C-terminal citrullinated peptide alters antigen-specific APC:T cell interactions leading to breach of immune tolerance.

In rheumatoid arthritis, the emergence of anti-citrullinated autoimmunity is associated with HLA-antigen-T cell receptor complexes. The precise mechanisms underpinning this breach of tolerance are not well understood. Porphyromonas gingivalis expresses an enzyme capable of non-endogenous C-terminal citrullination with potential to generate citrullinated autoantigens. Here we document how C-terminal citrullination of ovalbumin peptide323-339 alters the interaction between antigen-presenting cells and OTII T cells to induce functional changes in responding T cells. These data reveal that C-terminal citrullination is sufficient to breach T cell peripheral tolerance in vivo and reveal the potential of C-terminal citrullination to lower the threshold for T cell activation. Finally, we demonstrate a role for the IL-2/STAT5/CD25 signalling axis in breach of tolerance. Together, our data identify a tractable mechanism and targetable pathways underpinning breach of tolerance in rheumatoid arthritis and provide new conceptual insight into the origins of anti-citrullinated autoimmunity.

Understanding the Prevalence of Prediabetes and Diabetes in Patients With Cancer in Clinical Practice: A Real-World Cohort Study.

BACKGROUND: This study aimed to understand the prevalence of prediabetes (preDM) and diabetes mellitus (DM) in patients with cancer overall and by tumor site, cancer treatment, and time point in the cancer continuum. METHODS: This cohort study was conducted at Huntsman Cancer Institute at the University of Utah. Patients with a first primary invasive cancer enrolled in the Total Cancer Care protocol between July 2016 and July 2018 were eligible. Prevalence of preDM and DM was based on ICD code, laboratory tests for hemoglobin A1c, fasting plasma glucose, nonfasting blood glucose, or insulin prescription. RESULTS: The final cohort comprised 3,512 patients with cancer, with a mean age of 57.8 years at cancer diagnosis. Of all patients, 49.1% (n=1,724) were female. At cancer diagnosis, the prevalence of preDM and DM was 6.0% (95% CI, 5.3%-6.8%) and 12.2% (95% CI, 11.2%-13.3%), respectively. One year after diagnosis the prevalence was 16.6% (95% CI, 15.4%-17.9%) and 25.0% (95% CI, 23.6%-26.4%), respectively. At the end of the observation period, the prevalence of preDM and DM was 21.2% (95% CI, 19.9%-22.6%) and 32.6% (95% CI, 31.1%-34.2%), respectively. Patients with myeloma (39.2%; 95% CI, 32.6%-46.2%) had the highest prevalence of preDM, and those with pancreatic cancer had the highest prevalence of DM (65.1%; 95% CI, 57.0%-72.3%). Patients who underwent chemotherapy, radiotherapy, or immunotherapy had a higher prevalence of preDM and DM compared with those who did not undergo these therapies. CONCLUSIONS: Every second patient with cancer experiences preDM or DM. It is essential to foster interprofessional collaboration and to develop evidence-based practice guidelines. A better understanding of the impact of cancer treatment on the development of preDM and DM remains critical.

MODELING STREAMFLOW AND WATER QUALITY SENSITIVITY TO CLIMATE CHANGE AND URBAN DEVELOPMENT IN 20 U.S. WATERSHEDS.

Watershed modeling in 20 large, United States (U.S.) watersheds addresses gaps in our knowledge of streamflow, nutrient (nitrogen and phosphorus), and sediment loading sensitivity to mid-21st Century climate change and urban/residential development scenarios. Use of a consistent methodology facilitates regional scale comparisons across the study watersheds. Simulations use the Soil and Water Assessment Tool. Climate change scenarios are from the North American Regional Climate Change Assessment Program dynamically downscaled climate model output. Urban and residential development scenarios are from U.S. Environmental Protection Agency's Integrated Climate and Land Use Scenarios project. Simulations provide a plausible set of streamflow and water quality responses to mid-21st Century climate change across the U.S. Simulated changes show a general pattern of decreasing streamflow volume in the central Rockies and Southwest, and increases on the East Coast and Northern Plains. Changes in pollutant loads follow a similar pattern but with increased variability. Ensemble mean results suggest that by the mid-21st Century, statistically significant changes in streamflow and total suspended solids loads (relative to baseline conditions) are possible in roughly 30-40% of study watersheds. These proportions increase to around 60% for total phosphorus and total nitrogen loads. Projected urban/residential development, and watershed responses to development, are small at the large spatial scale of modeling in this study.

