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Purpose: Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. Experimental Design: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT. Results: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo. Conclusions: Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Objectives Thoracic CT is commonly performed in cats and has been shown to be more sensitive than radiography for the detection of lymphadenopathy. However, no studies to date have reported the normal CT appearance of feline intrathoracic lymph nodes. The aim of this study was to describe the distribution, number, size and attenuation of presumed normal intrathoracic lymph nodes in cats. Methods Retrospective analysis of the imaging archive in a teaching hospital identified cats that had undergone thoracic CT and were deemed to have no evidence of either intra- or extrathoracic disease that would influence the size of the intrathoracic lymph nodes. Lymph nodes were classified into lymph centres based on anatomical references. Descriptions of the position of each node, number of nodes in each centre, size of each node and attenuation pre- and post-contrast were recorded. Statistical analysis aimed to assess for correlations between these factors and the age, weight and sex of the subjects. Results Twenty cats were included in the study. One or more lymph nodes were identified in the sternal (n = 19/20), cranial mediastinal (n = 14/20) and tracheobronchial (n = 15/20) centres, while none were identified in other locations. Size varied between lymph centres and within lymph centres where more than one lymph node was identified. Tracheobronchial and sternal nodes were consistent in location, while mediastinal nodes were variable. All nodes were round to ellipsoid in shape and showed moderate enhancement post-contrast. No significant correlations were found with age, weight or sex. Conclusions and relevance This study describes the CT characteristics of presumed normal intrathoracic lymph nodes in cats, and only identified nodes in the sternal, cranial mediastinal and tracheobronchial locations. This study serves as a reference point for CT analysis of feline thoracic lymph nodes.

OBJECTIVES To describe the CT appearance of anal sac adenocarcinoma lesions in a population of dogs including the relations between primary tumour, and locoregional and distant metastasis. MATERIALS AND METHODS Retrospective review of dogs with confirmed anal sac adenocarcinoma and available CT images of the thorax, abdomen and pelvis. RESULTS A population of 70 dogs were included in the study. No association was found between anal sac mass size and presence or absence of iliosacral lymph node enlargement. The prevalence of local metastatic disease characterised by iliosacral lymphadenomegaly in this study was 71%, with pulmonary metastases identified in 11% of cases. There were no cases of distant pulmonary metastasis without concurrent locoregional lymphadenomegaly. CLINICAL SIGNIFICANCE In our population of dogs local metastatic spread of anal sac adenocarcinoma was common, with a relatively low prevalence of pulmonary metastasis. The study demonstrates the importance of thorough rectal examination and/or imaging to assess the iliosacral lymph centre in this disease irrespective of the size of the anal sac mass.

Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment 1, oocytes, corona radiata, cumulus, and granulosa cells were collected from mares classified as young (4-12 years) or old (> 20 years). Transcript abundance was evaluated for connexins -37 (GJA4) and -43 (GJA1); zona pellucida glycoproteins 1, 2, 3, and 4 (ZP1, ZP2, ZP3, ZP4); Tubulin (TUBA1A), and equine chorionic gonadotropin β. For experiment 2, oocytes that failed to cleave following intracytoplasmic sperm injection (ICSI) were stored in salt solution for up to 4 years and used for sperm binding assays. Transcript abundance for GJA1 was decreased in oocytes, corona radiata, and granulosa cells while GJA4 was decreased in cumulus cells from old compared to young mares. Additionally, ZP1 tended to be decreased in corona radiata and cumulus cells from old mares. Oocytes from old mares tended to bind less spermatozoa compared young mares. Oocytes that failed to cleave following ICSI can be used for sperm binding studies for up to 2 years without losses in sperm binding. Our findings suggest that maternal age may contribute to changes in cellular communication and the ZP that could impact sperm binding.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Exposure to hog barn organic dust contributes to occupational lung diseases, which are mediated by inflammatory and oxidative stress pathways. Isoprostanes—a family of eicosanoids produced by oxidation of phospholipids by oxygen radicals—are biomarkers of pulmonary oxidative stress. Importantly, 8-isoprostane has been implicated as a key biomarker and mediator of oxidative stress because it is a potent pulmonary vasoconstrictor. Antioxidants found in fruits and vegetables hold promise for preventing or reducing effects of oxidative stress-related diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). Here, we investigated 8-isoP and oxidant production by organic dust-exposed airway epithelial cells and the inhibitory effects of an extract from calyces of the sorrel plant, Hibiscus sabdariffa, on oxidant-producing pathways. Confluent cultures of normal human tracheobronchial epithelial cells were pretreated or not with 1% sorrel extract prior to 5% dust extract (DE) exposure. Following DE treatments, live cells, cell-free supernatants, or cell extracts were evaluated for the presence of 8-isoprostane, superoxide, hydrogen peroxide, nitric oxide, hydroxyl radical, peroxynitrite, and catalase activity to evaluate sorrel’s inhibitory effect on oxidative stress. The well-known radical scavenging antioxidant, N-acetyl cysteine (NAC), was used for comparisons with sorrel. DE exposure augmented the production of all radicals measured including 8-isoprostane (p value < 0.001), which could be inhibited by NAC or sorrel. Among reactive oxygen and nitrogen species generated in response to DE exposure, sorrel had no effect on H2O2 production and NAC had no significant effect on NO⋅ production. The observations reported here suggest a possible role for sorrel in preventing 8-isoprostane and oxidant-mediated stress responses in bronchial epithelial cells exposed to hog barn dust. These findings suggest a potential role for oxidative stress pathways in mediating occupational lung diseases and antioxidants within sorrel and NAC in reducing dust-mediated oxidative stress within the airways of exposed workers.

