Inflammasome expression is higher in ovarian tumors than in normal ovary

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Luborsky, J., Barua, A., Edassery, S., Bahr, J. M., & Edassery, S. L. Inflammasome expression is higher in ovarian tumors than in normal ovary. Plos One, 15(1), (2020): e0227081, doi:10.1371/journal.pone.0227081.Chronic inflammation fundamentally influences cancer risk and development. A mechanism of chronic inflammation is the formation of inflammasome complexes which results in the sustained secretion of the pro-inflammatory cytokines IL1β and IL18. Inflammasome expression and actions vary among cancers. There is no information on inflammasome expression in ovarian cancer (OvCa). To determine if ovarian tumors express inflammasome components, mRNA and protein expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat family, pyrin domain containing 3), caspase-1, IL1β, and IL18 expression in hen and human OvCa was assessed. Chicken (hen) OvCa a valid model of spontaneous human OvCa. Hens were selected into study groups with or without tumors using ultrasonography; tumors were confirmed by histology, increased cellular proliferation, and expression of immune cell marker mRNA. mRNA expression was higher for hallmarks of inflammasome activity (caspase-1, 5.9x increase, p = 0.04; IL1β, 4x increase, p = 0.04; and IL18, 7.8x increase, p = 0.0003) in hen OvCa compared to normal ovary. NLRP3, caspase-8 and caspase-11 mRNA did not differ significantly between tumor and non-tumor containing ovaries. Similar results occurred for human OvCa. Protein expression by immunohistochemistry paralleled mRNA expression and was qualitatively higher in tumors. Increased protein expression of caspase-1, IL1β, and IL18 occurred in surface epithelium, tumor cells, and immune cells. The aryl hydrocarbon receptor (AHR), a potential tumor suppressor and NLRP3 regulator, was higher in hen (2.4x increase, p = 0.002) and human tumors (1.8x increase, p = 0.038), suggesting a role in OvCa. Collectively, the results indicate that inflammasome expression is associated with hen and human OvCa, although the NLR sensor type remains to be determined.This research was made possible by NIH grant NCI R03CA182120 (JL), DOD grant W81XWH-08-1-0203 (JL) and Swim Across America (AB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The hen model of human ovarian cancer develops anti-mesothelin autoantibodies in response to mesothelin expressing tumors

<p>Abstract</p> <p>Objective</p> <p>Study of the hen immune system led to seminal contributions to basic immunological principles. Recent studies of spontaneous ovarian cancer in the laying hen show strikingly similar tumor types and antigen expression compared to human ovarian cancer, suggesting hens would be valuable for studies of tumor immunology and pre-clinical vaccine development. Circulating mesothelin is a relatively specific marker for human ovarian cancer and autoantibodies to mesothelin were reported. We hypothesized that hen tumors express mesothelin and that circulating anti-mesothelin antibodies occur in response to tumors.</p> <p>Methods</p> <p>Mesothelin mRNA expression was analyzed by RT-PCR in hen ovarian tumors and normal ovaries. Mesothelin protein expression was evaluated by immunohistochemistry (IHC) and two-dimensional SDS-PAGE Western blots. Anti-mesothelin antibodies were assessed by immunoassay of sera from hens with normal ovaries and with ovarian tumors.</p> <p>Results</p> <p>Significant mesothelin mRNA expression was observed in 57% (12/21) of hen ovarian tumors but not in normal ovaries and was found predominantly in serous tumors as in humans. Mesothelin protein was detected in tumors with mesothelin mRNA by IHC and 2D Western blots, but not in normal ovaries or tumors without mesothelin mRNA. Circulating anti-mesothelin antibodies occurred in 44% (n = 4/9) of hens with ovarian tumors which express mesothelin mRNA and were not found in hens with tumors that did not express mesothelin (n = 0/5) or normal ovaries (n = 0/5).</p> <p>Conclusion</p> <p>The results support the utility of the hen as a novel model for preclinical studies of mesothelin as a biomarker and a target for immunotherapy.</p

A clinically relevant sheep model of orthotopic heart transplantation 24 h after donor brainstem death

