consecutive series of 202 vertical growth phase melanomas and 68 corresponding metastases and compared the results with Ki-67 expression, p53 expression, clinicopathological variables, and survival data. Forty-five percent of the primary tumors showed absent or minimal nuclear staining for p16 protein. These cases were significantly associated with high Ki-67 expression (P < 0.0001), ulceration (P = 0.001), and vascular invasion (P = 0.03). Further loss of p16 expression was observed in metastatic lesions (77% were negative; P < 0.0001). Absent/minimal nuclear p16 staining significantly predicted poor patient survival (log-rank test, P = 0.0003), with 37% and 67% estimated 10-year survival rates for cases with absent or present p16 expression, respectively. In multivariate analysis, p16 staining was an independent prognostic factor (hazard ratio, 2.5; 95% confidence interval, 1.5-4.2; P = 0.0008), along with p53 expression, Ki-67 expression, anatomical site, Clark's level of invasion, and vascular invasion. Our findings indicate that loss of nuclear p16 protein expression in vertical growth phase melanomas is associated with increased tumor cell proliferation (Ki-67) and independently predicts decreased patient survival. Cases without p53 expression had improved survival.