Inhibition of mast cell degranulation by novel small molecule MRGPRX2 antagonists.

BACKGROUND: Mas-related G protein-coupled receptor X2 (MRGPRX2) is a promiscuous receptor on mast cells that mediates IgE-independent degranulation and has been implicated in multiple mast cell-mediated disorders, including chronic urticaria, atopic dermatitis, and pain disorders. Although it is a promising therapeutic target, few potent, selective, small molecule antagonists have been identified, and functional effects of human MRGPRX2 inhibition have not been evaluated in vivo. OBJECTIVE: We sought to identify and characterize novel, potent, and selective orally active small molecule MRGPRX2 antagonists for potential treatment of mast cell-mediated disease. METHODS: Antagonists were identified using multiple functional assays in cell lines overexpressing human MRGPRX2, LAD2 mast cells, human peripheral stem cell-derived mast cells, and isolated skin mast cells. Skin mast cell degranulation was evaluated in Mrgprb2em(-/-) knockout and Mrgprb2em(MRGPRX2) transgenic human MRGPRX2 knock-in mice by assessment of agonist-induced skin vascular permeability. Ex vivo skin mast cell degranulation and associated histamine release was evaluated by microdialysis of human skin tissue samples. RESULTS: MRGPRX2 antagonists potently inhibited agonist-induced MRGPRX2 activation and mast cell degranulation in all mast cell types tested in an IgE-independent manner. Orally administered MRGPRX2 antagonists also inhibited agonist-induced degranulation and resulting vascular permeability in MRGPRX2 knock-in mice. In addition, antagonist treatment dose dependently inhibited agonist-induced degranulation in ex vivo human skin. CONCLUSIONS: MRGPRX2 small molecule antagonists potently inhibited agonist-induced mast cell degranulation in vitro and in vivo as well as ex vivo in human skin, supporting potential therapeutic utility as a novel treatment for multiple human diseases involving clinically relevant mast cell activation.

Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats.

Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 8 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (> 7 years), measuring glutathione, and markers of oxidative stress. Whole blood and erythrocyte total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared with their younger counterparts (P ≤ 0·02). The inclusion level study identified 1·5 % free GLY for the subsequent dry diet feeding study. Significant increases in erythrocyte total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P ≤ 0·03; maximum difference of 1·23 µM). Oxidative stress markers were also significantly different between groups at 8 (P = 0·004; difference of 0·68 nG/ml in 8-hydroxy-2'-deoxyguanosine) and 12 weeks (P ≤ 0·049; maximum difference of 0·62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared with younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1·5 % free GLY for 12 weeks elevated initial erythrocyte glutathione and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.

AN OUTBREAK OF FELINE INFECTIOUS PERITONITIS IN THREE RELATED SAND CATS (FELIS MARGARITA) IN HUMAN CARE.

Feline infectious peritonitis (FIP) is a systemic disease in felid species caused by infection with mutated forms of feline coronavirus (FCoV), and outbreaks can devastate exotic felid populations in human care. Feline infectious peritonitis was diagnosed in three of four related juvenile sand cats (Felis margarita) from a single institution over a 6-wk period. Case 1 was a 7-mon-old male found deceased with no premonitory signs. Case 2, an 8-mon-old male (littermate to Case 1), and Case 3, a 6-mon-old male (from a different litter with identical parentage), were evaluated for lethargy and anorexia 1 mon after Case 1. Both exhibited transient anisocoria and progressive lethargy, anorexia, and dehydration despite antibiotic and supportive treatment. Approximately 1 wk after initial presentation, Case 2 was humanely euthanized, and Case 3 was found deceased. Necropsy findings included intrathoracic and/or intra-abdominal lymphadenopathy (3/3 cases), bicavitary effusion (2/3), multifocal tan hepatic and intestinal nodules (1/3), and multifocal yellow renal nodules (1/3). Histologically, all cats had severe pyogranulomatous vasculitis in multiple organs, and the presence of FCoV antigen was confirmed using immunohistochemical staining. Next-generation sequencing of the virus from Case 3's affected kidney demonstrated ∼93% homology to the UG-FH8 virus, a serotype 1 feline alphacoronavirus isolated from Denmark. Future research will focus on comparative viral genomic sequencing with the goals of identifying potential sources of FCoV infection and identifying features that may have contributed to the development of FIP in this species.