Objective To determine whether the sensitivity of clinical examination for assessing upper airway disease severity in 3 breeds of brachycephalic dogs can be improved by incorporating an exercise test (ET) or by auscultation of a laryngeal stridor to predict laryngeal collapse. Study design Prospective clinical study. Animals Client-owned brachycephalic dogs (n = 44 ET; n = 57 laryngeal stridor assessment). Methods In the first part of the study, clinical examinations were performed at rest and after 5-minute walk and 3-minute trot tests, and a grade reflective of brachycephalic obstructive airway syndrome (BOAS) severity was assigned. Whole-body barometric plethysmography was used as a comparative, objective measure of disease severity. In the second part of the study, the degree of laryngeal collapse present in dogs undergoing BOAS surgery was compared to pre-exercise and postexercise laryngeal stridor detected during functional testing. Results The sensitivity of clinical examination for BOAS diagnosis was 56.7% pre-ET, 70% after a 5-minute walk test, and 93.3% after a 3-minute trot test. The sensitivity of laryngeal stridor as a predictor of laryngeal collapse was improved after exercise (70%) compared with before exercise (60%). Specificity of laryngeal stridor for laryngeal collapse was 100% (pre-exercise and postexercise). Conclusion The sensitivity of clinical examination for BOAS diagnosis was improved by inclusion of an ET, particularly the 3-minute trot test. Audible laryngeal stridor was highly specific but only moderately sensitive for laryngeal collapse. Clinical significance Inclusion of a 3-minute trot test and careful auscultation for laryngeal stridor are recommended during BOAS assessment of brachycephalic dogs.

Grazing can induce changes in both plant productivity and nutritional quality, which may subsequently influence herbivore carrying capacity. While research on Soay sheep (Ovis aries L.) dynamics on Hirta Island in the St. Kilda archipelago has elucidated the complexity of population drivers, including parasites, the role of herbivore-generated feedbacks as an intrinsic regulating factor remains unclear. The sheep lack large predators and every 3-9 years undergo population crashes (overcompensatory mortality). We investigated the effects of grazing on (1) sward productivity and (2) quality (toxicity) of the primary forage species, red fescue (Festuca rubra L.), which is highly infected by an alkaloid-synthesizing fungal endophyte. Grazing had a negative impact on both forage quantity and quality. At higher sheep densities, impacts on sward growth were magnified, resulting in a nonlinear relationship with plant productivity. Simultaneously, endophyte hyphal load (and by inference, toxicity) peaked close to the time of a crash. A greenhouse experiment showed that alkaloid concentration in F. rubra increased in response to artificial defoliation. We conclude that at high sheep densities, grazing-mediated reductions in productivity, together with sustained alkaloid production, are likely to influence sheep dynamics. Future research should consider the interactive effects of forage toxicity, quantity, and nutritional content.

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 days and a uterine biopsy at 16 days (0 day = ovulation). With the exception of 8 day embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry. Obese mares had elevated concentrations of insulin, leptin, and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates that increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.Summary SentenceExcess adiposity is sufficient to increase transcript abundance of inflammatory cytokines in the uterine endometrium and alters embryonic lipid content, and gene expression for lipid metabolism, endoplasmic reticulum stress, and mitochondrial function.

Objective APELA is a small, secreted peptide that can function as a ligand for the G-protein coupled receptor, Apelin Receptor (APLNR, APJ). APELA plays an essential role in endoderm differentiation and cardiac development during embryogenesis. We investigated whether APELA exerts any functions in cancer progression. Methods The Cancer Genome Atlas (TCGA) RNA sequencing datasets, microarray from an OCCC mouse model, and RNA isolated from fresh frozen and FFPE patient tissue were used to assess APELA expression. APELA knockout ovarian clear cell carcinoma (OCCC) cell lines were generated using CRISPR/Cas9. Results APELA was expressed in various ovarian cancer histotypes and was especially elevated in OCCC. Disruption of APELA expression in OCCC cell lines suppressed cell growth and migration, and altered cell-cycle progression. Moreover, addition of human recombinant APELA peptide to the OCCC cell line OVISE promoted cell growth and migration. Interestingly, OVISE cells do not express APLNR, suggesting that APELA can function through an APLNR-independent pathway. Furthermore, APELA affected cell growth and cell cycle progression in a p53-dependent manner. In addition, APELA knockdown induced p53 expression in cancer cell lines. Conclusions Our findings uncover a potential oncogenic role for APELA in promoting ovarian tumour progression and provide a possible therapeutic strategy in ovarian cancer by targeting APELA.

Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.