BACKGROUND: Heart transplantation (HTx) from brainstem dead (BSD) donors is the gold-standard therapy for severe/end-stage cardiac disease, but is limited by a global donor heart shortage. Consequently, innovative solutions to increase donor heart availability and utilisation are rapidly expanding. Clinically relevant preclinical models are essential for evaluating interventions for human translation, yet few exist that accurately mimic all key HTx components, incorporating injuries beginning in the donor, through to the recipient. To enable future assessment of novel perfusion technologies in our research program, we thus aimed to develop a clinically relevant sheep model of HTx following 24 h of donor BSD. METHODS: BSD donors (vs. sham neurological injury, 4/group) were hemodynamically supported and monitored for 24 h, followed by heart preservation with cold static storage. Bicaval orthotopic HTx was performed in matched recipients, who were weaned from cardiopulmonary bypass (CPB), and monitored for 6 h. Donor and recipient blood were assayed for inflammatory and cardiac injury markers, and cardiac function was assessed using echocardiography. Repeated measurements between the two different groups during the study observation period were assessed by mixed ANOVA for repeated measures. RESULTS: Brainstem death caused an immediate catecholaminergic hemodynamic response (mean arterial pressure, p = 0.09), systemic inflammation (IL-6 - p = 0.025, IL-8 - p = 0.002) and cardiac injury (cardiac troponin I, p = 0.048), requiring vasopressor support (vasopressor dependency index, VDI, p = 0.023), with normalisation of biomarkers and physiology over 24 h. All hearts were weaned from CPB and monitored for 6 h post-HTx, except one (sham) recipient that died 2 h post-HTx. Hemodynamic (VDI - p = 0.592, heart rate - p = 0.747) and metabolic (blood lactate, p = 0.546) parameters post-HTx were comparable between groups, despite the observed physiological perturbations that occurred during donor BSD. All p values denote interaction among groups and time in the ANOVA for repeated measures. CONCLUSIONS: We have successfully developed an ovine HTx model following 24 h of donor BSD. After 6 h of critical care management post-HTx, there were no differences between groups, despite evident hemodynamic perturbations, systemic inflammation, and cardiac injury observed during donor BSD. This preclinical model provides a platform for critical assessment of injury development pre- and post-HTx, and novel therapeutic evaluation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40635-021-00425-4

Acompanhamento de pacientes submetidos à cirurgia bariátrica : aspectos laboratoriais nos períodos pré e pós-operatório

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2017A obesidade é uma doença crônica e endócrino-metabólica caracterizada pelo acúmulo excessivo de triacilgliceróis no tecido adiposo, capaz de ser revertida ou amenizada através de intervenção cirúrgica. Epidemiologicamente têm sido descritas associações entre o excesso de peso, resistência à insulina e processo inflamatório crônico. Além disso, nas últimas décadas o sistema complemento foi associado a doenças metabólicas e cardiovasculares e intimamente relacionado com a obesidade e resistência à insulina. Sendo assim, a melhora do estado metabólico e a remissão da inflamação em pacientes obesos submetidos à cirurgia bariátrica foram avaliadas, bem como a associação dos fatores 3 e 4 (C3 e C4) do sistema complemento com a sensibilidade à insulina e a perda de peso após a cirurgia bariátrica. Para isso, a presença de comorbidades e as concentrações séricas de leptina, adiponectina, resistina e grelina foram avaliados em pacientes obesos mórbidos antes, 1, 3 e 6 meses após a cirurgia bariátrica. Também foram medidas as concentrações de IL-1ß, IL-6, TNF-a, proteína amiloide sérica A (SAA), proteína quimiotática de monócitos 1 (MCP-1), C3, C4, glicose, insulina, colesterol total, triacilglicerol, LDL- colesterol, HDL-colesterol e foi realizado o cálculo do modelo de avaliação da homeostase da resistência à insulina (HOMA-IR) durante o seguimento da cirurgia, bem como em comparação com um grupo de indivíduos não-obesos. Como resultado, observou-se uma redução significativa de peso acompanhada de melhora do perfil lipídico, da sensibilidade à insulina e das comorbidades. Ainda, houve diminuição de leptina e aumento de adiponectina no período pós-cirúrgico. IL-1ß, IL-6, TNF-a, MCP-1 e SAA não mostraram diferença no acompanhamento da cirurgia, porém SAA correlacionou-se com o IMC e apresentou-se muito mais alto no grupo de pacientes obesos. Além disso, C3 e C4 foram significativamente maiores em indivíduos obesos quando comparados aos indivíduos não-obesos e C3 e C4 foram positivamente correlacionados com HOMA-IR e as concentrações de C3 foram significativamente diminuídas após a cirurgia. Com base nesses resultados, a cirurgia bariátrica mostrou melhorar o estado metabólico melhorando as comorbidades associadas à obesidade e os biomarcadores de adiposidade leptina e adiponectina, mas não os demais hormônios e citocinas inflamatórias e C3 e C4 foram fortemente associados à sensibilidade à insulina.Abstract: Obesity is a chronic and endocrine-metabolic disease characterized by triacylglycerol accumulation in the adipose tissue, which can be reversed or improved through surgical intervention. Epidemiologically, associations between overweight, insulin resistance and chronic inflammatory process have been described. Furthermore, in the last decades the complement system was associated with metabolic and cardiovascular diseases and related to obesity and insulin resistance. Thus, metabolic status improvement and inflammation remission in obese patients undergoing bariatric surgery were evaluated, as well as the association of complement system factors 3 and 4 (C3 and C4) with insulin sensitivity and weight loss after bariatric surgery. For this, comorbidities and leptin, adiponectin, resistin and ghrelin serum concentrations were evaluated in morbidly obese patients before, 1, 3 and 6 months after bariatric surgery. IL-1ß, IL-6, TNF-a, serum amyloid A protein (SAA), monocyte chemotactic protein 1 (MCP-1), C3, C4, glucose, insulin, total cholesterol, triacylglycerol, LDL-cholesterol, HDL-cholesterol concentrations and the calculation of the homeostasis model of insulin resistance (HOMA-IR) were performed during the surgery follow-up, as well in a group of non-obese individuals. As a result, significant weight loss followed by improvement in lipid profile, insulin sensitivity and comorbidities were observed. Still, there was a decrease in leptin and an increase in adiponectin in the postoperative period. IL-1ß, IL-6, TNF-a, MCP-1 and SAA showed no difference after surgery, but SAA correlated with BMI and was much higher in obese patients. In addition, both C3 and C4 were significantly higher in obese individuals when compared to lean individuals and positively correlated with HOMA-IR. C3 concentrations were significantly decreased after surgery. Based on these results, bariatric surgery has been shown to improve metabolic status by improving obesity-associated comorbidities and adiposity biomarkers leptin and adiponectin but not the other hormones and inflammatory cytokines and C3 and C4 were strongly associated with insulin sensitivity

Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret

The mechanism of implantation in carnivores is poorly understood. However, a previously unidentified 60-kDa protein has been shown to be necessary for embryo implantation in ferrets. Here we identify this protein as glucose-6-phosphate isomerase (GPI). GPI is expressed by the corpus luteum on days 6–9 of pregnancy, the time at which implantation-promoting activity has been found in corpora lutea. Passive immunization against GPI reduced the number of implantation sites in pregnant ferrets in a dose-dependent manner. GPI is a multifunctional protein. Although first identified for its role in glycolysis, GPI has since been implicated in neural growth, lymphocyte maturation, and metastasis. This study demonstrates a previously uncharacterized function of this protein that may represent the natural motility-stimulating activity that has been co-opted by tumor cells

Culture of Chicken Granulosa Cells from Small Yellow Follicles: A Suitable Culture System

In the chicken ovary, one small yellow follicle (SYF) is selected daily to enter the hierarchy and ovulates a few days later. Cellular changes that occur in these follicles may provide an understanding of the mechanism of follicular selection. It was reported that granulosa cells isolated from SYF undergo apoptosis within 6 h in suspension culture. In this study, a culture method in which granulosa cells from SYF remain viable in vitro for 24 h is reported. Granulosa layers were isolated from SYF (Gr-SYF) and from the largest preovulatory follicle (Gr-F1) and dispersed in collagenase (1.5 mg/ml) for 15 min. Cells (1&times;106/500&mu;l for Gr-SYF and 2&times;105/500&mu;l for Gr-F1) were cultured either in 24 well plates in an incubator with 5% CO2/95% O2 at 39&deg;C or in suspension in falcon tubes and incubated in water bath at 37C, for 0, 6, 12 and 24 h. Theca cell explants (8 mm diameter) were cultured in 1 ml of the same medium for 6 and 12 h. Cell proliferation (n=6) at 6, 12 and 24 h, DNA integrity and membrane permeability (n=4) were determined by a colorimetric assay, gel electrophoresis and flow cytometry, respectively. Proliferation increased in Gr-SYF cultured in a plate at 12 and 24 h whereas it did not change in Gr-F1 cells at any time of culture. DNA fragmentation was observed in Gr-SYF cultured in suspension at 12 h and theca cells at 6 and 12 h but not in Gr-SYF cultured in the plate. Cell permeability and DNA stainability (indicative of apoptosis) were only increased in Gr-SYF cultured in suspension. In conclusion, we have identified culture conditions that will allow the culture of granulosa cells obtained from SYF, the stage prior to selection