Does anesthesia duration or number of cases per patient predict safety events?

BACKGROUND: The need for dental rehabilitation under general anesthesia is increasing, with varying needs between patients. Mortality has been found to be a rare event in these patients; however other perioperative events can and do occur. Previous studies have established increased incidence of perioperative events with younger, sicker children, and longer anesthetics, however, no studies to date have evaluated if the incidence of perioperative events is more closely associated with one long anesthetic or multiple anesthetics per patient. AIMS: To evaluate the association of perioperative events related to single anesthetic duration or number of anesthetics per patient for dental rehabilitation. METHODS: After Children's Wisconsin Human Research Protection Program determined this quality activity did not meet the definition of human subjects research, we performed an epidemiologic observational evaluation by extracting all dental related cases (dental alone or with oral surgeon vs. dental with other specialties) with an associated general anesthesia encounter from Children's Wisconsin electronic data warehouse from June 1, 2015 to December 31, 2021. These cases occurred at a free-standing children's hospital or associated pediatric-only ambulatory surgery center. The risk of perioperative safety events was analyzed for previously identified risk groups such as American Society of Anesthesiologists Physical Status (ASA-PS), patient age, anesthesia case time with the addition of number of dental cases per patient. RESULTS: In this study, 8468 procedures were performed on 8082 patients. Of this cohort, 7765 patients underwent one procedure for dental care while 317 patients underwent a total of 703 dental-related procedures, ranging from two to five procedures per patient. Multivariable logistic regression identified increased risk of perioperative events in patients with ASA-PS 3 (n = 1459, rate 1.78%, p value .001, OR 5.7, CI 2.1-15.5) and ASA-PS 4 (n = 86, rate 5.8%, p < .001, OR 17.2, CI 4.4-67.3), anesthesia duration (p < .001, OR 1.46, CI 1.21-1.76), but no increased risk with number of anesthetics per patient (p value .54, OR 0.81, CI 0.4-1.61). CONCLUSIONS: Limiting dental care under general anesthesia to multiple short cases may decrease the risk of perioperative events when compared to completing all treatment in one long operative session.

Rethinking the association between overweight/obesity and ADHD in children: a longitudinal and psychosocial perspective.

OBJECTIVE: This study examines the association between attention-deficit/hyperactivity disorder (ADHD) and overweight/obesity in a large-scale longitudinal study of children, while controlling for a range of psychosocial factors. METHOD: Data were obtained from Growing Up in Ireland, a nationally representative and longitudinal study of approximately 6500 children who were assessed at 9 and 13 years of age. Body mass index (BMI) was determined using measured height and weight, ADHD status was determined by parent reports of professional diagnoses and ADHD symptoms were measured using the Strengths and Difficulties Questionnaire (SDQ). RESULTS: The associations between ADHD status, ADHD symptoms (SDQ) and BMI category at age 9 and 13 years were evaluated using logistic regression. Adjustments were made for child factors (sex, developmental coordination disorder, emotional symptoms, conduct problems, birth weight and exercise) and parental factors (socio-economic status, parental BMI, parental depression, and maternal smoking and alcohol use during pregnancy). Logistic regression indicated that ADHD status was not associated with BMI category at 9 or at 13 years of age, but children with ADHD at 9 years were significantly more likely to be overweight/obese at 13 years than those without ADHD. However, when other child and parental factors were adjusted for, ADHD status was no longer significantly associated with weight status. Female sex, low levels of exercise, overweight/obese parents and prenatal smoking during pregnancy consistently increased the odds of childhood overweight/obesity. CONCLUSIONS: While ADHD and overweight/obesity co-occur in general populations, this relationship is largely explained by a variety of psychosocial factors.

Patient-reported outcomes after oesophagectomy in the multicentre LASER study.

BACKGROUND: Data on the long-term symptom burden in patients surviving oesophageal cancer surgery are scarce. The aim of this study was to identify the most prevalent symptoms and their interactions with health-related quality of life. METHODS: This was a cross-sectional cohort study of patients who underwent oesophageal cancer surgery in 20 European centres between 2010 and 2016. Patients had to be disease-free for at least 1 year. They were asked to complete a 28-symptom questionnaire at a single time point, at least 1 year after surgery. Principal component analysis was used to assess for clustering and association of symptoms. Risk factors associated with the development of severe symptoms were identified by multivariable logistic regression models. RESULTS: Of 1081 invited patients, 876 (81.0 per cent) responded. Symptoms in the preceding 6 months associated with previous surgery were experienced by 586 patients (66.9 per cent). The most common severe symptoms included reduced energy or activity tolerance (30.7 per cent), feeling of early fullness after eating (30.0 per cent), tiredness (28.7 per cent), and heartburn/acid or bile regurgitation (19.6 per cent). Clustering analysis showed that symptoms clustered into six domains: lethargy, musculoskeletal pain, dumping, lower gastrointestinal symptoms, regurgitation/reflux, and swallowing/conduit problems; the latter two were the most closely associated. Surgical approach, neoadjuvant therapy, patient age, and sex were factors associated with severe symptoms. CONCLUSION: A long-term symptom burden is common after oesophageal cancer surgery.

Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Plasma microRNA levels in male and female children with cystic fibrosis.

A gender gap exists in cystic fibrosis (CF). Here we investigate whether plasma microRNA expression profiles differ between the sexes in CF children. MicroRNA expression was quantified in paediatric CF plasma (n = 12; six females; Age range:1-6; Median Age: 3; 9 p.Phe508del homo- or heterozygotes) using TaqMan OpenArray Human miRNA Panels. Principal component analysis indicated differences in male versus female miRNA profiles. The miRNA array analysis revealed two miRNAs which were significantly increased in the female samples (miR-885-5p; fold change (FC):5.07, adjusted p value: 0.026 and miR-193a-5p; FC:2.6, adjusted p value: 0.031), although only miR-885-5p was validated as increased in females using specific qPCR assay (p < 0.0001). Gene ontology analysis of miR-885-5p validated targets identified cell migration, motility and fibrosis as processes potentially affected, with RAC1-mediated signalling featuring significantly. There is a significant increase in miR-885-5p in plasma of females versus males with CF under six years of age.

The Effect of Hydroxyurea Therapy on Adverse Clinical Events and Haematological Indices in Paediatric Patients with Sickle Cell Anaemia / Efecto de la Terapia con Hidroxiurea en los Eventos Clínicos Adversos y los Indices Haematológicos de Pacientes Paediátricos con Aanemia de Células Falciformes

ABSTRACT Objective: The aim of this study was to determine the effect of hydroxyurea on adverse clinical events and haematological indices in paediatric patients with sickle cell anaemia. Method: This study compared the same cohort of patients before and after hydroxyurea therapy, monitoring the rate of adverse events, pre- and post-treatment and haematological indices. Results: Of the 40 patients, the incidence rate of painful crises post-treatment was 80% lower than pre-treatment. Post-treatment incidence rates of painful crises managed at home, requiring emergency department care or requiring admission to the ward were also lower - 79%, 81% and 84%, respectively. There was no significant difference in the incidence of other clinical events. The haemoglobin concentration increased within the first month and plateaued while the mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) continued to increase until six months before plateauing out. The white blood cell count (WBC) and absolute neutrophil count (ANC) decreased over the first month before levels stabilized. The reticulocyte percentage and the absolute reticulocyte count (ARC) decreased over the first three months before plateauing while the platelet count remained stable. Conclusion: Hydroxyurea significantly reduced the incidence of painful crises. There were significant increases in haemoglobin, MCV and MCHC with decreases in WBC, ANC, ARC, and reticulocyte percentage while the platelet count remained relatively stable.

RESUMEN Objetivo: El objetivo de este estudio fue determinar el efecto de la hidroxiurea sobre los eventos clínicos adversos y los índices hematológicos en pacientes pediátricos con anemia falciforme. Método: Este estudio comparó una misma cohorte de pacientes antes y después del tratamiento con hidroxiurea, monitoreando la tasa de eventos adversos, el tratamiento previo y posterior, y los índices hematológicos. Resultados: En los 40 pacientes, la tasa de incidencia de postratamiento de crisis dolorosas fue 80% inferior a la del pretratamiento. Las tasas de incidencia de postratamientos de crisis dolorosas que fueron tratadas en el hogar, atendidas en el departamento de emergencias, o requirieron ingreso hospitalario, fueron también menores -79%, 81%y 84%, respectivamente. No hubo diferencias significativas en la incidencia de otros eventos clínicos. La concentración de hemoglobina aumentó en el primer mes y se estabilizó, mientras que el volumen corpuscular medio (VCM) y la concentración de hemoglobina corpuscular media (CHCM) continuaron aumentando hasta seis meses antes de estabilizarse. nivelarse. El conteo de glóbulos blancos (CGB) y el conteo absoluto de neutrófilos (CAN) disminuyeron durante el primer mes antes de que los niveles se estabilizaran. El porcentaje de reticulocitos y el conteo absoluto de reticulocitos (CAR) disminuyeron durante los primeros tres meses antes de estabilizarse, mientras que el conteo de plateletas permaneció estable. Conclusión: La hidroxiurea redujo significativamente la incidencia de crisis dolorosas. Hubo aumentos significativos de hemoglobina, VCM y CHCM con disminuciones de CGB, CAN, CAR, y porcentaje de reticulocitos mientras que el conteo plaquetario permaneció relativamente estable.

Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia.

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible 'knockdown' mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3-4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin-5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder.

Evaluation of the ability of Acinetobacter baumannii to form biofilms on six different biomedical relevant surfaces.

UNLABELLED: The human opportunistic pathogen, Acinetobacter baumannii, has the propensity to form biofilms and frequently cause medical device-related infections in hospitals. However, the physio-chemical properties of medical surfaces, in addition to bacterial surface properties, will affect colonization and biofilm development. The objective of this study was to compare the ability of A. baumannii to form biofilms on six different materials common to the hospital environment: glass, porcelain, stainless steel, rubber, polycarbonate plastic and polypropylene plastic. Biofilms were developed on material coupons in a CDC biofilm reactor. Biofilms were visualized and quantified using fluorescent staining and imaged using confocal laser scanning microscopy (CLSM) and by direct viable cell counts. Image analysis of CLSM stacks indicated that the mean biomass values for biofilms grown on glass, rubber, porcelain, polypropylene, stainless steel and polycarbonate were 0·04, 0·26, 0·62, 1·00, 2·08 and 2·70 µm(3) /µm(2) respectively. Polycarbonate developed statistically more biofilm mass than glass, rubber, porcelain and polypropylene. Viable cell counts data were in agreement with the CLSM-derived data. In conclusion, polycarbonate was the most accommodating surface for A. baumannii ATCC 17978 to form biofilms while glass was least favourable. Alternatives to polycarbonate for use in medical and dental devices may need to be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: In the hospital environment, Acinetobacter baumannii is one of the most persistent and difficult to control opportunistic pathogens. The persistence of A. baumannii is due, in part, to its ability to colonize surfaces and form biofilms. This study demonstrates that A. baumannii can form biofilms on a variety of different surfaces and develops substantial biofilms on polycarbonate - a thermoplastic material that is often used in the construction of medical devices. The findings highlight the need to further study the in vitro compatibility of medical materials that could be colonized by A. baumannii and allow it to persist in hospital settings.

Delayed hematopoietic recovery after auto-SCT in patients receiving arsenic trioxide-based therapy for acute promyelocytic leukemia: a multi-center analysis.

A potential link between arsenic (ATO)-based therapy and delayed hematopoietic recovery after autologous hematopoietic SCT (HSCT) for acute promyelocytic leukemia (APL) has previously been reported. We retrospectively reviewed the clinical histories of 58 patients undergoing autologous HSCT for APL at 21 institutions in the United States and Japan. Thirty-three (56%) of the patients received ATO-based therapy prior to stem cell collection. Delayed neutrophil engraftment occurred in 10 patients (17%): 9 of the 10 patients (90%) received prior ATO (representing 27% of all ATO-treated patients), compared with 1 of the 10 patients (10%) not previously treated with ATO (representing 4% of all ATO-naïve patients; P<0.001). Compared with ATO-naïve patients, ATO-treated patients experienced significantly longer times to ANC recovery (median 12 days vs 9 days, P<0.001). In multivariate analysis, the only significant independent predictor of delayed neutrophil engraftment was prior treatment with ATO (hazard ratio 4.87; P<0.001). Of the available stem cell aliquots from APL patients, the median viable post-thaw CD34+ cell recovery was significantly lower than that of cryopreserved autologous stem cell products from patients with non-APL AML. Our findings suggest that ATO exposure prior to CD34+ cell harvest has deleterious effects on hematopoietic recovery after autologous HSCT.

Innate immunity in cystic fibrosis lung disease.

Chronic lung disease determines the morbidity and mortality of cystic fibrosis (CF) patients. The pulmonary immune response in CF is characterized by an early and non-resolving activation of the innate immune system, which is dysregulated at several levels. Here we provide a comprehensive overview of innate immunity in CF lung disease, involving (i) epithelial dysfunction, (ii) pathogen sensing, (iii) leukocyte recruitment, (iv) phagocyte impairment, (v) mechanisms linking innate and adaptive immunity and (iv) the potential clinical relevance. Dissecting the complex network of innate immune regulation and associated pro-inflammatory cascades in CF lung disease may pave the way for novel immune-targeted therapies in CF and other chronic infective lung diseases.