Envenomation by the Eastern coral snake is rare but may be associated with significant morbidity. While effective, acquisition of North American Coral Snake Antivenin (NACSAV) is difficult because production was discontinued for many years.The purpose of this study is to characterize coral snake exposures in Florida and determine the effects of varying treatment paradigms on patient outcomes.This study is an observational case series of cases received at Florida poison centers. Included cases were Eastern coral snake exposures occurring between January 1, 1998 and October 31, 2010. Excluded cases included those found to be unrelated or those not followed for at least 24 h post envenomation. Case comments were reviewed to obtain data. Comparisons were made between asymptomatic patients receiving empiric antivenom therapy (empiric group) and those asymptomatic patients who received antivenom upon developing signs of systemic envenomation (withhold group).Of the 553 cases identified, 387 were included in the final analysis. According to case comments, 56.3% of patients had no reported systemic symptoms. Most commonly, patients were reported to have pain (40.6%), paresthesias (28.4%), nausea (12.7%), and emesis (11.4%). NACSAV was administered to 252 patients (65%). Of those patients receiving NACSAV, 18.25% were reported to have had an adverse reaction. Patients in the withhold group (n = 106) had significantly fewer minor, moderate, and major outcomes than patients in the empiric group (n = 134, p0.01).While patients in the withhold group had favorable outcomes compared with those in the empiric group, this strategy cannot be applied to all patients presenting asymptomatic to healthcare facilities due to study limitations.Further studies are needed to determine what treatment strategy is most appropriate for asymptomatic patients presenting to healthcare facilities.

A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. miR-200c increases sensitivity to taxanes in vitro by targeting class III β-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately before cytotoxic chemotherapy may allow for a better response or lower effective dose. Mol Cancer Ther; 11(12); 2556–65. ©2012 AACR.

Synthetic grafts comprised of a porous scaffold in the size and shape of the natural tracheobronchial tree, and autologous stem cells have shown promise in the ability to restore the structure and function of a severely damaged airway system. For this specific application, the selected scaffold material should be biocompatible, elicit limited cytotoxicity, and exhibit sufficient mechanical properties. In this research, we developed composite nanofibers of polycaprolactone (PCL) and depolymerized chitosan using the electrospinning technique and assessed the properties of the fibers for its potential use as a scaffold for regenerating tracheal tissue. Water-soluble depolymerized chitosan solution was first prepared and mixed with polycaprolactone solution making it suitable for electrospinning. Morphology and chemical structure analysis were performed to confirm the structure and composition of the fibers. Mechanical testing of nanofibers demonstrated both elastic and ductile properties depending on the ratio of PCL to chitosan. To assess biological potential, porcine tracheobronchial epithelial (PTBE) cells were seeded on the nanofibers with composition ratios of PCL/chitosan: 100/0, 90/10, 80/20, and 70/30. Transwell inserts were modified with the nanofiber membrane and cells were seeded according to air-liquid interface culture techniques that mimics the conditions found in the human airways. Lactase dehydrogenase assay was carried out at different time points to determine cytotoxicity levels within PTBE cell cultures on nanofibers. This study shows that PCL/chitosan nanofiber has sufficient structural integrity and serves as a potential candidate for tracheobronchial tissue engineering.

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. These changes can lead tumor cells to acquire characteristics that enable movement from the primary site of origin when conditions become unfavorable. Such characteristics include gain of front-rear polarity, increased migration/invasion, and resistance to anoikis, which facilitate tumor survival during metastasis. An epithelial to mesenchymal transition (EMT) constitutes one way that cancer cells can gain traits that promote tumor progression and metastasis. Two microRNA (miRNA) families, the miR-200 and miR-221 families, play crucial opposing roles that affect the differentiation state of breast cancers. These two families are differentially expressed between the luminal A subtype of breast cancer as compared to the less well-differentiated triple negative breast cancers (TNBCs) that exhibit markers indicative of an EMT. The miR-200 family promotes a well-differentiated epithelial phenotype, while high miR-221/222 results in a poorly differentiated, mesenchymal-like phenotype. This review focuses on the mechanisms (specific proven targets) by which these two miRNA families exert opposing effects on cellular plasticity during breast tumorigenesis and metastasis.

Fear and anxiety behaviors are underpinned by neuronal changes within the amygdala. Here, the effects of exposure to natural and synthetic cat odor on behavior and amygdala plasticity were determined. Exposure to natural odor elicited typical and persistent anxiety-related behaviors, such as avoidance, freezing, and flat-back approach; however, synthetic odorant evoked no significant alteration in behavior. Furthermore, ex vivo induction of long-term potentiation within the medial nucleus of the amygdala, a principal area involved in olfactory perception, was significantly reduced after exposure to natural, but not synthetic, odor. Data presented here suggests that the synthetic odorant utilized may lack the constituents that are required to indicate predator presence in rodents and also the capacity to modulate neuronal plasticity within the medial nucleus of the amygdala.

