Roles of tumor necrosis factor alpha on sperm acrosin activity and acrosome reaction

Aim: To study the roles of tumor necrosis factor alpha (TNF-a)on the sperm acrosin activity and acrosome reaction. Methods:The sperm acrosin activity was tested by the method of BAEE/ADH Unity and the acrosome reaction by the Triple-stain technique. Results: TNF-a decreased the sperm acrosin activityand acrosome reaction (P＜0.01, P＜0.01, respectively);     

Effect of recombinant β-defensin 1 protein on human sperm motility and viability

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of “sperm maturation” during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is “β-defensin 1” which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO₂ atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.     

Cortistatin is endogenous to the human intervertebral disc and exerts in vitro mitogenic effects on annulus cells and a downregulatory effect on TNF-α expression

Background contextCortistatin (CST) is a recently discovered cyclic neuropeptide with biologic anti-inflammatory properties relevant to disc degeneration.PurposeTo test whether CST is present in the disc tissue, whether its expression is influenced by tumor necrosis factor-α (TNF-α), and whether it influences cell proliferation.Study designInstitutional review board–approved study using immunohistochemistry on human disc tissue, in vitro annulus cultures to determine the effect of CST on cell proliferation, and the effect of TNF-α on CST gene expression.Patient sampleDiscs from 12 subjects used for immunohistochemistry, four annulus specimens used for cell culture with proinflammatory cytokines, and 11 used for cell proliferation analyses.Outcome measuresImmunohistochemical localization of CST, gene expression of CST, and cell proliferation analyses.MethodsImmunohistochemistry localized CST in disc tissue. Microarray analysis measured CST gene expression. Human annulus cells were exposed to CST for proliferation tests or cultured for the effect of TNF-α on CST expression. Standard statistical analyses were performed.ResultsImmunohistochemistry identified CST in outer annulus, inner annulus, and nucleus tissue. Annulus cells exposed to TNF-α revealed significantly lower CST expression (p=.013). Exposure to CST significantly increased proliferation. Quantitative real-time polymerase chain reaction also confirmed expression of CST in vitro.ConclusionsData provide the first evidence that CST is present in the human disc. Addition of CST significantly increased cell proliferation. Cortistatin expression was significantly downregulated by TNF-α exposure in vitro. Findings suggest possible in vivo reduction of the anti-inflammatory actions of CST because of elevated proinflammatory cytokines during degenerating disc.     

Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

OBJECTIVE

To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes.

RESEARCH DESIGN AND METHODS

Muscle precursor cells were isolated from four age-matched subject groups: 1) nonobese, normal glucose tolerant; 2) obese, normal glucose tolerant; 3) obese, impaired glucose tolerant; and 4) obese, type 2 diabetes (T2D). The level of inflammation (nuclear factor-κB [NF-κB] signaling) and effect of pharmacological AMPK activation was assessed by Western blots, enzyme-linked immunosorbent assay, and radioactive assays (n = 5 for each subject group).

RESULTS

NF-κB-p65 DNA binding activity was significantly elevated in myocytes from obese T2D patients compared with nonobese control subjects. This correlated to a significant increase in tumor necrosis factor-α concentration in cell culture media. In addition, insulin-stimulated glucose uptake was completely suppressed in myocytes from obese impaired glucose tolerant and T2D subjects. It is interesting that activation of AMPK by A769662 attenuated NF-κB-p65 DNA binding activity in obese T2D cells to levels measured in nonobese myocytes; however, this had no effect on insulin sensitivity of the cells.

