p53 Protein Expression in a Population-Based Series of Primary Vulval Squamous Cell Carcinoma and Immediate Adjacent Field Change

Objectives.Our aim was to study correlations between survival, disease recurrence, and p53 protein expression in a well defined population-based series of vulval squamous cell carcinoma and immediate adjacent epithelial skin changes.Methods.One hundred fifteen vulval squamous cell carcinoma were studied. Epithelial skin changes immediately adjacent to tumor were classified into nonneoplastic epithelial disorders (NNED) or vulval intraepithelial neoplasia (VIN). Archival specimens containing primary tumor and immediate adjacent skin were immunostained with a mouse monoclonal antibody to p53 protein.Results.p53 overexpression, defined as greater than 10% nuclear epithelial staining, was observed in 68% of tumors. Tumor immunostaining did not correlate with actuarial survival or disease-free interval. p53 overexpression was associated with a nonsignificant trend toward shorter disease-free interval in those tumors with nodal metastatic disease at diagnosis (P= 0.07). The only clinicopathological variable found to correlate with p53 expression was tumor grade (P= 0.002). Immediate adjacent abnormal skin changes were associated with p53 overexpression in 32% of cases. Adjacent normal skin did not immunostain for p53. p53 overexpression was most likely to occur in adjacent epithelial changes incorporating both NNED and high grade VIN (P= 0.005). Patterns of epithelial p53 overexpression in adjacent abnormal skin were either basal or full thickness. Full thickness epithelial p53 overexpression was most likely to occur in those disorders containing VIN (P< 0.0001). Positive immunostaining of adjacent skin abnormalities did not predict local tumor recurrence.Conclusions.This study demonstrates that although vulval squamous tumor p53 expression is not of prognostic significance, distinct immunostaining patterns can be observed in immediate adjacent skin. Vulval epithelial skin disorders displaying histological features of both NNED and VIN III may contain a profile of underlying molecular change which is of significance in subsequent tumor development.     

Pneumococcal meningitis: development of a new animal model.

HYPOTHESIS: The rat is a suitable animal to establish a model for the study of pneumococcal meningitis postcochlear implantation. BACKGROUND: There has been an increase in the number of cases of cochlear implant-related meningitis. The most common organism identified was Streptococcus pneumoniae. Whether cochlear implantation increases the risk of pneumococcal meningitis in healthy subjects without other risk factors remains to be determined. Previous animal studies do not focus on the pathogenesis and risk of pneumococcal meningitis postimplantation and are based on relatively small animal numbers, making it difficult to assess the cause-and-effect relationship. There is, therefore, a need to develop a new animal model allowing direct examination of the pathogenesis of meningitis in the presence of a cochlear implant. METHODS: Eighteen nonimplanted rats were infected with 1 x 10 and 1 x 10 colony-forming units (CFU) of a clinical isolate of S. pneumoniae via three different inoculation routes (middle ear, inner ear, and i.p.) to examine for evidence of meningitis during 24 hours. Six implanted rats were infected with the highest amount of bacteria possible for each route of inoculation (4 x 10 CFU i.p., 3 x 10 CFU middle ear, and 1 x 10 CFU inner ear) to examine for evidence of meningitis with the presence of an implant. The histological pattern of cochlear infections for each of the three different inoculating routes were examined. RESULTS: Pneumococcal meningitis was evident in all 6 implanted animals for each of the three different routes of inoculation. Once in the inner ear, bacteria were found to enter the central nervous system via either the cochlear aqueduct or canaliculi perforantes of the osseous spiral lamina, reaching the perineural and perivascular space then the internal acoustic meatus. The rate, extent, and pattern of infection within the cochleae depended on the route of inoculation. Finally, there was no evidence of pneumococcal meningitis observed in 18 nonimplanted rats inoculated at a lower concentration of S. pneumoniae when observed for 24 hours postinoculation. CONCLUSION: Meningitis in implanted rats after inoculation with a clinical isolate of S. pneumoniae is possible via all three potential routes of infection via the upper respiratory tract. The lack of meningitis observed in the 18 nonimplanted rats suggests that longer postinoculation monitoring periods are required to ensure whether or not meningitis will develop. Based on this work, we have developed a new animal model that will allow quantitative risk assessment of meningitis postcochlear implantation, and the assessment of the efficacy of potential interventional strategies in future studies.     

