Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Through chemical screening, we identified a pyrazolone that reversibly blocked the activation of phagocyte oxidase (phox) in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spared activation of phox by phorbol ester or bacteria, bacterial killing, TNF-induced granule exocytosis and phox assembly, and endothelial transmigration. We traced the pyrazolone's mechanism of action to inhibition of TNF-induced intracellular Ca2+ elevations, and identified a nontransmembrane ("soluble") adenylyl cyclase (sAC) in neutrophils as a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazolone's effect on phox. Both compounds blocked TNF-induced activation of Rap1A, a phox-associated guanosine triphosphatase that is regulated by cAMP. Thus, TNF turns on phox through a Ca2+-triggered, sAC-dependent process that may involve activation of Rap1A. This pathway may offer opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function.     

Regional distribution of antibodies to herpes simplex virus type 1 (HSV-1) and HSV-2 in men and women in Ontario,Canada

This study estimated the regional and age- and gender-specific seroprevalences of herpes simplex virus type 1 (HSV-1) and HSV-2 in Ontario, Canada. Stored serum specimens from subjects aged 15 to 44 years, including men (n = 979), women not under prenatal care (n = 638), and women under prenatal care (n = 701) submitted for routine viral serology were randomly selected according to regional population size from public health laboratories. HSV-1 and HSV-2 testing was done with the MRL enzyme immunoassay (EIA) (Focus Technologies), and HSV-2 was also tested by the Gull/Meridian EIA. Specimens discordant for HSV-2 antibodies between the two EIAs were resolved by a recombinant immunoblot assay (Focus Technologies). The overall age- and gender-standardized seroprevalences of HSV-1 and HSV-2 were 51.1% (95% confidence interval [CI], 50.1 to 52.1) and 9.1% (95% CI, 8.6 to 9.7), respectively. The seroprevalence of HSV-1 antibodies increased from 26.9 to 54.7% in men between 15 to 16 and 40 to 44 years of age, from 32.0 to 88.7% in women not under prenatal care, and from 55.2 to 69.2% in women under prenatal care. The seroprevalence of HSV-2 increased from 3.8 to 21.3% in men between 15 to 16 and 40 to 44 years of age, from 0 to 18.9% in women not under prenatal care, and from 3.4 to 23.1% in women under prenatal care. HSV-2 results were discordant for 3.3% (76 of 2,318) of specimens. Both types of HSV antibodies appeared to be acquired earlier among women under prenatal care than among men and women not under prenatal care. Antibodies were more prevalent among people in northern Ontario (72.9% of subjects [range, 68.4 to 77.4%] for HSV-1 and 13.7% of subjects [95% CI, 10.2 to 17.2%] for HSV-2) than elsewhere.     

Normalization of the tumor microenvironment: evidence for tissue inhibitor of metalloproteinase-2 as a cancer therapeutic

Matrix metalloproteinases (MMPs) are members of the Metzincin family of proteases responsible for degrading the extracellular matrix (ECM). In early studies, MMP degradation of the sub-epithelial basement membrane was thought to be tumor cell autonomous and contribute to the invasive behavior of malignant cells. It is now recognized that MMPs have multiple roles that can either promote or inhibit tumor progression and metastasis. The endogenous inhibitors of the MMPs are the tissue inhibitors of metalloproteinases (TIMPs). Early studies on the tumor microenvironment revealed TIMP function to be principally through the inhibition of MMPs, thereby blocking tumor cell migration and invasion. However, data from a number of laboratories are now reporting that TIMPs have direct cellular functions, independent of their MMP inhibitory activity. The TIMPs can modulate normal tissue physiology and development, as well as pathology and progression in a variety of acute and chronic disease states. In this review, we briefly describe the role of MMPs and TIMPs in ECM turnover and formation of the tumor microenvironment. Based on the evidence presented, we postulate that TIMP-2 and other soluble components of the normal ECM may provide a novel therapeutic approach to cancer treatment through “normalization” of the tumor microenvironment.     

Chemical inhibitors of TNF signal transduction in human neutrophils point to distinct steps in cell activation

Chemical screening identified three small compounds that selectively inhibited activation of the respiratory burst (RB) of human neutrophils in response to tumor necrosis factor (TNF) and formylated peptide but not phorbol ester and spared the ability of neutrophils to kill bacteria. These compounds partially inhibited TNF-triggered cytoskeletal rearrangements without blocking adhesion or transmigation of polymorphonuclear neutrophils through TNF-activated monolayers of endothelial cells. The compounds were nontoxic to neutrophils and endothelial cells. They had no direct inhibitory effect on the tyrosine kinases Src, Syk, or Pyk2. However, their differential effects on cell spreading, bacteria-induced RB, TNF-induced degranulation, TNF-induced protein tyrosine phosphorylation, and TNF-induced Syk activation suggested that each may act on different elements of neutrophil signaling pathways.     

Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages

The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha were validated using QPCR primers and probes which were generated for the equine IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these conditions the amplification efficiencies of the primers and probes ranged from 99% to 101%. The mean coefficient of variation (CV) across five sets of plasmid DNA for both intra-assay and inter-assay variation was 0.63% (range 0.2% to 1.8%). Amplification efficiency was also determined using 2-fold dilutions of cDNA and under these conditions amplification efficiency ranged from 83% to 95%. The specificity of amplification was confirmed by DNA sequencing of reaction products. The QPCR assays were also evaluated using three sets of cDNA from equine monocyte derived macrophages (EMDM) stimulated for 1 h with lipopolysaccharide (LPS). The general trend was the same for all three samples with IL-1alpha showing the greatest induction and IL-6 the lowest induction. The range of cytokine induction was greater than has previously been reported with values ranging from 12-fold to 30,000-fold. We present a set of QPCR primers and probes that are suitable for quantitation of expression of a set of equine cytokines. The primers and probes have been rigorously analyzed, and we demonstrate that they are specific for the desired genes, have a high amplification efficiency and the assays are highly reproducible.     

Impact of innervation and exercise on muscle regeneration in neovascularized muscle grafts in rats.

The direction of known staged process of regeneration of free muscle grafts was inverted in our experimental rat model from a centripetal to a centrifugal by central implantation of blood vessels into isolated free muscle grafts. The effects of innervation, reinnervation and exercise on muscle fiber regeneration were analyzed at various intervals from 4 to 90 days by morphological and morphometric methods. Reinnervation occurred as well in grafts with the motor nerve left intact as it did in grafts with a severed and reimplanted nerve. Reinnervation proved to be prerequisite for a lasting muscle regeneration. Denervated muscle grafts even after neovascularization underwent irreversible fibrosis. A positive effect of exercise on the early states (30 days) of muscle regeneration was revealed by morphometrical analysis. In the long term (90 days) fiber diameter assimilated in all groups. The animal model mimics a clinical situation of flap prefabrication demonstrating the relationship of functional tissue regeneration and neovascularization. It can be transferred into the acute clinical situation as well as in tissue engineering.     

Maternal self-medication and provision of nevirapine to newborns by women in Rakai, Uganda

To assess the effectiveness of maternal self-administration of nevirapine for prevention of mother-to-child transmission (MTCT) of HIV, we conducted a program to provide maternal and newborn doses of nevirapine to pregnant women in rural Uganda. Women provided blood for HIV testing and were offered voluntary counseling and testing (VCT) during annual community HIV surveys. HIV-positive women who accepted VCT were offered nevirapine tablets and syrup. Blood samples were collected postpartum from women and their babies. Infants were tested for HIV by polymerase chain reaction (PCR), and a subsample of maternal and infant blood was assayed for nevirapine. Among the 981 women tested for HIV, 900 (91.7%) accepted VCT, of whom 105 (11.7%) were HIV-positive. Ninety-three women accepted nevirapine, of whom 81 (87.1%) were followed postpartum; 75 (92.6%) reported receipt of the drug, and 69 reported taking the tablets (85.2%). There were 81 liveborn babies (3 sets of twins), and 67 (84.8%) received the syrup. In a subsample of 25 mothers reporting receipt of the drug, nevirapine was detected in 22 (88.0%) and 24 (96.0%) babies tested. PCR of 67 infant blood samples identified 5 HIV-positive (MTCT rate = 7.5%, 95% confidence interval [CI]: 0.3%-16.6%). Mothers can administer nevirapine to themselves and their newborns and can achieve low rates of perinatal HIV infection.     

Effect of mortality from COVID-19 on inpatient outcomes

When hospitals first encountered coronavirus disease 2019 (COVID-19), there was a dearth of therapeutic options and nearly 1 in 3 patients died from the disease. By the summer of 2020, as deaths from the disease declined nationally, multiple single-center studies began to report declining mortality of patients with COVID-19. To evaluate the effect of COVID-19 on hospital-based mortality, we searched the Vizient Clinical Data Base for outcomes data from approximately 600 participating hospitals, including 130 academic medical centers, from January 2017 through December 2020. More than 32 million hospital admissions were included in the analysis. After an initial spike, mortality from COVID-19 declined in all regions of the country to under 10% by June 2020 and remained constant for the remainder of the year. Despite this, inpatient, all-cause mortality has increased since the beginning of the pandemic, even those without respiratory failure. Inpatient mortality has particularly increased in elderly patients and in those requiring intubation for respiratory failure. Since June 2020, COVID-19 kills one in every 10 patients admitted to the hospital with this diagnosis. The addition of this new disease has raised overall hospital mortality especially those who require intubation for respiratory failure.