MicroRNAs (miRNAs) are small non-coding RNAs that serve as post-transcriptional regulators of gene expression. They work predominantly by binding to complementary sequences in target messenger RNA (mRNA) 3' untranslated regions (UTRs) where they prevent translation or cause degradation of the message. Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate genes in steroid responsive tissues. Recent studies demonstrate that SHRs regulate miRNAs, and in turn, miRNAs can regulate SHR expression and function. Mounting evidence indicates that miRNAs are intimately involved with SHRs, as they are with other transcription factors, often in double negative feedback loops. Investigators are just beginning to expose the details of these complex relationships and reveal the extent to which miRNAs are involved with SHRs in normal physiology and the pathobiology of steroid hormone responsive tissues.

The spirochetal agent of Lyme disease, Borrelia burgdorferi , is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was ( i ) infectious in mice when introduced by needle inoculation, ( ii ) acquired by larval ticks feeding on infected mice, and ( iii ) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.

Summary Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasome-dependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition., Graphical Abstract, Highlights • Mutation of Arc ubiquitination sites slows Arc degradation • Arc knockin mice have enhanced mGluR-LTD • The persistence of Arc reduces cognitive flexibility, Ubiquitin-dependent proteasomal degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We generated a mouse disrupting the temporal control of Arc removal (ArcKR). ArcKR mice exhibit enhanced mGluR-LTD and have impaired cognitive flexibility.

Modulation of synaptic strength may underlie stress-induced changes in cognitive ability. Long-term memory formation underpinning fear and anxiety behaviors, such as those seen in post-traumatic stress and phobic disorders, is thought to be dependent on amygdalo-hippocampal interactions. In most models, however, painful stimuli are used to induced stress and anxiety. Here, the effects of a novel conflict model, developed to generate a more naturalistic model of anxiety, utilizing two non-noxious stressors (predator (cat) odor and light), on hippocampus plasticity were determined. Exposure to the external stimuli elicited typical, stimulus-specific, anxiety-related behaviors. Dual presentation of the stressors evoked an increase in the variability of behaviors, suggesting that the animals were experiencing conflicting drives. Induction of long-term potentiation (LTP) within the CA1 region of the hippocampus was reduced following exposure to light stress, independent of presence, or absence, of odor. However, after a single presentation, LTP was reduced following either odor presentation or dual presentation of the stressors. Furthermore, LTP in ex vivo tissue obtained from conflict-exposed animals showed differential hemispheric responses, suggesting that long-term contextual-related components of anxiety behavior are dependent on modification of hippocampal circuitry.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of β 1 integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein–protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

Transforming growth factor (TGF)-beta1 plays an important role in the development of pulmonary fibrosis. In this study we examined the relationship between TGF-beta1 stimulation and the expression of heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) in cultured normal human lung fibroblasts (NHLFs) and in murine lungs in vivo. By removing 6-O-sulfates from specific HS intrachain sites on the cell surface, Sulf1 has been shown to modulate the activities of many HS binding growth factors and morphogens including fibroblast growth factor (FGF)-2. Real time reverse transcription-PCR analysis revealed that TGF-beta1 increased Sulf1 expression in NHLFs in a dose- and time-dependent manner which was accompanied by a decrease in 6-O-sulfated disaccharides as revealed by high performance liquid chromatography analysis. Decreased ERK activation after FGF-2 stimulation was observed in TGF-beta1-treated NHLFs compared with control cells without changes in HS-dependent FGF-2 binding or FGF-2.FR1c complex formation. To study the function of Sulf1, negative control or Sulf1-specific small interference RNA (siRNA)-transfected NHLFs were stimulated with TGF-beta1. Enhanced Smad2/3 phosphorylation and elevated total Smad2 protein level were observed in Sulf1 siRNA-transfected cells and were accompanied by enhanced expression of alpha-smooth muscle actin and fibronectin. In addition, Sulf1 siRNA transfection enhanced the anti-proliferative effect of TGF-beta1. Finally Sulf1 expression was up-regulated in the lungs of mice treated with adenovirus encoding active TGF-beta1. Taken together, our data indicate that Sulf1 is a TGF-beta1-responsive gene both in vitro and in vivo and may function as a negative regulator of TGF-beta1-induced fibrogenesis.

Borrelia burgdorferi , the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the σ N -σ S regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.

To describe the anatomic and histopathologic outcomes using different incision techniques with transconjunctival 23-G and 25-G vitrectomy systems.New Zealand rabbits were randomized to either 23-G or 25-G vitrectomy surgeries using angled incisions and straight incisions. After pars plana vitrectomy, the cannulas were removed and 0.1% trypan blue was injected to evaluate for leakage. The animals were killed on day 7 and the eyes enucleated for gross analysis and histopathologic analysis by frozen section.Leakage of trypan blue was noted from 10.8% and 5.7% of straight and angled incisions, respectively. There was no difference between 23-G and 25-G incisions (8.3%). On gross examination, the 25-G system resulted in 58% and 24% open external wounds for straight and angled incisions, respectively (P = 0.04). The 23-G system resulted in 83% and 39% open external wounds with straight and angled incisions, respectively (P = 0.017). The average wound area after the 23-G surgery was 223.1 microm(2) and 115.7 microm(2) for straight versus angled incisions, respectively (P = 0.02). The average wound area formed after the 25-G surgery was 160.3 microm(2) and 85.2 microm(2) for straight versus angled incisions, respectively (P = 0.001).Outcomes were similar for 23-G angled incisions, 25-G straight incisions, and 25-G angled incisions.

Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cε (PKCε), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCε expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCε mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCε. The interaction of PKCε with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCε. Localization of PKCε with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCε with Stat3 was PKCε isoform specific. Inhibition of PKCε protein expression in DU145 cells using specific PKCε small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCε activation is essential for constitutive activation of Stat3 and prostate cancer progression. [Cancer Res 2007;67(18):8828–38]

Clusterin mRNA levels were shown to increase dramatically in rat ventral prostate following castration, and clusterin was therefore originally thought to be repressed by androgens. It was later discovered that the increased clusterin levels are most likely due to castration-induced apoptosis of the prostatic epithelium rather than direct action of the androgen receptor (AR). In the studies presented here, LNCaP cells in culture and rat prostate organ culture were treated with androgens. Clusterin mRNA and protein are shown to increase with androgen treatment in a time- and dose-dependent manner. This induction of clusterin requires AR and can be inhibited by casodex, an AR antagonist. We have found that the first intron of the clusterin gene contains putative androgen response elements. The intronic region is shown to be bound by AR in chromatin immunoprecipitation assays and is transactivated by AR in reporter assays. Two isoforms of clusterin result from alternate transcriptional start sites. Both isoforms are cytoprotective; however, Isoform 1 has the capacity to produce a splice variant that is apoptotic. Real time PCR was used to determine the response of the two isoforms to androgens. Intriguingly, these results illustrated that Isoform 2 was up-regulated, whereas Isoform 1 was down-regulated by androgens. Isoform 2 was also increased as the LNCaP xenograft tumor progressed to androgen-independence, whereas Isoform 1 was unaltered. This androgen regulation of clusterin may underline the cytoprotective role of androgens in normal prostate physiology as well as play an antiapoptotic role in prostate cancer progression.

The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple-negative breast cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen-positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage-independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3-dioxygenase (TDO2), in an NF-κB–dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR), an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor–treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor–negative status, and shorter overall survival. Our results define an NF-κB–regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype. Cancer Res; 75(21); 4651–64. ©2015 AACR.

Obesity is associated with disrupted reproductive cycles in mares, but the impact of obesity on follicles and oocytes has received minimal attention. We investigated the impact of obesity on 1) expression of selected genes in follicle cells for carbohydrate metabolism, inflammatory cytokines, lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function; 2) follicular fluid content of metabolic hormones and metabolites; and 3) lipid fingerprint of oocytes. Mares (9-13 yr) were classified as control (n = 8, normal weight, body condition score [BCS] 5.1, 10.4% body fat) or obese (n = 9, BCS 7.9, 16.2% body fat). Gene expression from granulosa cells (GC) and cumulus cells (CC) was evaluated by RT-PCR. Serum and follicular fluid were evaluated for insulin, leptin, adiponectin, and metabolite profiling. Oocyte lipid fingerprints were acquired using matrix-assisted laser desorption/ionization mass spectrometry. Several genes for lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function were different between groups in GC and CC. Obese had (P0.05) or tended to have (0.05P0.1) lower insulin sensitivity and higher insulin and leptin in serum and follicular fluid. Many metabolites differed between control and obese in serum and/or follicular fluid and correlated with BCS and/or insulin sensitivity. Oocytes from control had greater concentrations of lipids consistent with phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins, while lipids consistent with triglycerides tended to be higher in obese. These findings suggest that maternal obesity causes alterations in the follicle and oocyte; the extent to which these alterations impact the conceptus and offspring is still to be determined.

The aim of this investigation was to test the hypothesis that fasting-induced augmented lysosomal autophagic turnover of cellular proteins and organelles will reduce potentially harmful lipofuscin (age-pigment) formation in cells by more effectively removing oxidatively damaged proteins. An animal model (marine snail--common periwinkle, Littorina littorea) was used to experimentally test this hypothesis. Snails were deprived of algal food for 7 days to induce an augmented autophagic response in their hepatopancreatic digestive cells (hepatocyte analogues). This treatment resulted in a 25% reduction in the cellular content of lipofuscin in the digestive cells of the fasting animals in comparison with snails fed ad libitum on green alga (Ulva lactuca). Similar findings have previously been observed in the digestive cells of marine mussels subjected to copper-induced oxidative stress. Additional measurements showed that fasting significantly increased cellular health based on lysosomal membrane stability, and reduced lipid peroxidation and lysosomal/cellular triglyceride. These findings support the hypothesis that fasting-induced augmented autophagic turnover of cellular proteins has an anti-oxidative cytoprotective effect by more effectively removing damaged proteins, resulting in a reduction in the formation of potentially harmful proteinaceous aggregates such as lipofuscin. The inference from this study is that autophagy is important in mediating hormesis. An increase was demonstrated in physiological complexity with fasting, using graph theory in a directed cell physiology network (digraph) model to integrate the various biomarkers. This was commensurate with increased health status, and supportive of the hormesis hypothesis. The potential role of enhanced autophagic lysosomal removal of damaged proteins in the evolutionary acquisition of stress tolerance in intertidal molluscs is discussed and parallels are drawn with the growing evidence for the involvement of autophagy in hormesis and anti-ageing processes.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial–melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell–cell interactions at the lymphatic–tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

