High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×106 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×108 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 106 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Effects of Pharmacological Inhibitors of NADPH Oxidase on Myogenic Contractility and Evoked Vasoactive Responses in Rat Resistance Arteries.

Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide, are reported to contribute to the dynamic regulation of contractility in various arterial preparations, however, the situation in pressurized, myogenically active resistance arteries is much less clear. In the present study, we have utilized established pharmacological inhibitors of NADPH oxidase activity to examine the potential contribution of ROS to intrinsic myogenic contractility in adult Sprague-Dawley rat resistance arteries and responses to vasoactive agents acting via the endothelium (i.e., acetylcholine, SKA-31) or smooth muscle (i.e., sodium nitroprusside, phenylephrine). In cannulated and pressurized cremaster skeletal muscle and middle cerebral arteries, the NOX inhibitors 2-acetylphenothiazine (2-APT) and VAS2870, selective for NOX1 and NOX2, respectively, evoked concentration-dependent inhibition of basal myogenic tone in a reversible and irreversible manner, respectively, whereas the non-selective inhibitor apocynin augmented myogenic contractility. The vasodilatory actions of 2-APT and VAS2870 occurred primarily via the vascular endothelium and smooth muscle, respectively. Functional responses to established endothelium-dependent and -independent vasoactive agents were largely unaltered in the presence of either 2-APT or apocynin. In cremaster arteries from Type 2 Diabetic (T2D) Goto-Kakizaki rats with endothelial dysfunction, treatment with either 2-APT or apocynin did not modify stimulus-evoked vasoactive responses, but did affect basal myogenic tone. These same NOX inhibitors produced robust inhibition of total NADPH oxidase activity in aortic tissue homogenates from control and T2D rats, and NOX isozymes 1, 2 and 4, along with superoxide dismutase 1, were detected by qPCR in cremaster arteries and aorta from both species. Based on the diverse effects that we observed for established, chemically distinct NOX inhibitors, the functional contribution of vascular NADPH oxidase activity to stimulus-evoked vasoactive signaling in myogenically active, small resistance arteries remains unclear.

Human mesenchymal stem cells inhibit the differentiation and effector functions of monocytes.

Although monocytes represent an essential part of the host defence system, their accumulation and prolonged stimulation could be detrimental and may aggravate chronic inflammatory diseases. The present study has explored the less-understood immunomodulatory effects of mesenchymal stem cells on monocyte functions. Isolated purified human monocytes were co-cultured with human umbilical cord-derived mesenchymal stem cells under appropriate culture conditions to assess monocytes' vital functions. Based on the surface marker analysis, mesenchymal stem cells halted monocyte differentiation into dendritic cells and macrophages and reduced their phagocytosis functions, which rendered an inability to stimulate T-cell proliferation. The present study confers that mesenchymal stem cells exerted potent immunosuppressive activity on monocyte functions such as differentiation, phagocytosis and Ag presentation; hence, they promise a potential therapeutic role in down-regulating the unwanted monocyte-mediated immune responses in the context of chronic inflammatory diseases.

Human Mesenchymal Stem Cells-mediated Transcriptomic Regulation of Leukemic Cells in Delivering Anti-tumorigenic Effects.

Treatment of leukemia has become much difficult because of resistance to the existing anticancer therapies. This has thus expedited the search for alternativ therapies, and one of these is the exploitation of mesenchymal stem cells (MSCs) towards control of tumor cells. The present study investigated the effect of human umbilical cord-derived MSCs (UC-MSCs) on the proliferation of leukemic cells and gauged the transcriptomic modulation and the signaling pathways potentially affected by UC-MSCs. The inhibition of growth of leukemic tumor cell lines was assessed by proliferation assays, apoptosis and cell cycle analysis. BV173 and HL-60 cells were further analyzed using microarray gene expression profiling. The microarray results were validated by RT-qPCR and western blot assay for the corresponding expression of genes and proteins. The UC-MSCs attenuated leukemic cell viability and proliferation in a dose-dependent manner without inducing apoptosis. Cell cycle analysis revealed that the growth of tumor cells was arrested at the G0/G1 phase. The microarray results identified that HL-60 and BV173 share 35 differentially expressed genes (DEGs) (same expression direction) in the presence of UC-MSCs. In silico analysis of these selected DEGs indicated a significant influence in the cell cycle and cell cycle-related biological processes and signaling pathways. Among these, the expression of DBF4, MDM2, CCNE2, CDK6, CDKN1A, and CDKN2A was implicated in six different signaling pathways that play a pivotal role in the anti-tumorigenic activity exerted by UC-MSCs. The UC-MSCs perturbate the cell cycle process of leukemic cells via dysregulation of tumor suppressor and oncogene expression.