Development of UK recommendations on treatment for post-surgical erectile dysfunction.

AIM: To develop a management strategy (rehabilitation programme) for postsurgical erectile dysfunction (ED) among men experiencing ED associated with treatment of prostate, bladder or rectal cancer that is suitable for use in a UK NHS healthcare context. METHODS: PubMed literature searches of ED management together with a survey of 13 experts in the management of ED from across the UK were conducted. RESULTS: Data from 37 articles and completed questionnaires were collated. The results discussed in this study demonstrate improved objective and subjective clinical outcomes for physical parameters, sexual satisfaction, and rates of both spontaneous erections and those associated with ED treatment strategies. CONCLUSION: Based on the literature and survey analysis, recommendations are proposed for the standardisation of management strategies employed for postsurgical ED.

Evaluation of a capacity building clinical educational model for oral health clinicians treating very young children.

OBJECTIVE: There are significant levels of dental caries in Australian school-aged children, with children aged five years having a mean dmft of 1.3. It has also been identified that, in general, oral health clinicians lack confidence to treat very young children and this study aimed to increase capacity of public sector oral health clinicians to treat preschool children. BASIC RESEARCH DESIGN: An educational program was developed, implemented and evaluated for its capability to increase the confidence and knowledge of oral health clinicians and dental assistants in providing oral care for children aged 12 months to 5 years. RESULTS: In 2011 and 2012, the course was delivered to 36 clinicians (22 dentists, 12 dental therapists, and two oral health therapists) and showed increases in their confidence and knowledge for participants when providing dental procedures to preschool children. CONCLUSIONS: The educational program that was developed and implemented has met its objective of increasing the knowledge and confidence of practicing oral health clinicians and dental assistants in the management of preschool children. Strategies to further enhance the outcomes of this educational program have been proposed.

Modeling the Effects of Future Hydroclimatic Conditions on Microbial Water Quality and Management Practices in Two Agricultural Watersheds.

Anticipated future hydroclimatic changes are expected to alter the transport and survival of fecally-sourced waterborne pathogens, presenting an increased risk of recreational water quality impairments. Managing future risk requires an understanding of interactions between fecal sources, hydroclimatic conditions and best management practices (BMPs) at spatial scales relevant to decision makers. In this study we used the Hydrologic Simulation Program FORTRAN to quantify potential fecal coliform (FC - an indicator of the potential presence of pathogens) responses to a range of mid-century climate scenarios and assess different BMP scenarios (based on reduction factors) for reducing the risk of water quality impairment in two, small agricultural watersheds - the Chippewa watershed in Minnesota, and the Tye watershed in Virginia. In each watershed, simulations show a wide range of FC responses, driven largely by variability in projected future precipitation. Wetter future conditions, which drive more transport from non-point sources (e.g. manure application, livestock grazing), show increases in FC loads. Loads typically decrease under drier futures; however, higher mean FC concentrations and more recreational water quality criteria exceedances occur, likely caused by reduced flow during low-flow periods. Median changes across the ensemble generally show increases in FC load. BMPs that focus on key fecal sources (e.g., runoff from pasture, livestock defecation in streams) within a watershed can mitigate the effects of hydroclimatic change on FC loads. However, more extensive BMP implementation or improved BMP efficiency (i.e., higher FC reductions) may be needed to fully offset increases in FC load and meet water quality goals, such as total maximum daily loads and recreational water quality standards. Strategies for managing climate risk should be flexible and to the extent possible include resilient BMPs that function as designed under a range of future conditions.