American Society for Veterinary Clinical Pathology-Recommended Clinical Pathology Competencies for Graduating Veterinarians.

Given the move toward competency-based veterinary education and the subsequent reevaluation of veterinary curricula, there is a need for specialties to provide guidance to veterinary college administrators and educators on the core knowledge and skills pertaining to their specialty to ensure their inclusion in revised or redesigned curricula. The American Society for Veterinary Clinical Pathology (ASVCP) Education Committee sought to create a list of competencies specific to clinical pathology expected of graduating veterinarians. The stimulus for this project was the American Veterinary Medical Association Council on Education Standards of Accreditation for Colleges of Veterinary Medicine, further driven by the 2018 publication of the Association of American Veterinary Medical Colleges Competency-Based Veterinary Education Working Group framework. The recommendations made in this document are the culmination of the 2016 ASVCP Education Forum for Discussion, multiple remote subcommittee communications, and feedback obtained from ASVCP membership. The final framework includes 8 clinical pathology-focused domains of competence with 20 clinical pathology competencies and 61 clinical pathology illustrative sub-competencies. The clinical pathology-focused domains of competence are: the pre-analytical phase of testing, laboratory medicine and instrumentation, principles of test selection and interpretation, hematology and hemostasis, chemistry, endocrinology, urinalysis, and cytology. These are not intended to replace the nine established AAVMC domains of competence with supportive competencies and illustrative sub-competencies but to guide institutions for how clinical pathology aligns within the competency-based veterinary education (CBVE) framework for the practice-ready veterinary graduate. This clinical pathology competency framework may prove useful and empowering during discussions of curriculum revisions and redesigns.

Multiple Myeloma with Aberrant CD3 Expression in a Red-Lored Amazon Parrot (Amazona autumnalis).

A 20-year-old, female, red-lored Amazon parrot (Amazona autumnalis) was presented for a 2-week history of weakness. On physical examination, the bird was quiet, fluffed, weak, and had a distended coelom. Radiographic and ultrasound imaging revealed coelomic distention, increased pulmonary parenchymal opacity, renomegaly, dilated intestines, and a thickened ventricular wall. The results of a complete blood cell count indicated the patient was anemic (28%) and had intermediate to large lymphocytes with immature chromatin that were suspected to be neoplastic. Immunocytochemistry on peripheral blood determined that the suspected circulating neoplastic cells were cluster of differentiation (CD) 3+ and occasionally expressed multiple myeloma oncogene 1 (MUM1). Abnormalities from a plasma biochemistry panel were moderate hyperphosphatemia (6.8 mg/dL), marked hyperproteinemia (13.6 g/L), analbuminemia (0 g/dL), and marked hyperglobulinemia (13.6 g/dL). Agarose gel plasma protein electrophoresis documented the presence of albumin (1.2 g/dL) and monoclonal bands which, on reduced lithium dodecyl sulfate polyacrylamide gel electrophoresis, resolved as 60-kd and ∼25-kd bands consistent with immunoglobulin Y heavy and light chains. On the basis of these findings, multiple myeloma was diagnosed. Because of a poor prognosis, the bird was euthanized for postmortem examination. Bone marrow cytology from samples collected during the postmortem examination revealed 17.4% plasma cells and 24% large immature cells with occasional plasmacytoid features. Histopathologic findings included aggregates of neoplastic plasma cells in the bone marrow, spleen, kidney, liver, gastrointestinal tract, muscle, ovary, and brain. The neoplastic cells were strongly immunoreactive for MUM1 and cluster of differentiation 3 (CD3), but negative for CD79a, paired box protein 5, and CD20. This confirmed the clinical diagnosis of multiple myeloma. This report describes an avian immunoglobulin Y-secreting multiple myeloma with aberrant CD3 expression and pseudoanalbuminemia. Aberrant CD3 expression by avian multiple myeloma may explain previously published cases of birds with a monoclonal gammopathy and apparent T-cell lymphoma diagnosed by CD3 immunoreactivity.

