Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Maternal Outcomes Following Active vs. Expectant Management of Previable Preterm Pre-Labor Rupture of Membranes: A Meta-Analysis.

The diagnosis of previable preterm pre-labor rupture of membranes (PROM) is known to be associated with poor outcomes for both the mother and the fetus. Following previable preterm PROM, patients are generally offered either active management through the termination of the pregnancy or expectant management to increase the chances of fetal survival. It is difficult to counsel patients because there is a lack of data directly comparing maternal outcomes following active vs. expectant management. Using the data in the current literature, the goal of the present meta-analysis was to determine if there were any differences in terms of maternal risks when active versus elective management was chosen. PubMed, Google Scholar, EMBASE, and Scopus were searched. We found four studies accounting for a total of 506 patients. The risk ratio (RR) of chorioamnionitis in active vs. expectant management was 0.30 (with a 95% confidence interval, CI, of 0.09-1.02). The heterogeneity of the study results was 81% (I2). A sub-analysis of two included studies revealed an RR of postpartum hemorrhage in active vs. expectant management of 0.75 (95% CI 0.27-2.07) and an RR of maternal sepsis of 0.23 (95% CI 0.04-1.28). The heterogeneity of the study results for this sub-analysis was 68% (I2) for postpartum hemorrhage and 0% (I2) for maternal sepsis. Overall, there was no statistically significant difference in the risk of chorioamnionitis, postpartum hemorrhage, or maternal sepsis when active management was chosen over expectant management in previable preterm PROM at <24 weeks. The scarcity and the high heterogeneity of the available data likely contributed to the lack of statistical significance and calls for further work directly comparing maternal outcomes following active vs. expectant management.

Splenocyte transfer from hypertensive donors eliminates premenopausal female protection from ANG II-induced hypertension.

Premenopausal females are protected from angiotensin II (ANG II)-induced hypertension following the adoptive transfer of T cells from normotensive donors. For the present study, we hypothesized that the transfer of hypertensive T cells (HT) or splenocytes (HS) from hypertensive donors would eliminate premenopausal protection from hypertension. Premenopausal recombination-activating gene-1 (Rag-1)-/- females received either normotensive (NT) or hypertensive cells 3 wk before ANG II infusion (14 days, 490 ng/kg/min). Contrary to our hypothesis, no increase in ANG II-induced blood pressure was observed in the NT/ANG or HT/ANG groups. Flow cytometry demonstrated that renal FoxP3+ T regulatory cells were significantly decreased, and immunohistochemistry showed an increase in renal F4/80+ macrophages in the HT/ANG group, suggesting a shift in the renal inflammatory environment despite no change in blood pressure. Renal mRNA expression of macrophage chemoattractant protein-1 (MCP-1), endothelin-1 (ET-1), and G protein-coupled estrogen receptor-1 (GPER-1) was significantly decreased in the HT/ANG group. The adoptive transfer of hypertensive splenocytes before ANG II infusion (HS/ANG) eliminated premenopausal protection from hypertension and significantly decreased splenic FoxP3+ T regulatory cells compared with females that received normotensive splenocytes (NS/ANG). Expression of macrophage inflammatory protein 1α/chemokine (C-C motif) ligand 3 (MCP-1/CCL3), a potent macrophage chemokine, was elevated in the HS/ANG group; however, no increase in renal macrophage infiltration occurred. Together, these data show that in premenopausal females, T cells from hypertensive donors are not sufficient to induce robust ANG II-mediated hypertension; in contrast, transfer of hypertensive splenocytes (consisting of T/B lymphocytes, dendritic cells, and macrophages) is sufficient. Further work is needed to understand how innate and adaptive immune cells and estrogen signaling coordinate to cause differential hypertensive outcomes in premenopausal females.NEW & NOTEWORTHY Our study is the first to explore the role of hypertensive T cells versus hypertensive splenocytes in premenopausal protection from ANG II-induced hypertension. We show that the hypertensive status of T cell donors does not impact blood pressure in the recipient female. However, splenocytes, when transferred from hypertensive donors, significantly increased premenopausal recipient blood pressure following ANG II infusion, highlighting the importance of further investigation into estrogen signaling and immune cell activation in females.

Menopause and FOXP3+ Treg cell depletion eliminate female protection against T cell-mediated angiotensin II hypertension.

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1-/- mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3+ T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1-/- menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females.NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

Sex-Specific Mechanisms in Inflammation and Hypertension.

PURPOSE OF REVIEW: Despite enhanced screening and therapeutic management, hypertension remains the most prevalent chronic disease in the United States and the leading cause of heart disease, chronic kidney disease, and stroke in both men and women. It is widely accepted that hypertension is a pro-inflammatory disease and that the immune system plays a vital role in mediating hypertensive outcomes and end organ damage. Despite known discrepancies in the risk of hypertension development between men and women, preclinical models of immune-mediated hypertension were historically developed solely in male animals, leading to a lack of sex-specific clinical practice guidelines or therapeutic targets. RECENT FINDINGS: Following the NIH policy on the consideration of sex as a biological variable in 2015, significant advancements have been made into sex-specific disease mechanisms in inflammation and hypertension. This review article serves to critically evaluate recent advancements in the field of sex-specific immune-mediated hypertension.