AGRICULTURAL BEST MANAGEMENT PRACTICE SENSITIVITY TO CHANGING AIR TEMPERATURE AND PRECIPITATION.

Agricultural best management practices (BMPs) reduce non-point source pollution from cropland. Goals for BMP adoption and expected pollutant load reductions are often specified in water quality management plans to protect and restore waterbodies; however, estimates of needed load reductions and pollutant removal performance of BMPs are generally based on historic climate. Increasing air temperatures and changes in precipitation patterns and intensity are anticipated throughout the U.S. over the 21st century. The effects of such changes on agricultural pollutant loads have been addressed by several authors, but how these changes will affect the performance of widely promoted BMPs has received limited attention. We use the Soil and Water Assessment Tool (SWAT) to investigate potential changes in the effectiveness of conservation tillage, no-till, vegetated filter strips, grassed waterways, nutrient management, winter cover crops, and drainage water management practices under potential future temperature and precipitation patterns. We simulate two agricultural watersheds in the Minnesota Corn Belt and the Georgia Coastal Plain with different hydro-climatic settings, under recent conditions (1950-2005) and multiple potential future mid-century (2030-2059) and late-century (2070-2099) climate scenarios. Results suggest future increases in agricultural source loads of sediment, nitrogen and phosphorous. Most BMPs continue to reduce loads, but removal efficiencies generally decline due to more intense runoff events, biological responses to changes in soil moisture and temperature, and exacerbated upland loading. The coupled effects of higher upland loading and reduced BMP efficiencies suggest that wider adoption, resizing, and/or combining practices may be needed in the future to meet water quality goals for agricultural lands.

Misoprostol is superior to combined omeprazole-sucralfate for the treatment of equine gastric glandular disease.

BACKGROUND: Previous studies have demonstrated a poor response to healing of gastric glandular lesions with oral omeprazole and other medications. OBJECTIVES: To evaluate the efficacy of two novel treatments (misoprostol [M] and combined omeprazole-sucralfate [OS]) in horses for gastric glandular disease. STUDY DESIGN: Prospective, clinical study. METHODS: Sixty-three sports horses with grade >1/4 glandular disease were identified by gastroscopy. Horses received either 5 µg/kg of misoprostol per os BID 1 h prior to feeding or a combination of 4 mg/kg enteric coated omeprazole per os SID and 12 mg/kg sucralfate per os BID where drugs were given 1 h prior to feeding and sucralfate given 60 min after omeprazole; allocation was dependent upon centre. Gastroscopy was repeated at 28-35 days. Evaluators of the gastroscopy images were blinded to the treatments the horses received and images were reviewed independently. RESULTS: The most common presenting sign in both treatment groups was poor performance (Overall - 65.1%; M - 60.5% and OS - 75%). Overall healing (P<0.001; OR = 11 [2.8-45]) and improvement (P = 0.006; OR = 11 [1.9-59]) of lesions were associated with resolution of clinical signs. Misoprostol was shown to be superior to combined omeprazole-sucralfate both for healing (M - 72% [95% CI 43-67] and OS - 20% [95% CI 7-41]; P<0.001) and improvement (M - 98% [95% CI 90-100] and OS - 65% [95% CI 43-83]; P<0.001). MAIN LIMITATIONS: Relatively small, clinical study, reliance on client questionnaire data, clients not blinded to the treatments the horse received, diet could have affected drug pharmacodynamics although mimics clinical practice and no validated scoring system available for glandular lesions. CONCLUSIONS: These results suggest that gastric glandular disease does indeed result in clinical signs. In this population of horses, misoprostol was superior to omeprazole and sucralfate and warrants further evaluation in a large scale, multi-centre trial.

Enrichment of Innate Lymphoid Cell Populations in Gingival Tissue.