Presentation, management and outcomes of ileoanal pouch cancer: a single-centre experience.

AIM: This study aimed to determine the clinical presentation, management and outcomes for patients with ileoanal pouch cancer. METHOD: Patients who were diagnosed with ileoanal pouch cancer were identified from our polyposis registry (1978-2019) and operative and referral records (2006-2019). Details of presentation, endoscopic surveillance, cancer staging and management were retrieved from hospital records. RESULTS: Eighteen patients were identified (12 with ulcerative colitis, one with Crohn's disease, three with familial adenomatous polyposis [FAP], two with dual diagnosis of FAP and inflammatory bowel disease). The median time from pouch formation to cancer diagnosis was 16.5 years (range 5-34 years) and the median age of the patient at pouch cancer diagnosis was 54 years (range 35-71 years). Eleven of the 18 patients were undergoing surveillance. Four of five FAP patients developed pouch cancer whilst on surveillance. Eight patients were asymptomatic at the time of pouch cancer diagnosis. Two patients had complete clinical response following chemoradiotherapy. Fourteen patients underwent pouch excision surgery (eight with exenteration). Median survival was 54 months; however, only eight patients had outcomes available beyond 24 months follow-up. CONCLUSIONS: Pouch cancer can occur in patients despite routine surveillance and without symptoms, and survival is poor. Centralization of 'high-risk' patients who require surveillance is recommended and a low threshold for referral to centres that can provide expert investigation and management is advised.

Virtual Microscopy Is More Effective Than Conventional Microscopy for Teaching Cytology to Veterinary Students: A Randomized Controlled Trial.

Virtual microscopy (VM) using scanned slides and imaging software is increasingly used in medical curricula alongside instruction in conventional microscopy (CM). Limited reports suggest that VM is useful in the veterinary education setting, and generally well-received by students. Whether students can apply knowledge gained through VM to practical use is unknown. Our objective was to determine whether instruction using VM, compared to CM, is a successful method of training veterinary students for the application of cytology in practice (i.e., using light microscopes). Seventy-one veterinary students from Colorado State University who attended a voluntary 3-hour cytology workshop were randomized to receive the same instruction with either VM (n = 35) or CM (n = 36). We compared these students to a control group (n = 22) of students who did not attend a workshop. All students took a post-workshop assessment involving the interpretation of four cases on glass slides with CM, designed to simulate the use of cytology in general practice. Students also took an 18-question survey related to the effectiveness of the workshop, providing their opinions on cytology instruction in the curriculum and their learning preference (VM or CM). The mean assessment score of the VM group (14.18 points) was significantly higher than the control group (11.33 points, p = .003), whereas the mean of the CM group (12.77 points) was not statistically significantly different from controls (p = .170). Not only is VM an effective method of teaching cytology to veterinary students that can be translated to a real-world case scenario, but it outperformed CM instruction in this study.

The Measure of DAMPs and a role for S100A8 in recruiting suppressor cells in breast cancer lung metastasis.

To determine whether there was a relationship between damage associated molecular pattern molecule (DAMP) expression and recruitment of suppressor cells to sites of metastasis we measured relative expression of DAMPs, regulatory T cells (Tregs), and myeloid derived suppressor cells (MDSC) in mice at various stages of breast cancer progression using the 4T1 model. Although S100A8 was expressed at relatively low levels in the tumor cells, expression was 100-fold higher in the lung and liver which are common sites of metastasis for this tumor. Despite the relatively high level of S100A8 expression in the lungs of naïve mice, the level of expression increased further and was significantly elevated after only 7 days of tumor growth. The same pattern was observed for MDSC, and both S100A8 and MDSC expression peaked in the lungs of mice following 21 days of tumor growth. Characterization of MDSC from the lungs revealed expression of RAGE, and the cells were capable of migrating in a dose-dependent manner toward S100A8. In addition, the MDSC expressed low levels of MHC Class I, MHC Class II, CD80, and secreted TGF-ß. Taken together, these data suggest that expression of S100A8 in the lungs may facilitate recruitment of MDSC, which may in turn aid in establishing a metastatic niche capable of suppressing a localized immune response.