Background . Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab ’ ) 2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab ’ ) 2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods . We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab ’ ) 2 with maintenance doses [F(ab ’ ) 2 /F(ab ’ ) 2 ], or F(ab ’ ) 2 with placebo maintenance doses [F(ab ’ ) 2 /placebo], versus Fab with maintenance doses [Fab/Fab]. The primary effi cacy endpoint was coagulopathy (platelet count 150 K/mm 3 , fi brinogen level 150 mg/dL) between end of maintenance dosing and day 8. Results . 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p 0.05) in the F(ab ’ ) 2 /F(ab ’ ) 2 cohort and 2/38 (5.3%, p 0.05) in the F(ab ’ ) 2 /placebo cohort. The lowest heterologous protein exposure was with F(ab ’ ) 2 /placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions . In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab ’ ) 2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation.

Synchronized and rhythmic activity within the amygdala is thought to play a pivotal role in the generation of fear- and anxiety-related behaviour. The aim here was to determine the validity of the in vitro amygdala slice preparation to investigate the generation of rhythmic activity similar to that observed in vivo. Extracellular population activity recorded from the lateral nucleus of the amygdala in vitro showed significant enhancement of activity within the theta-band frequency (3-9 Hz) in the presence of kainic acid (100 nM; n = 18). Alterations in the patterns of oscillatory activity within the gamma frequency band (20-40 Hz) were observed in the presence of (RS)-3,5-dihydroxyphenylglycine (10 mu M; n = 7) or carbachol (50 mu M; n = 5). Theta frequency oscillatory activity was blocked in the presence of the gap junction blocker carbenoxolone (100 mm), whereas gamma frequency oscillatory activity showed increased variability in the dominant frequency of rhythmic activity. The results suggest that the neuronal circuitry of the amygdala in vitro is capable of generating and sustaining rhythmic activity and that intercellular communication via gap junctions may play a role in the synchronization of population activity underlying this oscillatory activity.

Background Despite recent recognition that visceral pain differs from somatic pain in its neurophysiologic basis and treatment modalities, most laboratory studies of postoperative pain use a model of superficial somatic injury, and there is no model of postoperative pain after gynecologic surgery. We describe spontaneous behavior in rats after laparotomy with or without noxious stimulation of the uterus and cervix to more specially address pain after gynecologic surgery. Methods Female Sprague-Dawley rats received inhaled anesthesia only, anesthesia with laparotomy, or laparotomy plus 60 min of tonic distension of the lower uterine segment and cervix, followed by video observation of spontaneous behavior. Results Compared with anesthesia alone, laparotomy decreased some spontaneous behaviors (drinking water, grooming, and exploration). Laparotomy plus uterocervical manipulation further decreased these behaviors and increased abnormal behaviors (licking of the lower abdomen and squashing posture of the pelvis to the floor). Intrathecal and systemic morphine restored spontaneous behavior and reduced abnormal behaviors, with minor differences between routes of administration. Conclusions These data suggest that specific behaviors may distinctly reflect somatic and visceral components of postoperative gynecologic pain, and that this model may be used to test novel therapies to relieve pain in this setting.

We have identified two novel mouse orexin type-2 receptor (OX2R) splice variants: OX2alphaR and OX2betaR. Here, we have mapped in detail the differential gene expression of OX2alphaR and OX2betaR in the mouse brain. Using RT-PCR and in situ hybridization, we show that OX2alphaR is expressed in various areas of the mouse brain and has low expression in caudal part of cerebellum. OX2betaR is expressed in detectable levels, with the exception of striatum, midbrain and anterior part of cerebellum/pons. Our novel findings demonstrate the differential expression of OX2R splice variants in the mouse brain.

PDGF isoforms are a family of polypeptides that bind to cell surface receptors and induce fibroblast proliferation and chemotaxis. PDGF-A and -B chain isoforms have previously been shown to be involved in murine lung development. A new PDGF polypeptide, PDGF-C, was recently recognized and differs from the PDGF-A and -B isoforms in that it requires proteolytic cleavage before it can bind and activate the PDGF alpha receptor. In these studies PDGF-C was over-expressed during embryogenesis using the lung specific surfactant protein C promoter. PDGF-C transgenic pups died from respiratory insufficiency within minutes following birth. At E18.5, nontransgenic lungs exhibited lung morphology consistent with the saccular stage of lung development. In contrast, E18.5 transgenic lungs retained many features of the canalicular stage of lung development and had abundant numbers of large poorly differentiated mesenchymal cells. These results suggest that PDGF-C is activated during lung development and is a potent growth factor for mesenchymal cells in vivo.