Innate lymphoid cells (ILCs) are a population of lymphocytes that act as the first line of immunologic defense at mucosal surfaces. The ILC family in the skin, lungs, and gastrointestinal tissues has been investigated, and there are reports of individual subsets of ILCs in the oral tissues. We sought to investigate the whole ILC population (group 1, 2, and 3 subsets) in the murine gingivae and the lymph nodes draining the oral cavity. We show that ILCs made up a greater proportion of the whole CD45+ lymphocyte population in the murine gingivae (0.356% ± 0.039%) as compared with the proportion of ILCs in the draining lymph nodes (0.158% ± 0.005%). Cytokine profiling of the ILC populations demonstrated different proportions of ILC subsets in the murine gingivae versus the regional lymph nodes. The majority of ILCs in the draining lymph nodes expressed IL-5, whereas there were equal proportions of IFN-γ- and IL-5 expressing ILCs in the oral mucosa. The percentage of IL-17+ ILCs was comparable between the murine gingivae and the oral draining lymph nodes. These data suggest an enrichment of ILCs in the murine gingivae, and these ILCs reflect a cytokine profile discrepant to that of the local draining lymph nodes. These studies indicate diversity and enrichment of ILCs at the oral mucosal surface. The function of ILCs in the oral cavity remains to be determined; here, we provide a premise of ILC populations that merits future consideration in investigations of mouse models and human tissues.

The effects of downscaling method on the variability of simulated watershed response to climate change in five U.S. basins.

Simulations of future climate change impacts on water resources are subject to multiple and cascading uncertainties associated with different modeling and methodological choices. A key facet of this uncertainty is the coarse spatial resolution of GCM output compared to the finer-resolution information needed by water managers. To address this issue, it is now common practice to apply spatial downscaling techniques, using either higher-resolution regional climate models or statistical approaches applied to GCM output to develop finer-resolution information for use in water resources impacts assessments. Downscaling, however, can also introduce its own uncertainties into water resources impacts assessments. This study uses watershed simulations in five U.S. basins to quantify the sources of variability in streamflow, nitrogen, phosphorus, and sediment loads associated with the underlying GCM compared to the choice of downscaling method (both statistically and dynamically downscaled GCM output). We also assess the specific, incremental effects of downscaling by comparing watershed simulations based on downscaled and non-downscaled GCM model output. Results show that the underlying GCM and the downscaling method each contribute to the variability of simulated watershed responses. The relative contribution of GCM and downscaling method to the variability of simulated responses varies by watershed and season of the year. Results illustrate the potential implications of one key methodological choice in conducting climate change impacts assessments for water - the selection of downscaled climate change information.

Mast Cells Contribute to Porphyromonas gingivalis-induced Bone Loss.