Mechanisms of protein misfolding in conformational lung diseases.

Genetic or environmentally-induced alterations in protein structure interfere with the correct folding, assembly and trafficking of proteins. In the lung the expression of misfolded proteins can induce a variety of pathogenetic effects. Cystic fibrosis (CF) and alpha-1 antitrypsin (AAT) deficiency are two major clinically relevant pulmonary disorders associated with protein misfolding. Both are genetic diseases the primary causes of which are expression of mutant alleles of the cystic fibrosis transmembrane conductance regulator (CFTR) and SERPINA1, respectively. The most common and best studied mutant forms of CFTR and AAT are ΔF508 CFTR and the Glu342Lys mutant of AAT called ZAAT, respectively. Non-genetic mechanisms can also damage protein structure and induce protein misfolding in the lung. Cigarette-smoke contains oxidants and other factors that can modify a protein's structure, and is one of the most significant environmental causes of protein damage within the lung. Herein we describe the mechanisms controlling the folding of wild type and mutant versions of CFTR and AAT proteins, and explore the consequences of cigarette-smoke-induced effects on the protein folding machinery in the lung.

CFTR mutation analysis and haplotype associations in CF patients.

Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies.

Vitamin D receptor agonists inhibit pro-inflammatory cytokine production from the respiratory epithelium in cystic fibrosis.

BACKGROUND: 1,25-Dihydroxycholecalciferol (1,25(OH)(2)D(3)) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF. METHODS: CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH)(2)D(3) or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH)(2)D(3). RESULTS: Pretreatment with 1,25(OH)(2)D(3) was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH)(2)D(3) treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH)(2)D(3) was associated with an increase in hCAP18/LL-37 mRNA and protein levels. CONCLUSIONS: Both 1,25(OH)(2)D(3) and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF.

High concentrations of pepsin in bronchoalveolar lavage fluid from children with cystic fibrosis are associated with high interleukin-8 concentrations.

BACKGROUND: Gastro-oesophageal reflux is common in children with cystic fibrosis (CF) and is thought to be associated with pulmonary aspiration of gastric contents. The measurement of pepsin in bronchoalveolar lavage (BAL) fluid has recently been suggested to be a reliable indicator of aspiration. The prevalence of pulmonary aspiration in a group of children with CF was assessed and its association with lung inflammation investigated. METHODS: This was a cross-sectional case-control study. BAL fluid was collected from individuals with CF (n=31) and healthy controls (n=7). Interleukin-8 (IL-8), pepsin, neutrophil numbers and neutrophil elastase activity levels were measured in all samples. Clinical, microbiological and lung function data were collected from medical notes. RESULTS: The pepsin concentration in BAL fluid was higher in the CF group than in controls (mean (SD) 24.4 (27.4) ng/ml vs 4.3 (4.0) ng/ml, p=0.03). Those with CF who had raised pepsin concentrations had higher levels of IL-8 in the BAL fluid than those with a concentration comparable to controls (3.7 (2.7) ng/ml vs 1.4 (0.9) ng/ml, p=0.004). Within the CF group there was a moderate positive correlation between pepsin concentration and IL-8 in BAL fluid (r=0.48, p=0.04). There was no association between BAL fluid pepsin concentrations and age, sex, body mass index z score, forced expiratory volume in 1 s or Pseudomonas aeruginosa colonisation status. CONCLUSIONS: Many children with CF have increased levels of pepsin in the BAL fluid compared with normal controls. Increased pepsin levels were associated with higher IL-8 concentrations in BAL fluid. These data suggest that aspiration of gastric contents occurs in a subset of patients with CF and is associated with more pronounced lung inflammation.

Evaluation of brain tissue or cerebrospinal fluid with broadly reactive polymerase chain reaction for Ehrlichia, Anaplasma, spotted fever group Rickettsia, Bartonella, and Borrelia species in canine neurological diseases (109 cases).