Owner experience and veterinary involvement with unlicensed GS-441524 treatment of feline infectious peritonitis: a prospective cohort study.

Introduction: Feline Infectious Peritonitis (FIP) has historically been a fatal coronavirus disease in cats. In recent years, the therapeutic agent GS-441524, developed by Gilead Sciences, was found to be a successful treatment for FIP in most patients in clinical trials. However, this particular drug has remained stalled in the therapeutic pipeline, leaving patients and cat owners without a licensed medication. In the meantime, online social media platforms began to emerge, connecting cat owners with a community of citizen non-veterinary professionals sourcing unlicensed GS-441524. Methods: This study prospectively followed participants (N = 141) that successfully completed 12 weeks of treatment, capturing their treatment experiences with self-administered GS-441524-like medication. A one-time survey was administered to enrolled participants with mixed format of questions (open-ended and multiple-choice) asking about treatment administration techniques, observed side effects of GS-441524, accrued cost, veterinarian involvement, impact on the cat-human bond, and social media usage. Results: Our results show cat owners experienced a shift in treatment modality from injectable GS-441524 to pill formulation across the treatment period. The average total cost of medication has decreased since 2021 to approximately USD 3100, and participants reported the human-animal bond being affected negatively. Additionally, there was an increased trend in veterinarian awareness of GS-441524-like therapeutics and monitoring of clients undergoing treatment. Social media usage was reported as being important at the beginning of treatment to establish treatment administration but lessened by the end of treatment. Discussion: This study is the first detailed, prospective account of owner experiences with unlicensed GS-441524, raising an important discussion surrounding citizen veterinary medicine.

Preliminary evaluation of ionized magnesium levels in dogs on long-term esomeprazole administration.

OBJECTIVE: To investigate the level of whole-blood ionized magnesium (iMg) in dogs with long-term use of proton pump inhibitors (PPIs). METHODS: The study included 10 client-owned dogs with esomeprazole administration over 6 months and 62 healthy dogs to determine de novo reference interval (RI) of iMg. Dogs that received esomeprazole for 6 months or longer were retrospectively reviewed to determine the incidence of hypo- or hypermagnesemia based on the de novo RI. Additionally, the iMg levels from the study group were compared with those of 20 age-, sex-, and body weight-matched controls from the 62 dogs. RESULTS: The median (range) duration of esomeprazole usage was 26 months (6 to 94). The de novo RI for iMg was determined as 0.73 (90% CI, 0.58 to 0.87) to 1.43 mg/dL (90% CI, 1.33 to 1.46). Based on the RI, none of the dogs with long-term esomeprazole developed hypo- or hypermagnesemia. The iMg from the matched control group was 1.17 mg/dL (90% CI, 0.83 to 1.46). The lowest iMg after 6 months of esomeprazole administration (90% CI, 0.96 mg/dL, 0.87 to 1.41) was significantly lower than the control group (P = .031). The iMg measured at the end of long-term esomeprazole treatment was 1.03 mg/dL (90% CI, 0.87 to 1.41) and not significantly different from the control group (P = .179). CONCLUSIONS: Ionized hypomagnesemia was not observed after long-term use of esomeprazole in the small number of dogs included in this study. Robust RI needs to be determined in future studies to investigate the incidence of hypomagnesemia in dogs with long-term use of PPIs. CLINICAL RELEVANCE: Future studies in a larger number of dogs are warranted to confirm the findings from the present study and to determine whether the long-term use of esomeprazole in dogs is at risk of developing ionized hypomagnesemia.

Unlicensed antiviral products used for the at-home treatment of feline infectious peritonitis contain GS-441524 at significantly different amounts than advertised.