Periodontitis is a chronic inflammatory and bone-destructive disease. Development of periodontitis is associated with dysbiosis of the microbial community, which may be caused by periodontal bacteria, such as Porphyromonas gingivalis Mast cells are sentinels at mucosal surfaces and are a potent source of inflammatory mediators, including tumor necrosis factors (TNF), although their role in the pathogenesis of periodontitis remains to be elucidated. This study sought to determine the contribution of mast cells to local bone destruction following oral infection with P. gingivalis Mast cell-deficient mice (Kit(W-sh/W-sh)) were protected from P. gingivalis-induced alveolar bone loss, with a reduction in anti-P. gingivalis serum antibody titers compared with wild-type infected controls. Furthermore, mast cell-deficient mice had reduced expression of Tnf, Il6, and Il1b mRNA in gingival tissues compared with wild-type mice. Mast cell-engrafted Kit(W-sh/W-sh) mice infected with P. gingivalis demonstrated alveolar bone loss and serum anti-P. gingivalis antibody titers equivalent to wild-type infected mice. The expression of Tnf mRNA in gingival tissues of Kit(W-sh/W-sh) mice was elevated following the engraftment of mast cells, indicating that mast cells contributed to the Tnf transcript in gingival tissues. In vitro, mast cells degranulated and released significant TNF in response to oral bacteria, and neutralizing TNF in vivo abrogated alveolar bone loss following P. gingivalis infection. These data indicate that mast cells and TNF contribute to the immunopathogenesis of periodontitis and may offer therapeutic targets.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: Equine gastric glandular disease (EGGD) is a common condition, for which the underlying pathophysiology is undetermined. Endoscopic mucosal biopsies have been proposed as a method for adapting therapy. OBJECTIVES: To evaluate diagnostic information obtained from endoscopic mucosal biopsies. STUDY DESIGN: Prospective, experimental study. METHODS: Twenty-one horses undergoing elective humane slaughter were subjected to gross examination of the glandular mucosa. Glandular pathology was graded using EGUS Council guidelines from digital camera images. Mucosal biopsies were obtained using a 'single-bite' (1.8 mm; A and 2.4 mm; B) or 'double bite' technique (2.4 mm; C) using endoscopic biopsy instruments. Tissue was formalin fixed, processed and stained using standard protocols. Inflammatory infiltrates visualised histologically were graded (mild, moderate or severe) and compared with ulcer grade. Full thickness biopsies were also obtained adjacent to the biopsy site and of other visual lesions and inflammatory cell counts were compared with mucosal biopsies using ICC. RESULTS: Full thickness samples were artefact free and allowed visualisation of all layers. Mucosal biopsy samples contained mucosa in all samples, submucosa in 55% (C), 61% (A) and 66% (B) of samples and glands in 50% (B), 66% (A) and 100% (C). Samples from A were too small for histological assessment (33%) and tissue damage was commonly seen in A and B (n = 8 and n = 10) when compared with C (n = 3). Horses with normal glandular appearance (grade 0; n = 7) mostly demonstrated mild gastritis (n = 5). Severe gastritis was identified in mild EGGD (grade 1/2), whilst mild and moderate gastritis was identified in all EGGD grades. There was no histological evidence of ulceration or erosion. There was poor agreement between cell numbers and sampling techniques (ICC<0.29). CONCLUSIONS: These data show lack of ulcerative pathology and instead inflammation in EGGD. Lesion appearance is a poor indicator of underlying severity. Mucosal biopsies offer limited value in predicting underlying disease. Ethical animal research: This study was approved by the University of Nottingham Ethics and Welfare Committee. The study was performed on material collected at an abattoir. SOURCE OF FUNDING: None. Competing interests: None declared.

Periodontitis in the absence of B cells and specific anti-bacterial antibody.

Periodontitis (PD) results from complex interactions between a dysbiotic oral microbiota and a dysregulated host immune response. The inflammatory infiltrate in the gingiva of PD patients includes an abundance of B cells, implicating these cells in the immunopathology. We sought to investigate the role of B cells in PD using a murine model. Wild-type or B-cell-deficient (µMT) mice were orally infected with Porphyromonas gingivalis. One or six weeks following infection, lymphocyte populations in the gingiva and cervical draining lymph nodes (dLN) were analysed by flow cytometry; serum anti-P. gingivalis IgG antibody titers were measured by enzyme-linked immunosorbent assay, and alveolar bone loss was determined. In wild-type mice, the percentage of gingival B cells expressing receptor activator of nuclear factor-κB ligand (RANKL) was significantly increased 1 week post-infection (5.36% control versus 11% PD, P < 0.01). The percentage of Fas(+) GL7(+) germinal centre B cells in the dLN was significantly increased at both 1 week (2.03% control versus 6.90% PD, P < 0.01) and 6 weeks (4.45% control versus 8.77% PD, P < 0.05) post-infection. B-cell-deficient mice were protected from P. gingivalis-induced alveolar bone loss, with a lack of B-cell proliferation and lack of CD4(+) CD44(+) CD62L(-) T-cell generation in the dLN, and absence of serum anti-P. gingivalis antibodies. Our data imply a pathological role for B cells in PD, and that selective targeting of this immune axis may have a role in treating severe periodontal disease.

IL-33 Exacerbates Periodontal Disease through Induction of RANKL.