SKA-31, an activator of Ca2+-activated K+ channels, improves cardiovascular function in aging.

Aging represents an independent risk factor for the development of cardiovascular disease, and is associated with complex structural and functional alterations in the vasculature, such as endothelial dysfunction. Small- and intermediate-conductance, Ca2+-activated K+ channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacological activators of these channels induce robust vasodilation upon acute exposure in isolated arteries and intact animals. However, the effects of prolonged in vivo administration of such compounds are unknown. In our study, we hypothesized that such treatment would ameliorate aging-related cardiovascular deficits. Aged (∼18 months) male Sprague Dawley rats were treated daily with either vehicle or the KCa channel activator SKA-31 (10 mg/kg, intraperitoneal injection; n = 6/group) for 8 weeks, followed by echocardiography, arterial pressure myography, immune cell and plasma cytokine characterization, and tissue histology. Our results show that SKA-31 administration improved endothelium-dependent vasodilation, reduced agonist-induced vascular contractility, and prevented the aging-associated declines in cardiac ejection fraction, stroke volume and fractional shortening, and further improved the expression of endothelial KCa channels and associated cell signalling components to levels similar to those observed in young male rats (∼5 months at end of study). SKA-31 administration did not promote pro-inflammatory changes in either T cell populations or plasma cytokines/chemokines, and we observed no overt tissue histopathology in heart, kidney, aorta, brain, liver and spleen. SKA-31 treatment in young rats had little to no effect on vascular reactivity, select protein expression, tissue histology, plasma cytokines/chemokines or immune cell properties. Collectively, these data demonstrate that administration of the KCa channel activator SKA-31 improved aging-related cardiovascular function, without adversely affecting the immune system or promoting tissue toxicity.

Pharmacological Targeting of KCa Channels to Improve Endothelial Function in the Spontaneously Hypertensive Rat.

Systemic hypertension is a major risk factor for the development of cardiovascular disease and is often associated with endothelial dysfunction. KCa2.3 and KCa3.1 channels are expressed in the vascular endothelium and contribute to stimulus-evoked vasodilation. We hypothesized that acute treatment with SKA-31, a selective activator of KCa2.x and KCa3.1 channels, would improve endothelium-dependent vasodilation and transiently lower mean arterial pressure (MAP) in male, spontaneously hypertensive rats (SHRs). Isolated vascular preparations exhibited impaired vasodilation in response to bradykinin (i.e., endothelial dysfunction) compared with Wistar controls, which was associated with decreased bradykinin receptor expression in mesenteric arteries. In contrast, similar levels of endothelial KCa channel expression were observed, and SKA-31 evoked vasodilation was comparable in vascular preparations from both strains. Addition of a low concentration of SKA-31 (i.e., 0.2-0.3 µM) failed to augment bradykinin-induced vasodilation in arteries from SHRs. However, responses to acetylcholine were enhanced. Surprisingly, acute bolus administration of SKA-31 in vivo (30 mg/kg, i.p. injection) modestly elevated MAP compared with vehicle injection. In summary, pharmacological targeting of endothelial KCa channels in SHRs did not readily reverse endothelial dysfunction in situ, or lower MAP in vivo. SHRs thus appear to be less responsive to endothelial KCa channel activators, which may be related to their vascular pathology.

Maternal Cognitive Impairment Associated with Gestational Diabetes Mellitus-A Review of Potential Contributing Mechanisms.