OBJECTIVE: To analyze the content of unlicensed GS-441524-like products being used as a largely successful at-home treatment for cats suspected to have FIP. The remdesivir content and pH were also measured. SAMPLE: 127 injectable and oral samples from 30 of the most popular brands of black market producers. METHODS: Unlicensed GS-441524-like products were procured through donations and tested for GS-441524 and remdesivir content by liquid chromatography with tandem mass spectrometry. A pH meter measured the pH of injectable samples. RESULTS: Of the 87 injectable formulations, 95% contained more (on average 39% more) GS-441524 than expected based on the producer's marketed concentrations. The average pH (1.30 pH) was well below the physiologic pH conditions recommended for SC injections. The oral formulations were more variable, with 43% containing more GS-441524 (on average 75% more) than expected and 58% containing less (on average 39% less) than the expected content. There was minimal variability in GS-441524 content between replicate samples in the injectables formulations (measured by coefficient of variation). One injectable and 2 oral samples additionally contained remdesivir. CLINICAL RELEVANCE: All unlicensed products used for the at-home treatment of FIP that we tested contain GS-441524. The injectables generally contain significantly more drug than advertised at a below-physiologic pH. Unlicensed oral products vary more widely in drug content and suffer from unconventional dosing and labeling. These data should highlight the need for regulation of these products and the development of legal pathways to procure GS-441524.

An Aptamer-Based Proteomic Analysis of Plasma from Cats (Felis catus) with Clinical Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Guidelines for resident training in veterinary clinical pathology. IV: Laboratory quality management-Teaching domains, competencies, and suggested learning outcomes.

BACKGROUND: The 2019 ASVCP Education Committee Forum for Discussion, presented at the annual ASVCP/ACVP meeting, identified a need to develop recommendations for teaching laboratory quality management principles in veterinary clinical pathology residency training programs. OBJECTIVES: To present a competency-based framework for teaching laboratory quality management principles in veterinary clinical pathology residency training programs, including entrustable professional activities (EPAs), domains of competence, individual competencies, and learning outcomes. METHODS: A joint subcommittee of the ASVCP Quality Assurance and Laboratory Standards (QALS) and Education Committees executed this project. A draft guideline version was reviewed by the ASVCP membership and shared with selected ACVP committees in early 2022, and a final version was voted upon by the full QALS and Education Committees in late 2022. RESULTS: Eleven domains of competence with relevant individual competencies were identified. In addition, suggested learning outcomes and resource lists were developed. Domains and individual competencies were mapped to six EPAs. CONCLUSIONS: This guideline presents a framework for teaching principles of laboratory quality management in veterinary clinical pathology residency training programs and was designed to be comprehensive yet practical. Guidance on pedagogical terms and possible routes of implementation are included. Recommendations herein aim to improve and support resident training but may require gradual implementation, as programs phase in necessary expertise and resources. Future directions include the development of learning milestones and assessments and consideration of how recommendations intersect with the American College of Veterinary Pathologists training program accreditation and certifying examination.

The molecular evolution of cancer associated genes in mammals.

Cancer is a disease that many multicellular organisms have faced for millions of years, and species have evolved various tumour suppression mechanisms to control oncogenesis. Although cancer occurs across the tree of life, cancer related mortality risks vary across mammalian orders, with Carnivorans particularly affected. Evolutionary theory predicts different selection pressures on genes associated with cancer progression and suppression, including oncogenes, tumour suppressor genes and immune genes. Therefore, we investigated the evolutionary history of cancer associated gene sequences across 384 mammalian taxa, to detect signatures of selection across categories of oncogenes (GRB2, FGL2 and CDC42), tumour suppressors (LITAF, Casp8 and BRCA2) and immune genes (IL2, CD274 and B2M). This approach allowed us to conduct a fine scale analysis of gene wide and site-specific signatures of selection across mammalian lineages under the lens of cancer susceptibility. Phylogenetic analyses revealed that for most species the evolution of cancer associated genes follows the species' evolution. The gene wide selection analyses revealed oncogenes being the most conserved, tumour suppressor and immune genes having similar amounts of episodic diversifying selection. Despite BRCA2's status as a key caretaker gene, episodic diversifying selection was detected across mammals. The site-specific selection analyses revealed that the two apoptosis associated domains of the Casp8 gene of bats (Chiroptera) are under opposing forces of selection (positive and negative respectively), highlighting the importance of site-specific selection analyses to understand the evolution of highly complex gene families. Our results highlighted the need to critically assess different types of selection pressure on cancer associated genes when investigating evolutionary adaptations to cancer across the tree of life. This study provides an extensive assessment of cancer associated genes in mammals with highly representative, and substantially large sample size for a comparative genomic analysis in the field and identifies various avenues for future research into the mechanisms of cancer resistance and susceptibility in mammals.