CONCLUSIONS

This work provides solid evidence that differentiated human muscle precursor cells maintain in vivo phenotypes of inflammation and insulin resistance and that obesity alone may not be sufficient to establish inflammation in these cells. It is important that we demonstrate an anti-inflammatory role for AMPK in these human cells. Despite attenuation of NF-κB activity by AMPK, insulin resistance in obese T2D cells remained, suggesting factors in addition to inflammation may contribute to the insulin resistance phenotype in muscle cells.It is increasingly recognized that chronic activation of inflammatory pathways in skeletal muscle is a major contributing factor in the pathophysiology of insulin resistance, obesity, and type 2 diabetes (T2D) (1–3). Subsequently, inflammation has been attributed to the increased morbidity and mortality associated with obesity. Skeletal muscle is responsible for 75–80% of glucose disposal in humans (4), and impaired insulin action in this tissue is considered the primary site of whole body insulin resistance. Therefore, development of strategies to reverse or prevent inflammation is important to successfully treat chronic diseases such as T2D. However, because the study of potential strategies in humans is difficult, it is essential that an optimal ex vivo model is established. Muscle precursor cell cultures, established from human muscle biopsies, have been shown to display numerous features of mature skeletal muscle (5) and have been used in a number of studies investigating muscle metabolism in T2D patients. It has been shown that myocytes isolated from individuals with T2D retain their donor phenotype, when differentiated into myocytes in vitro, in terms of deficient insulin signaling and phosphatidylinositol 3-kinase activity (6,7). However, the retention of other in vivo phenotypes such as obesity-associated inflammation has not been demonstrated in these cultures. It is important, therefore, to establish whether muscle precursor cells from obese and T2D volunteers retain their inflammatory phenotype in culture to establish this as a model of muscle inflammation and assess how this can be manipulated to treat metabolic diseases.In obesity, accumulation of fatty acids serves to increase circulating levels of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), via the activation of nuclear factor-κB (NF-κB), which has been shown to impair numerous other signaling pathways regulating skeletal muscle insulin signaling and fatty acid oxidative capacity (2,3,8). NF-κB is activated by multiple proinflammatory stimuli including TNF-α, lipopolysaccharide, and free fatty acids (9–11). NF-κB signaling, therefore, serves as an indicator of inflammatory tone in skeletal muscle. Given the increasing evidence that there is a causal link between inflammation and metabolic diseases, there is considerable interest in developing anti-inflammatory strategies to counteract the suppression in insulin sensitivity induced by obesity-associated inflammation. Recently, AMP-activated protein kinase (AMPK) has been implicated as a modulator of inflammatory responses based on the fact that treatment with AMPK activators suppresses lipopolysaccharide and palmitate-induced NF-κB activity and expression of proinflammatory cytokines in multiple cell types (12–16). The anti-inflammatory role of AMPK in skeletal muscle, however, is poorly defined. In this article, we examine the level of basal inflammation in muscle cells isolated from lean and obese subjects (representative of the whole glucose tolerance continuum) and assess the role that direct pharmacological activation of AMPK plays in attenuating inflammation in these cells.     

Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines,rendering them more susceptible to immune-mediated recognition and lysis

Background

Avelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFβ) receptor 2, which functions as a TGFβ “trap.” Advanced urothelial tumors have been shown to express TGFβ, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFβ from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines.

Darbepoetin alfa (KRN321) is safe and effective when administered subcutaneously once every 2 or 4 weeks to patients on peritoneal dialysis in Japan

Background  

Darbepoetin alfa (KRN321) is a recombinant protein that stimulates erythropoiesis by the same mechanism as endogenous erythropoietin. Due to its longer half-life and greater biological activity than recombinant human erythropoietin (rHuEPO), KRN321 maintains an effective hemoglobin (Hb) level at extended dose intervals compared with rHuEPO. The efficacy and safety of KRN321 administered subcutaneously to patients on peritoneal dialysis (PD) were tested.     

Effect of palbociclib plus endocrine therapy on time to chemotherapy across subgroups of patients with hormone receptor‒positive/human epidermal growth factor receptor 2‒negative advanced breast cancer: Post hoc analyses from PALOMA-2 and PALOMA-3

BackgroundPrevious analyses from the PALOMA-2 and PALOMA-3 studies showed that palbociclib (PAL) plus endocrine therapy (ET) prolongs time to first subsequent chemotherapy (TTC) versus placebo (PBO) plus ET in the overall population of patients with hormone receptor‒positive/human epidermal growth factor receptor 2‒negative (HR+/HER2−) advanced breast cancer (ABC). Here, we evaluated TTC in relevant patient subgroups.MethodsThese post hoc analyses evaluated TTC by subgroup using data from 2 randomized, phase 3 studies of women with HR+/HER2− ABC. In PALOMA-2, postmenopausal patients previously untreated for ABC were randomized 2:1 to receive PAL (125 mg/day, 3/1-week schedule) plus letrozole (LET; 2.5 mg/day; n = 444) or PBO plus LET (n = 222). In PALOMA-3, premenopausal or postmenopausal patients whose disease had progressed after prior ET were randomized 2:1 to receive PAL (125 mg/day, 3/1-week schedule) plus fulvestrant (FUL; 500 mg; n = 347) or PBO plus FUL (n = 174).ResultsFirst subsequent chemotherapy was received by 35.5% and 56.2% in PALOMA-2 and PALOMA-3 after progression on palbociclib plus ET or placebo plus ET. Across all subgroups analyzed, the median progression-free survival (PFS) was longer in the PAL plus ET arm than the PBO plus ET arm. TTC was longer with PAL plus ET versus PBO plus ET across the same patient subgroups in both studies.ConclusionsAcross all subgroups, PAL plus ET versus PBO plus ET had longer median PFS and resulted in prolonged TTC in both the PALOMA-2 and PALOMA-3 studies.Pfizer Inc (NCT01740427, NCT01942135).