Gestational diabetes mellitus (GDM) carries many risks, where high blood pressure, preeclampsia and future type II diabetes are widely acknowledged, but less focus has been placed on its effect on cognitive function. Although the multifactorial pathogenesis of maternal cognitive impairment is not completely understood, it shares several features with type 2 diabetes mellitus (T2DM). In this review, we discuss some key pathophysiologies of GDM that may lead to cognitive impairment, specifically hyperglycemia, insulin resistance, oxidative stress, and neuroinflammation. We explain how these incidents: (i) impair the insulin-signaling pathway and/or (ii) lead to cognitive impairment through hyperphosphorylation of τ protein, overexpression of amyloid-ß and/or activation of microglia. The aforementioned pathologies impair the insulin-signaling pathway primarily through serine phosphorylation of insulin receptor substances (IRS). This then leads to the inactivation of the phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT) signaling cascade, which is responsible for maintaining brain homeostasis and normal cognitive functioning. PI3K/AKT is crucial in maintaining normal cognitive function through the inactivation of glycogen synthase kinase 3ß (GSκ3ß), which hyperphosphorylates τ protein and releases pro-inflammatory cytokines that are neurotoxic. Several biomarkers were also highlighted as potential biomarkers of GDM-related cognitive impairment such as AGEs, serine-phosphorylated IRS-1 and inflammatory markers such as tumor necrosis factor α (TNF-α), high-sensitivity C-reactive protein (hs-CRP), leptin, interleukin 1ß (IL-1ß), and IL-6. Although GDM is a transient disease, its complications may be long-term, and hence increased mechanistic knowledge of the molecular changes contributing to cognitive impairment may provide important clues for interventional strategies.

Programming of Vascular Dysfunction in the Intrauterine Milieu of Diabetic Pregnancies.

With the rising global tide of obesity, gestational diabetes mellitus (GDM) burgeoned into one of the most common antenatal disorders worldwide. Macrosomic babies born to diabetic mothers are more likely to develop risk factors for cardiovascular disease (CVD) before they reach adulthood. Rodent studies in offspring born to hyperglycemic pregnancies show vascular dysfunction characterized by impaired nitric oxide (NO)-mediated vasodilation and increased production of contractile prostanoids by cyclooxygenase 2 (COX-2). Vascular dysfunction is a key pathogenic event in the progression of diabetes-related vascular disease, primarily attributable to glucotoxicity. Therefore, glucose-induced vascular injury may stem directly from the hyperglycemic intrauterine environment of GDM pregnancy, as evinced by studies showing endothelial activation and inflammation at birth or in childhood in offspring born to GDM mothers. This review discusses potential mechanisms by which intrauterine hyperglycemia programs dysfunction in the developing vasculature.

SKA-31, an activator of endothelial Ca2+-activated K+ channels evokes robust vasodilation in rat mesenteric arteries.

It is now well recognized that endothelial KCa2.3 and KCa3.1 channel activities contribute to dilation of resistance arteries via endothelium-mediated hyperpolarization and vascular smooth muscle relaxation. In this study, we have investigated the functional effect of the KCa channel activator SKA-31 in third order rat mesenteric arteries using arterial pressure myography. Isolated arteries were cannulated, pressurized intraluminally to 70 mmHg at 36 °C and then constricted with 1 µM phenylephrine. Acute bath exposure to SKA-31 evoked a robust and reversible inhibition of developed tone (IC50 = 0.22 µM). The vasodilatory effects of SKA-31 and acetylcholine were blunted in the presence of KCa2.3 and KCa3.1 channel antagonists, and were largely prevented following endothelial denudation. Western blot and q-PCR analyses of isolated mesenteric arteries revealed KCa2.3 and KCa3.1 channel expression at the protein and mRNA levels, respectively. Penitrem-A, an inhibitor of KCa1.1 channels, decreased vasodilatory responses to acetylcholine, sodium nitroprusside and NS-1619, but had little effect on SKA-31. Similarly, bath exposure to the eNOS inhibitor L-NAME did not alter SKA-31 and acetylcholine-mediated vasodilation. Collectively, these data highlight the major cellular mechanisms by which the endothelial KCa channel activator SKA-31 inhibits agonist-evoked vasoconstriction in rat small mesenteric arteries.

Pharmacologic targeting of endothelial Ca2+-activated K+ channels: A strategy to improve cardiovascular function.