We previously demonstrated that outer surface protein C (OspC) ofBorrelia burgdorferiis essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that aB. burgdorferi ospCmutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. TheospCmutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival ofB. burgdorferiwithin the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of theospCmutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown thatB. burgdorferiis prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. TheospCmutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

BACKGROUND Relaxin is a potent peptide hormone normally secreted by the prostate. This study characterized relaxin expression during prostate cancer progression to androgen independence (AI), and in response to androgens. METHODS The prostate cancer cell line, LNCaP, was assayed by microarrays and confirmatory Northern analysis to assess changes in relaxin levels due to androgen treatment and in LNCaP xenografts following castration. Relaxin protein levels were examined by immunohistochemistry (IHC) in tissue microarrays of human prostate cancer samples following androgen ablation. RESULTS Relaxin levels decreased in a time and concentration-dependent manner due to androgens in vitro, and increased in xenografts post-castration. Relaxin increased in radical prostatectomy specimens after 6 months of androgen ablation and in AI tumors, was highest in bone metastases. CONCLUSIONS Relaxin is negatively regulated by androgens in vitro and in vivo, which correlates to clinical prostate cancer specimens following androgen ablation. The role of relaxin in angiogenesis and tissue remodeling suggests it may contribute to prostate cancer progression. Prostate © 2006 Wiley-Liss, Inc.

We recently described a hitherto unrecognized family of autoantibodies that become unmasked (detectable) subsequent to oxidation-reduction (redox) reactions. These masked redox-reactive autoantibodies are not detectable by using conventional immunoassays. Additional experimentation has demonstrated that autoantibodies in the blood of patients with autoimmune diseases can be masked (become undetectable) by exposure to oxidizing agents. Simultaneous masking and unmasking of different autoantibodies in a given patient's serum or plasma is evidence that immune complexes are not the source of redox-reactive autoantibodies. We propose that a mechanism responsible for unmasking-masking antibody specificities requires nitrosylation of tyrosine residues in the hypervariable or complementarity determining regions of the antibodies' antigen-binding sites. Other laboratories, selected by us for their respective expertise, have studied our redox-reacted and control serum and/or antibody preparations and have found an expanding array of autoantibody specificities. The gathering data suggest that certain autoimmune diseases may involve redox disorders rather than a failure to deplete, suppress, tolerate or divert self-directed B cell activity. The persistence and fluctuation of redox-reactive autoantibodies suggest that they are manifestations of an as yet undefined natural selective pressure on the evolution of the immunological system. We propose that they are the "contrivances" suggested by Paul Ehrlich more than a hundred years ago, and that these antibodies perform important physiological and pathophysiological functions.

Age-related changes in follicle paracrine signalling are not defined, and follicular gene transcript abundance could predict oocyte viability. Granulosa cells from preovulatory follicles of mares considered Young (n = 12; 4–14 years), Mid-aged (n = 9; 15–19 years) and Old (n = 14; 20–27 years) were evaluated for transcript abundance related to systemic and follicle-specific pathways. Gene transcript abundance for receptors of insulin, adiponectin and peroxisome proliferating factor-γ were higher or tended to be higher in Mid-aged or Old than Young mares. Transcript abundance for interleukin (IL)-6 was elevated in Old versus Young mares, and IL-6 signal transducer was elevated in Old versus younger groups. Expression of tumour necrosis factor (TNF) receptor superfamily member 1A was higher in Mid-aged than Young mares, whereas TNF-inducible gene 6 protein mRNA tended to decrease in Mid-aged versus Young and Old mares. Genes for LH receptor and steroidogenic acute regulatory protein tended to be increased in Old versus Mid-aged and Young mares, respectively. Young and Old mares had higher mRNA for tissue-type plasminogen activator than Mid-aged mares. Thioredoxin-2 mRNA was higher in Old than younger groups. We observed age-related changes in mRNA of receptors for metabolic hormones, inflammatory processes, steroidogenic hormones, tissue remodelling and mitochondrial function, which could contribute to and/or mark alterations in follicular function and fertility.

Tumor necrosis factor (TNF)-alpha, a cardinal molecule in the cascade of sepsis-induced host injury, binds to two distinct receptors: tumor necrosis factor receptor (TNFR) 1 and TNFR2. We used the cecal ligation and puncture model of polymicrobial sepsis to elucidate the role of these receptors in sepsis pathogenesis. Mice lacking TNFR1 had prolonged survival with less hypothermia, whereas mice lacking TNFR2-/- had shortened survival and more profound hypothermia than wild-type mice. TNFR1-/- and TNFR2-/- mice had increased serum concentrations of interleukin (IL) 1beta and total TNF-alpha (free plus receptor bound) compared with wild-type mice, but there were no differences in IL6 or IL10 concentrations. Furthermore, free TNF-alpha was markedly elevated in the serum and peritoneal fluid of mice lacking TNFR2, supporting a role for this receptor in regulating the concentration of TNF-alpha. Lastly, apoptosis of ileal crypt epithelial cells was increased in mice lacking TNFR1, but there were no differences in lymphocyte apoptosis. These data suggest that in sepsis, TNFR1 mediates much of the TNF-alpha-induced pathology, whereas TNFR2 mediates protective effects.