Cytokines mediate the balance between protective and destructive immunity in periodontitis. We sought to investigate the role of IL-33 in periodontitis. The expression of IL-33 in gingival tissue from healthy controls (n = 10) and patients with chronic periodontitis (n = 17) was investigated. Based on a murine model of periodontal disease, the function of IL-33 was determined first by administration of exogenous IL-33 and second by inhibition of IL-33 signaling using mice deficient in the IL-33 receptor ST2. Alveolar bone level, serum antibody, and lymphocyte responses were assessed in the murine model. Expression of IL-33 and ST2 was elevated in gingival tissues from patients with chronic periodontitis as compared with healthy tissues (P < 0.05). Similarly, Il33 expression was higher in periodontal tissues of Porphyromonas gingivalis-infected mice as compared with sham-infected controls (P < 0.05). IL-33 treatment of P. gingivalis-infected mice significantly exacerbated alveolar bone loss when compared with infection or IL-33 treatment alone (P < 0.001). Conversely, P. gingivalis infection-induced alveolar bone loss was attenuated in mice lacking ST2. The percentages of T and B lymphocytes expressing nuclear factor κB ligand (RANKL) in the gingival tissues and T lymphocytes expressing RANKL in the cervical draining lymph nodes were higher in IL-33-treated P. gingivalis-infected mice versus phosphate buffered saline-treated P. gingivalis-infected controls (all P < 0.001). Targeting the RANKL pathway by osteoprotegerin administration abrogated periodontal bone destruction in P. gingivalis-infected, IL-33-treated mice. These data demonstrate a previously unrecognized role for IL-33 in exacerbating bone loss in a RANKL-dependent manner in the context of bacterial infection and suggest that this pathway may be amenable to manipulation as a novel therapeutic target in periodontitis.

Inhibition of human leukocyte elastase. 1. Inhibition by C-7-substituted cephalosporin tert-butyl esters.

Time-dependent inhibitors of the enzyme human leukocyte elastase have been developed based on the cephem nucleus. A series of cephalosporin tert-butyl esters has been examined, and the activity of these compounds has been found to be very sensitive to C-7 substituents, with small, alpha-oriented, electron-withdrawing groups showing greatest activity. Additionally, the oxidation state of the sulfur atom has been found to play a role in potency, with sulfones showing considerably greater activity than the corresponding sulfides or beta-sulfoxides. The alpha-sulfoxides were inactive.

Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

Effects of six weeks of chronic ethanol administration on lactic acid accumulation and high energy phosphate levels after experimental brain injury in rats.

The effects of 6 weeks of chronic ethanol administration on the lateral fluid percussion (FP) brain injury-induced regional accumulation of lactate and on the levels of total high-energy phosphates were examined in rats. In both the chronic ethanol diet (ethanol diet) and pair-fed isocaloric sucrose control diet (control diet) groups, tissue concentrations of lactate were elevated in the cortices and hippocampi of both the ipsilateral and contralateral hemispheres at 5 min after brain injury. In both diet groups, concentrations of lactate were elevated only in the injured left cortex and the ipsilateral hippocampus at 20 min after FP brain injury. No significant differences were found in the levels of lactate in the cortices and hippocampi of sham animals and brain-injured animals between the ethanol and control diet groups at 5 min and 20 min after injury. In the ethanol and control diet groups, tissue concentrations of total high-energy phosphates (ATP + PCr) were not affected in the cortices and hippocampi at 5 min and 20 min after lateral FP brain injury. No significant differences were found in the levels of total high-energy phosphates in the cortices and hippocampi of the sham and brain-injured animals between the ethanol and control diet groups at 5 min and 20 min after injury. Histologic studies revealed a similar extent of damage in the cortex and in the CA3 region of the ipsilateral hippocampus in both diet groups at 14 days after lateral FP brain injury. These findings suggest that 6 weeks of chronic ethanol administration does not alter brain injury-induced accumulation of lactate, levels of total high energy phosphates, and extent of morphological damage.

Effects of six weeks of chronic ethanol administration on the behavioral outcome of rats after lateral fluid percussion brain injury.