Endothelial small and intermediate-conductance, Ca2+-activated K+ channels (KCa2.3 and KCa3.1, respectively) play an important role in the regulation of vascular function and systemic blood pressure. Growing evidence indicates that they are intimately involved in agonist-evoked vasodilation of small resistance arteries throughout the circulation. Small molecule activators of KCa2.x and 3.1 channels, such as SKA-31, can acutely inhibit myogenic tone in isolated resistance arteries, induce effective vasodilation in intact vascular beds, such as the coronary circulation, and acutely decrease systemic blood pressure in vivo. The blood pressure-lowering effect of SKA-31, and early indications of improvement in endothelial dysfunction suggest that endothelial KCa channel activators could eventually be developed into a new class of endothelial targeted agents to combat hypertension or atherosclerosis. This review summarises recent insights into the activation of endothelial Ca2+ activated K+ channels in various vascular beds, and how tools, such as SKA-31, may be beneficial in disease-related conditions.

The vascular endothelium: A regulator of arterial tone and interface for the immune system.

As the primary interface between the blood and various tissues of the body, the vascular endothelium exhibits a diverse range of roles and activities, all of which contribute to the overall health and function of the cardiovascular system. In this focused review, we discuss several key aspects of endothelial function, how this may be compromised and subsequent consequences. Specifically, we examine the dynamic regulation of arterial contractility and distribution of blood flow through the generation of chemical and electrical signaling events that impinge upon vascular smooth muscle. The endothelium can generate a diverse range of vasoactive compounds and signals, most of which act locally to adjust blood flow in a dynamic fashion to match tissue metabolism. Disruption of these vascular signaling processes (e.g. reduced nitric oxide bioavailability) is typically referred to as endothelial dysfunction, which is a recognized risk factor for cardiovascular disease in patients and occurs early in the development and progression of hypertension, atherosclerosis and tissue ischemia. Endothelial dysfunction is also associated with type-2 Diabetes and aging and increased mechanistic knowledge of the cellular changes contributing to these effects may provide important clues for interventional strategies. The endothelium also serves as the initial site of interaction for immune cells entering tissues in response to damage and acts to facilitate the actions of both the innate and acquired immune systems to interact with the vascular wall. In addition to representing the main cell type responsible for the formation of new blood vessels (i.e. angiogenesis) within the vasculature, the endothelium is also emerging as a source of extracellular vesicle or microparticles for the transport of signaling molecules and other cellular materials to nearby, or remote, sites in the body. The characteristics of released microparticles appear to change with the functional status of the endothelium; thus, these microparticles may represent novel biomarkers of endothelial health and more serious cardiovascular disease.

Understanding the mode-of-action of Cassia auriculata via in silico and in vivo studies towards validating it as a long term therapy for type II diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: Cassia auriculata (CA) is used as an antidiabetic therapy in Ayurvedic and Siddha practice. This study aimed to understand the mode-of-action of CA via combined cheminformatics and in vivo biological analysis. In particular, the effect of 10 polyphenolic constituents of CA in modulating insulin and immunoprotective pathways were studied. MATERIALS AND METHODS: In silico target prediction was first employed to predict the probability of the polyphenols interacting with key protein targets related to insulin signalling, based on a model trained on known bioactivity data and chemical similarity considerations. Next, CA was investigated in in vivo studies where induced type 2 diabetic rats were treated with CA for 28 days and the expression levels of genes regulating insulin signalling pathway, glucose transporters of hepatic (GLUT2) and muscular (GLUT4) tissue, insulin receptor substrate (IRS), phosphorylated insulin receptor (AKT), gluconeogenesis (G6PC and PCK-1), along with inflammatory mediators genes (NF-κB, IL-6, IFN-γ and TNF-α) and peroxisome proliferators-activated receptor gamma (PPAR-γ) were determined by qPCR. RESULTS: In silico analysis shows that several of the top 20 enriched targets predicted for the constituents of CA are involved in insulin signalling pathways e.g. PTPN1, PCK-α, AKT2, PI3K-γ. Some of the predictions were supported by scientific literature such as the prediction of PI3K for epigallocatechin gallate. Based on the in silico and in vivo findings, we hypothesized that CA may enhance glucose uptake and glucose transporter expressions via the IRS signalling pathway. This is based on AKT2 and PI3K-γ being listed in the top 20 enriched targets. In vivo analysis shows significant increase in the expression of IRS, AKT, GLUT2 and GLUT4. CA may also affect the PPAR-γ signalling pathway. This is based on the CA-treated groups showing significant activation of PPAR-γ in the liver compared to control. PPAR-γ was predicted by the in silico target prediction with high normalisation rate although it was not in the top 20 most enriched targets. CA may also be involved in the gluconeogenesis and glycogenolysis in the liver based on the downregulation of G6PC and PCK-1 genes seen in CA-treated groups. In addition, CA-treated groups also showed decreased cholesterol, triglyceride, glucose, CRP and Hb1Ac levels, and increased insulin and C-peptide levels. These findings demonstrate the insulin secretagogue and sensitizer effect of CA. CONCLUSION: Based on both an in silico and in vivo analysis, we propose here that CA mediates glucose/lipid metabolism via the PI3K signalling pathway, and influence AKT thereby causing insulin secretion and insulin sensitivity in peripheral tissues. CA enhances glucose uptake and expression of glucose transporters in particular via the upregulation of GLUT2 and GLUT4. Thus, based on its ability to modulate immunometabolic pathways, CA appears as an attractive long term therapy for T2DM even at relatively low doses.