BACKGROUND: Vector-transmitted microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, Bartonella, and Borrelia are commonly suspected in dogs with meningoencephalomyelitis (MEM), but the prevalence of these pathogens in brain tissue and cerebrospinal fluid (CSF) of dogs with MEM is unknown. HYPOTHESIS/OBJECTIVES: To determine if DNA from these genera is present in brain tissue and CSF of dogs with MEM, including those with meningoencephalitis of unknown etiology (MUE) and histopathologically confirmed cases of granulomatous (GME) and necrotizing meningoencephalomyelitis (NME). ANIMALS: Hundred and nine dogs examined for neurological signs at 3 university referral hospitals. METHODS: Brain tissue and CSF were collected prospectively from dogs with neurological disease and evaluated by broadly reactive polymerase chain reaction (PCR) for Ehrlichia, Anaplasma, Spotted Fever Group Rickettsia, Bartonella, and Borrelia species. Medical records were evaluated retrospectively to identify MEM and control cases. RESULTS: Seventy-five cases of MUE, GME, or NME, including brain tissue from 31 and CSF from 44 cases, were evaluated. Brain tissue from 4 cases and inflammatory CSF from 30 cases with infectious, neoplastic, compressive, vascular, or malformative disease were evaluated as controls. Pathogen nucleic acids were detected in 1 of 109 cases evaluated. Specifically, Bartonella vinsonii subsp. berkhoffii DNA was amplified from 1/6 dogs with histopathologically confirmed GME. CONCLUSION AND CLINICAL IMPORTANCE: The results of this investigation suggest that microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, and Borrelia are unlikely to be directly associated with canine MEM in the geographic regions evaluated. The role of Bartonella in the pathogenesis of GME warrants further investigation.

Role of latent feline leukemia virus infection in nonregenerative cytopenias of cats.

BACKGROUND: Nonregenerative cytopenias such as nonregenerative anemia, neutropenia, and thrombocytopenia in cats with feline leukemia virus (FeLV) antigen are assumed to be caused by the underlying FeLV infection. In addition, cats with negative FeLV antigen-test results that have cytopenias of unknown etiology often are suspected to suffer from latent FeLV infection that is responsible for the nonregenerative cytopenias. OBJECTIVE: The purpose of this study was to assess the role of latent FeLV infection by polymerase chain reaction (PCR) in bone marrow of cats with nonregenerative cytopenias that had negative FeLV antigen test results in blood. ANIMALS: Thirty-seven cats were included in the patient group. Inclusion criteria were (1) nonregenerative cytopenia of unknown origin and (2) negative FeLV antigen test result. Antigenemia was determined by detection of free FeLV p27 antigen by ELISA in serum. Furthermore, 7 cats with positive antigen test results with nonregenerative cytopenia were included as control group I, and 30 cats with negative antigen test results without nonregenerative cytopenia were included as control group II. METHODS: Whole blood and bone marrow samples were tested by 2 different PCR assays detecting sequences of the envelope or long terminal repeat genes. FeLV immunohistochemistry was performed in bone marrow samples. RESULTS: Two of the 37 cats (5.4%) in the patient group were positive on the bone marrow PCR results and thus were latently infected with FeLV. CONCLUSIONS AND CLINICAL IMPORTANCE: The findings of this study suggest that FeLV latency is rare in cats with nonregenerative cytopenias.

Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Immunofluorescent synaptonemal complex analysis in azoospermic men.

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs.

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.

Association of IL-10 polymorphism with severity of illness in community acquired pneumonia.

BACKGROUND: The influence of genetic polymorphisms of interleukin (IL)-10, tumour necrosis factor (TNF)-alpha, and IL-6 gene promoters on severity of systemic inflammatory response syndrome (SIRS) associated with community acquired pneumonia (CAP) was studied. METHODS: Using PCR-RFLP analysis we analysed a -1082G/A single nucleotide polymorphism (SNP) of the anti-inflammatory IL-10 gene, a -308G/A SNP of the pro-inflammatory TNF-alpha gene and a -174G/C SNP of the IL-6 gene. Illness severity was stratified according to SIRS score, calculated by presence of up to four physiological indices: temperature, white blood cell count, heart rate and respiratory rate (non-SIRS, SIRS 2, SIRS 3, and SIRS 4). RESULTS: A statistically significant stepwise increase in frequency of the IL-10 G allele, associated with higher expression of the gene, was observed in patients with increasing severity of illness from non-SIRS (n=19) to SIRS 2 (n=17), SIRS 3 (n=33) and SIRS 4 (n=24). This was primarily due to a higher frequency of the GG genotype with increasing severity from non-SIRS through to SIRS 4. IL-10 G allele frequency was also increased in patients who died as a result of CAP (n=11) compared with CAP survivors (n=82) (p=0.01). No association was seen between the TNF-alpha -308G/A and IL-6 -174G/C SNPs and disease. Additionally, no interaction between all three SNP genotypes and disease severity was observed. CONCLUSIONS: A polymorphism affecting IL-10 expression may influence the severity of illness in patients with CAP.