Spinal noradrenaline (NA) released in response to noxious stimuli may play an important role in suppression of nociceptive transmission. Here, we investigated the efficacy of a competitive NA transporter inhibitor (reboxetine) and a noncompetitive NA transporter inhibitor peptide, Xen2174, isolated from the Pacific cone snail, to treat tactile hypersensitivity following paw incisional surgery. Male Sprague-Dawley rats were anesthetized, an incision of the plantar aspect of the hind paw was performed, and withdrawal threshold to von Frey filaments near the surgical site determined. Reboxetine (0.5-5 microg) and Xen2174 (0.3-100 microg) increased withdrawal threshold when injected 24h after paw incision, with a peak effect at 15-60 min, for Xen2174, an ED50 value of 0.64 microg. Administration of Xen2174 (3-30 microg) 15 min before incision also reduced hypersensitivity in a dose-dependent manner. Withdrawal threshold after the single 30 microg dose was greater than vehicle control even at 2, 3, and 5 days after incision. Doses

Pain after surgery results in significant morbidity, and systemic opioids often fail to provide adequate analgesia without marked sedation and respiratory depression. Intrathecal morphine provides better analgesia, but is limited by delayed respiratory depression. Intrathecal injection of the cyclooxygenase inhibitor, ketorolac, has recently entered clinical trials, and the current study examined the interaction between intrathecal morphine and ketorolac to treat postoperative pain. We also sought to compare these treatments on a commonly used assessment of withdrawal threshold and a new assessment of spontaneous behavior after surgery. Male Sprague Dawley rats and underwent hind paw incision or subcostal laparotomy surgery. Intrathecal morphine, ketorolac, or their combination were injected on the first postoperative day, with outcome measure being return to pre-surgery withdrawal threshold with von Frey filament testing of the paw after paw incision, or return to pre-surgery exploratory activity after laparotomy. Intrathecal morphine completely reversed the effects of surgery in both models, but intrathecal ketorolac only partially reversed them. Ketorolac enhanced the potency of morphine several fold in both models, and did so synergistically after paw incision. In all cases drug potency was greater for spontaneous than elicited responses. These data confirm that spinal opioid receptor and cyclooxygenase enzyme inhibition diminish elicited tactile hypersensitivity after surgery, and that they similarly return spontaneous behavior to normal. Differences in drug potency could reflect fundamental differences in outcome measures or in the surgical procedures themselves. These data support combination study of intrathecal morphine and ketorolac for postoperative pain.

Cyclooxygenase-2 (COX-2) activity in the spinal cord plays a key role in sensitization to sensory stimuli during acute inflammation. In contrast, intrathecal administration of COX-2 specific inhibitors has minimal analgesic effects in an incisional model of postoperative pain. We investigated the role of COX isoforms in this model by examining the expression of COX-1 and the effect of intrathecal COX inhibitors. A 1cm longitudinal incision was made through skin, fascia and muscles of the plantar aspect of the left paw in male rats, and withdrawal threshold to von Frey filaments measured. Rats were perfused at 1, 2, 3, 5, and 7 days after incision, and COX-1 immunohistochemistry was performed on L3 to S2 spinal cord and gracile nucleus sections. Other rats received intrathecally the COX-1 preferring inhibitor, ketorolac, the specific COX-1 inhibitor, SC-560, the COX-2 inhibitor, NS-398 or vehicle 1 day after surgery. Withdrawal threshold was measured at intervals up to 5 days later. COX-1 immunoreactivity increased in glia in the ipsilateral L4-L6 spinal dorsal horn and ipsilateral gracile nucleus after incision. Mechanical allodynia peaked on postoperative day 1, and COX-1 immunoreactivity increased on day 1, peaked on day 2, and declined thereafter. Ketorolac and SC-560 dose-dependently increased withdrawal threshold in this model, but NS-398 had no effect. These results suggest that COX-1 plays an important role in spinal cord pain processing and sensitization after surgery. Increased COX-1 activity could precede the up-regulation of COX-1 protein, and spinally administered specific COX-1 inhibitors may be useful to treat postoperative pain.

Systemic hypoxia produces a rapid microvascular inflammatory response characterized by increased reactive oxygen species (ROS) levels, leukocyte-endothelial adherence and emigration, and increased vascular permeability. The lipid inflammatory mediator leukotriene B4 (LTB4) is involved in the early hypoxia-induced responses (ROS generation and leukocyte adherence). Whether other lipid inflammatory mediators participate in this phenomenon is not known. The objective of these experiments was to study the role of platelet-activating factor (PAF) in the microvascular inflammatory response to hypoxia and its potential interactions with LTB4 in this response. Intravital microscopy was used to examine mesenteric venules of anesthetized rats. We found that WEB-2086, a PAF receptor antagonist, completely prevented the increase in ROS levels and leukocyte adherence during a brief reduction in inspired Po 2 to anesthetized rats; administration of either WEB-2086 or the LTB4 antagonist LTB4-DMA attenuated leukocyte emigration and the increase in vascular permeability to the same extent during prolonged systemic hypoxia in conscious rats. Furthermore, no additive effect was observed in either response when both antagonists were administered simultaneously. This study demonstrates a role for PAF in the rapid microvascular inflammatory response to hypoxia, as well as contributions of PAF and LTB4 to the slowly developing responses observed during sustained hypoxia. The incomplete blockade of the hypoxia-induced increases in vascular permeability and leukocyte emigration by combined administration of both antagonists indicates that factors in addition to LTB4 and PAF participate in these phenomena.