Fibroblasts influence muscle progenitor differentiation and alignment in contact independent and dependent manners in organized co-culture devices.

Myoblasts are precursor muscle cells that lie nascent to mature skeletal muscle. Once muscle is damaged, these cells migrate, fuse, and regenerate the muscle tissue. It is known that skeletal muscle can partially regenerate in vivo after muscle tissue damage. However, this regeneration does not always occur, especially in more severe injuries. Cellular therapy using tissue-engineering approaches has been shown to improve organ repair and function. To exploit potential benefits of using cell therapy as an avenue for skeletal muscle repair, it is important to understand the cellular dynamics underlying skeletal myocyte formation and growth. Cardiac fibroblasts have been shown to have a major influence on cardiomyocyte function, repair, and overall spatial distribution. However, little is known regarding fibroblasts' role on skeletal myocyte function. In this study, we utilized a reconfigurable co-culture device to understand the contact and paracrine effects of fibroblasts on skeletal myocyte alignment and differentiation using murine myoblast and fibroblast cell lines. We demonstrate that myotube alignment is increased by direct contact with fibroblasts, while myotube differentiation is reduced both in the gap and contact configurations with fibroblasts after 6 days of co-culture. Furthermore, neutralizing antibodies to FGF-2 can block these effects of fibroblasts on myotube differentiation and alignment. Finally, bi-directional signaling is critical to the observed myoblast-fibroblast interactions, since conditioned media could not reproduce the same effects observed in the gap configuration. These findings could have direct implications on cell therapies for repairing skeletal muscle, which have only utilized skeletal myoblasts or stem cell populations alone.

Flow Cytometry in Veterinary Practice.

This article summarizes the current applications of flow cytometry in clinical veterinary medicine, which is largely restricted to the diagnosis of hematopoietic neoplasms (lymphomas and leukemias) of domestic dogs, cats, and horses. A brief background on the technique of flow cytometry and fundamentals of data interpretation are included. Major emphasis is placed on clinical indications for flow cytometry, principles of sample collection and submission, and awareness of diagnostic and prognostic utility. Expectations regarding both the benefits and limitations of flow cytometry in a clinical setting, and its complementary nature with other types of testing, are also reviewed.

The Late Asymptomatic and Terminal Immunodeficiency Phases in Experimentally FIV-Infected Cats-A Long-Term Study.

Feline immunodeficiency virus (FIV) is a lentivirus in the family Retroviridae that infects domestic cats resulting in an immunodeficiency disease featuring a progressive and profound decline in multiple sets of peripheral lymphocytes. Despite compelling evidence of FIV-associated immunopathology, there are conflicting data concerning the clinical effects of FIV infection on host morbidity and mortality. To explore FIV-associated immunopathogenesis and clinical disease, we experimentally inoculated a cohort of four specific pathogen-free kittens with a biological isolate of FIV clade C and continuously monitored these animals along with two uninfected control animals for more than thirteen years from the time of inoculation to the humane euthanasia endpoint. Here, we report the results obtained during the late asymptomatic and terminal phases of FIV infection in this group of experimentally FIV-infected cats.

Xenon augmented hypothermia reduces early lactate/N-acetylaspartate and cell death in perinatal asphyxia.