The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M₁ muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M₁ and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M₁-mEGFP was expressed at levels equal to the M₁ receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M₁-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M₁-mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M₁-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Measuring and understanding the extent and potential significance of quaternary organization of members of the class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs) have both fascinated and frustrated researchers for many years (1, 2). Over time, a wide range of methods have been applied to address this question, and many different GPCRs have been examined. Outcomes have ranged from assertions that such receptors are monomeric and that results consistent with other conclusions reflect either artifacts of the method of measurement or that studies have been performed at nonphysiological levels of expression of the receptor being studied, to those that have suggested rather stable dimeric or tetrameric complexes (1). Only in the case of rhodopsin, the photon receptor expressed at very high levels (in the range of 24,000–30,000 molecules/µm²) in rod outer segments of the eye, have detailed studies been conducted in situ on a class A GPCR. In this example, various studies have shown that rhodopsin is organized as rows of dimers (3, 4). However, to our knowledge, no other GPCR is expressed natively at levels akin to rhodopsin. As such, although a substantial number of studies, generally performed in transfected cell lines or in artificial bilayer systems, have provided evidence that other GPCRs can and do form dimeric and/or higher-order quaternary complexes in a concentration-dependent manner (1, 2), how levels of expression required to observe such complexes relate to expression levels in native cells and tissues has been poorly defined, as is the stability of such complexes and whether they are regulated by ligand binding.Developments in fluorescence fluctuation analysis (FFA) have facilitated efforts to define the oligomeric status of transmembrane receptor proteins (5, 6). Unlike methods based on resonance energy transfer, only a single fluorophore-linked protein is required to be expressed to use FFA. It is, therefore, more practical to use such methods in native cells and tissues if linked to genome-editing approaches and/or the generation of transgenic “knock-in” animal models in which a receptor of interest is replaced with a fluorophore-tagged, modified form of the receptor. Moreover, the recent introduction of fluorescence intensity fluctuation (FIF) spectrometry (7–10) has overcome issues with other methods based on FFA that result in information being compressed due to averaging of oligomer-size data from interrogated regions of interest (RoIs) in which complex mixtures of oligomers of different sizes may be present (7, 8).To define whether the class A M₁ muscarinic acetylcholine receptor is present in hippocampal and cortical neurons as strict monomers or as a range of monomeric, dimeric, and, potentially, oligomeric complexes, we applied FIF spectrometry to images of such neurons isolated from a line of transgenic mice in which we replaced the M₁ receptor with a form of the receptor that includes C-terminally linked monomeric enhanced green fluorescent protein (mEGFP). We first show that both expression levels and function of the introduced M₁-mEGFP construct appear equivalent to the native M₁ receptor in wild-type (WT) mice, using a range of methods and measures ranging from [³H]ligand binding and cell signaling assays to locomotion. We then demonstrate in hippocampal and cortical neurons that in the basal state, the M₁-mEGFP construct is present as a mixture of monomers and dimeric or oligomeric complexes. We also show that the presence of either an agonist or an antagonist ligand promotes monomerization of the receptor. In these studies, we combined analysis of images of a fluorophore-modified receptor in situ with calculation of receptor oligomer complexity. The studies provide a clear and unambiguous answer to a long-standing question that has been the subject of considerable debate (11–13) but that has previously been restricted to studies performed on transfected cell lines. Moreover, these studies are a model for subsequent studies for researchers who plan to explore the topic of dimerization of rhodopsin-family GPCRs.     

Why MSM in Rural South African Communities Should be an HIV Prevention Research Priority

Research into HIV and men who have sex with men’s (MSM) health in South Africa has been largely confined to the metropolitan centres. Only two studies were located making reference to MSM in rural contexts or same-sex behaviors among men in the same. There is growing recognition in South Africa that MSM are not only disproportionately affected by HIV and have been underserved by the country’s national response, but that they contribute significantly to sustaining the high number of new infections recorded each year. We argue that to meet the objectives of the country’s national strategic plan for HIV, STI and TB it is important we know how these behaviours may be contributing to the sustained rural HIV epidemic in the youngest age groups and determine what constitutes appropriate and feasible programmatic response that can be implemented in the country’s public sector health services.     

Characterization of benign and metastatic vertebral compression fractures with quantitative diffusion MR imaging