This study examined the effects of 6 weeks of chronic ethanol administration on the behavioral outcome in rats after lateral fluid percussion (FP) brain injury. Rats were given either an ethanol liquid diet (ethanol diet-groups) or a pair-fed isocaloric sucrose control diet (control diet groups) for 6 weeks. After 6 weeks, the ethanol diet was discontinued for the ethanol diet rats and they were then given the control sucrose diet for 2 days. During those 2 days, the rats were trained to perform a beam-walking task and subjected to either lateral FP brain injury of low to moderate severity (1.8 atm) or to sham operation. In both the control diet and the ethanol diet groups, lateral FP brain injury caused beam-walking impairment on days 1 and 2 and spatial learning disability on days 7 and 8 after brain injury. There were no significant differences in beam-walking performance and spatial learning disability between brain injured animals from the control and ethanol diet groups. However, a trend towards greater behavioral deficits was observed in brain injured animals in the ethanol diet group. Histologic analysis of both diet groups after behavioral assessment revealed comparable ipsilateral cortical damage and observable CA3 neuronal loss in the ipsilateral hippocampus. These results only suggest that chronic ethanol administration, longer than six weeks of administration, may worsen behavioral outcome following lateral FP brain injury. For more significant behavioral and/or morphological change to occur, we would suggest that the duration of chronic ethanol administration must be increased.

Association of vitamin D receptors with the nuclear matrix of human and rat genitourinary tissues.

Calcitrol, 1,25 dihydroxyvitamin D3 (1,25-D3) has an important role in the antiproliferative and growth regulatory effects on normal and neoplastic cells (e.g. prostate cancer cells). 1,25-D3 binds to the vitamin D receptor (VDR), a member of the steroid receptor superfamily. Steroids, via intranuclear receptors, have been demonstrated to have high affinity binding to the nuclear matrix, the tissue specific scaffolding of the nucleus that is involved in the organization of DNA, replication and transcription. We hypothesized that the VDR interacts closely with the nuclear matrix in both human and rat tissues. In the studies described here, nuclear matrix proteins (NMP) were extracted from a number of rat and human tissues and immunoblot analysis performed using a rat anti-VDR antibody. The results from these studies reveal that the anti-VDR antibody detects six forms of the VDR in the NMP preparations: human testis demonstrated a protein of 57 and 52 kDa molecular weight compared with 57 and 37 kDa in the rat testis. Human prostate demonstrated proteins of 52 kDa compared to rat ventral (57 and 37 kDa) and dorsal prostate (52 and 26 kDa). Human and rat bladder NMP demonstrated a protein binding at 55 kDa and rat seminal vesicle NMP binding at 48 kDa. This is the first report of VDRs associated with the nuclear matrix. The varying molecular weight proteins reactive with the anti-VDR antibody within these tissues may represent different isoforms, proteolytic cleavage of a larger VDR or post-translational modification. The VDR-NMP interaction may be involved in the tissue specific actions of 1,25-D3 especially growth regulatory and antiproliferative effects.

The use of DNA probes in the identification of leishmanias: discrimination between isolates of the Leishmania mexicana and L. braziliensis complexes.

Kinetoplast DNA (kDNA), initially characterized by buoyant density, from ten reference isolates of the Leishmania braziliensis and L. mexicana complexes has been radio-actively labelled and used as hybridization probes. Filters containing endonuclease digested, electrophoresed, Southern transferred fragments of kDNA from reference and other isolates sent to us for DNA typing have been tested for kDNA sequence homology. We record a complete lack of sequence homology between kDNA of any isolate of the L. braziliensis complex and kDNA of any isolate of the L. mexicana complex. L. b. braziliensis, L. b. guyanensis and L. b. panamensis have kDNA sequences in common with each other and with a number of test isolates from Brazil, Panama, Venezuela and Peru. L. b. panamensis (1.695 g/ml) can be separated from L. b. braziliensis or L. b. guyanensis (1.691-1.693 g/ml) by differences in buoyant density of kDNA. L. m. mexicana and L. m. pifanoi have kDNA sequences in common with each other but kDNA of L. m. amazonensis has insignificant homology with kDNA of other reference isolates of the L. mexicana complex. We conclude that the kDNAs of species of the L. mexicana complex are sufficiently different from kDNA of species of the L. braziliensis complex to make kDNA sequence homology identification a feasible proposition.