OBJECTIVE: Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia-ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry. METHODS: Thirty-six newborn piglets were randomized (all groups n = 9), with intervention from 2 to 26 hours, to: (1) normothermia; (2) normothermia + 24 hours 50% inhaled xenon; (3) 24 hours hypothermia (33.5°C); or (4) 24 hours hypothermia (33.5°C) + 24 hours 50% inhaled xenon. Serial MRS was acquired before, during, and up to 48 hours after hypoxia-ischemia. RESULTS: Mean arterial blood pressure was lower in all treatment groups compared with normothermia (p < 0.01) (although >40mmHg); the combined therapy group required more fluid boluses (p < 0.05) and inotropes (p < 0.001). Compared with no intervention, both hypothermia and xenon-augmented hypothermia reduced the temporal regression slope magnitudes for phosphorus-MRS inorganic phosphate/exchangeable phosphate pool (EPP) and phosphocreatine/EPP (both p < 0.05); for lactate/N-acetylaspartate (NAA), only xenon-augmented hypothermia reduced the slope (p < 0.01). Xenon-augmented hypothermia also reduced transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)(+) nuclei and caspase 3 immunoreactive cells in parasagittal cortex and putamen and increased microglial ramification in midtemporal cortex compared with the no treatment group (p < 0.05). Compared with hypothermia, however, combination treatment did not reach statistical significance for any measure. Lactate/NAA showed a strong positive correlation with TUNEL; nucleotide triphosphate/EPP showed a strong negative correlation with microglial ramification (both p < 0.01). INTERPRETATION: Compared with no treatment, xenon-augmented hypothermia reduced cerebral MRS abnormalities and cell death markers in some brain regions. Compared with hypothermia, xenon-augmented hypothermia did not reach statistical significance for any measure. The safety and possible improved efficacy support phase II trials.

Examining organizational change in primary care practices: experiences from using ethnographic methods.

BACKGROUND: Qualitative methods are an important part of the primary care researcher's toolkit providing a nuanced view of the complexity in primary care reform and delivery. Ethnographic research is a comprehensive approach to qualitative data collection, including observation, in-depth interviews and document analysis. Few studies have been published outlining methodological issues related to ethnography in this setting. OBJECTIVE: This paper examines some of the challenges of conducting an ethnographic study in primary care setting in Canada, where there recently have been major reforms to traditional methods of organizing primary care services. METHODS: This paper is based on an ethnographic study set in primary care practices in Ontario, Canada, designed to investigate changes to organizational and clinical routines in practices undergoing transition to new, interdisciplinary Family Health Teams (FHTs). The study was set in six new FHTs in Ontario. This paper is a reflexive examination of some of the challenges encountered while conducting an ethnographic study in a primary care setting. RESULTS: Our experiences in this study highlight some potential benefits of and difficulties in conducting an ethnographic study in family practice. Our study design gave us an opportunity to highlight the changes in routines within an organization in transition. A study with a clinical perspective requires training, support, a mixture of backgrounds and perspectives and ongoing communication. CONCLUSIONS: Despite some of the difficulties, the richness of this method has allowed the exploration of a number of additional research questions that emerged during data analysis.

Diagnostic accuracy of cytology for the detection of bacterial infection in fluid samples from veterinary patients.

BACKGROUND: Bacterial sepsis is a relatively common, life-threatening condition with a high case fatality rate. The current primary diagnostic tools for detecting bacterial infection in fluids are bacterial culture and fluid cytology. While culture is the gold standard, it can take up to several days for results to be made available to clinicians, which can delay recognition of bacterial sepsis and negatively impact patient outcomes. OBJECTIVES: The aim of this study was to evaluate the diagnostic accuracy of cytology for detecting bacterial infection in body fluids. METHODS: We retrospectively reviewed 10 years of medical records at the Ohio State University's Veterinary Medical Center for mammalian patients with both cytology and bacterial culture of fluid samples, including body cavity fluids (abdominal and thoracic effusion), blood, joint fluid, and CSF. The overall sensitivity and specificity of cytology relative to the reference method of bacterial culture was recorded, as well as among the subcategories of fluid type. RESULTS: The overall sensitivity and specificity of cytology for the diagnosis of sepsis were 42.6% and 93.0%, respectively. Individual sensitivities and specificities were also calculated for each fluid type. Thoracic fluid cytology had relatively high sensitivity and low specificity, in contrast to the other fluid types analyzed. CONCLUSIONS: Overall, cytology is poorly sensitive but highly specific for the detection of bacterial infection in fluid samples. The results from this study will allow a better comparison between the diagnostic accuracy of cytology and emerging diagnostic tests for the detection of bacterial sepsis in mammalian patients.