BACKGROUND AND PURPOSE: Conventional imaging techniques cannot be used to unambiguously and reliably differentiate malignant from benign vertebral compression fractures. Our hypothesis is that these malignant and benign vertebral lesions can be better distinguished on the basis of tissue apparent diffusion coefficients (ADCs). The purpose of this study was to test this hypothesis by using a quantitative diffusion imaging technique. METHODS: Twenty-seven patients with known cancer and suspected metastatic vertebral lesions underwent 1.5-T conventional T1-weighted, T2-weighted, and contrast-enhanced T1-weighted imaging to identify the lesions. Diffusion-weighted images of the areas of interest were acquired by using a fast spin-echo diffusion pulse sequence with b values of 0-250 s/mm(2). The abnormal regions on the diffusion-weighted images were outlined by using the conventional images as guides, and the ADC values were calculated. On the basis of pathologic results and clinical findings, the cases were divided into two categories: benign compression fractures and metastatic lesions. The ADC values for each category were combined and plotted as histograms; this procedure was followed by statistical analysis. RESULTS: The patient group had 12 benign fractures and 15 metastases. The mean ADC values, as obtained from the histograms, were (1.9 +/- 0.3) x 10(-4) mm(2)/s and (3.2 +/- 0.5) x 10(-4) mm(2)/s for metastases and benign fractures, respectively. CONCLUSION: Our results indicate that quantitative ADC mapping, instead of qualitative diffusion-weighted imaging, can provide valuable information in differentiating benign vertebral fractures from metastatic lesions.     

Monitoring systemic donor lymphocyte macrochimerism to aid the diagnosis of graft-versus-host disease after liver transplantation

BACKGROUND: The diagnosis of graft-versus-host disease (GvHD) after liver transplantation can be difficult because early symptoms are often nonspecific. In this study, the presence of donor lymphocyte macrochimerism in recipient peripheral blood was examined as a diagnostic aid for GvHD after cadaveric donor liver transplantation. METHODS: Between 1996 and 2002, 33 liver transplant recipients with a clinical suspicion of GvHD (skin rash, diarrhea, pyrexia, pancytopenia, or anemia, without an obvious alternative cause) were investigated for peripheral blood donor lymphocyte macrochimerism. Donor macrochimerism was determined at the time of first clinical presentation by a low-sensitivity polymerase chain reaction (PCR) to detect donor human leukocyte antigen (HLA) alleles using genomic DNA extracted from recipient peripheral blood. Where donor HLA alleles were detected, the percentage of donor T cells was quantified by two-color flow cytometric analysis using antibodies specific for mismatched donor and recipient HLA alleles. The relationship between the presence or absence of donor lymphocyte macrochimerism and final diagnoses based on clinical and histological criteria was examined. RESULTS: Seven of the 33 patients were PCR positive for donor HLA alleles. All had macrochimerism, with donor T lymphocyte levels ranging from 4% to 50% of circulating lymphocytes. All seven patients had normal liver function tests, skin rash, and diagnosis of GvHD histologically confirmed by skin or gut biopsies. Twenty-six patients were PCR negative, and, in 23, an alternative diagnosis was eventually established. The remaining three patients made a rapid and spontaneous recovery with no further symptoms suggestive of GvHD. CONCLUSIONS: Donor lymphocyte macrochimerism was present in all patients in whom the diagnosis of GvHD was confirmed. In patients with symptoms consistent with GvHD and a negative PCR for donor HLA, an alternative diagnosis was eventually established or the patients recovered spontaneously. Detection of donor HLA alleles in recipient peripheral blood by PCR is a useful diagnostic tool for GvHD after liver transplantation.     

Prospective psychosocial monitoring of living kidney donors using the Short Form-36 health survey: results at 12 months

BACKGROUND: Lack of prospective psychosocial outcome studies on living kidney donors impedes identification of risk factors for poor outcome. METHODS: Psychiatric assessment of living kidney donors was performed preoperatively and at 4 and 12 months postoperatively using a semistructured interview, the Short Form (SF)-36 Health Survey, and Patient Health Questionnaire psychiatric assessment. A total of 48 of 51 consecutive donors (94%) over a 5-year period were available for follow-up and completed all assessments. RESULTS: At preoperative assessment, only 1 of the 48 donors (2%) had a Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition Axis I psychiatric disorder, but 15 (31%) developed a disorder during the 12 months, a 29% incidence. Disorders were depressive (12%), anxiety (6%), and adjustment (13%). Seven donors (15%) demonstrated a disorder at 12 months (depressive 10%, anxiety 2%, adjustment 2%). There was a corresponding decline in psychosocial function as measured by the SF-36 Mental Component Summary score; it decreased at both 4 and 12 months (P<0.01, P<0.05); for 19% of donors, this was a larger decrease than would be expected for the cohort (>2 standard error of measurement units). Scores for SF-36 scales of General Health and Vitality decreased significantly (P<0.05), as did those of Bodily Pain, indicating greater impairment from pain. Psychiatric disorder at 12 months was associated with donor psychosocial function (Mental Component Summary) and psychiatric disorder at 4 months (P<0.01), physical function (SF-36 Physical Component Summary score) at 4 and 12 months (P<0.01), and recipient psychiatric disorder at 12 months (P<0.05). CONCLUSIONS: Donors should be alerted to possible psychosocial impairment, assessed for risk factors, and monitored for at least 12 months. Treatment should be available.