Animal Model of Gestational Diabetes Mellitus with Pathophysiological Resemblance to the Human Condition Induced by Multiple Factors (Nutritional, Pharmacological, and Stress) in Rats.

This study attempts to develop an experimental gestational diabetes mellitus (GDM) animal model in female Sprague-Dawley rats. Rats were fed with high fat sucrose diet, impregnated, and induced with Streptozotocin and Nicotinamide on gestational day 0 (D0). Sleeping patterns of the rats were also manipulated to induce stress, a lifestyle factor that contributes to GDM. Rats were tested for glycemic parameters (glucose, C-peptide, and insulin), lipid profiles (total cholesterol, triglycerides, HDL, and LDL), genes affecting insulin signaling (IRS-2, AKT-1, and PCK-1), glucose transporters (GLUT-2 and GLUT-4), proinflammatory cytokines (IL-6, TNF-α), and antioxidants (SOD, CAT, and GPX) on D6 and D21. GDM rats showed possible insulin resistance as evidenced by high expression of proinflammatory cytokines, PCK-1 and CRP. Furthermore, low levels of IRS-2 and AKT-1 genes and downregulation of GLUT-4 from the initial to final phases indicate possible defect of insulin signaling. GDM rats also showed an impairment of antioxidant status and a hyperlipidemic state. Additionally, GDM rats exhibited significantly higher body weight and blood glucose and lower plasma insulin level and C-peptide than control. Based on the findings outlined, the current GDM animal model closely replicates the disease state in human and can serve as a reference for future investigations.

Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus.

AIM: To assess the amount and pattern of reactive oxygen species (ROS) production in diabetic patient-derived neutrophils. METHODS: Blood samples from type 2 diabetes mellitus (DM) patients and volunteers (controls) were subjected to neutrophil isolation and the assessment of neutrophil oxidative burst using chemiluminescence assay. Neutrophils were activated by using phorbol myristate acetate (PMA) and neutrophils without activation were kept as a negative control. The chemiluminescence readings were obtained by transferring cell suspension into a 1.5 mL Eppendorf tube, with PMA and luminol. Reaction mixtures were gently vortexed and placed inside luminometer for a duration of 5 min. RESULTS: Our results showed that in the resting condition, the secretion of ROS in normal non-diabetic individuals was relatively low compared to diabetic patients. However, the time scale observation revealed that the secreted ROS declined accordingly with time in non-diabetic individuals, yet such a reduction was not detected in diabetic patients where at all the time points, the secretion of ROS was maintained at similar magnitudes. This preliminary study demonstrated that ROS production was significantly higher in patients with DM compared to non-diabetic subjects in both resting and activated conditions. CONCLUSION: The respiratory burst activity of neutrophils could be affected by DM and the elevation of ROS production might be an aggravating factor in diabetic-related complications.

Enhanced CD4+CD25+ regulatory T cells with splenic proliferation and protection against oxidative stress by nicotinamide in gestational diabetes.

Gestational diabetes mellitus (GDM) is a common complication during pregnancy. Metabolic changes in GDM affect fetal development and fetal glucose homeostasis. Several complications of diabetes are related to increased intracellular oxidative stress where prooxidants exceed antioxidant capacity. The present study was initiated to evaluate the effects of nicotinamide on CD4+CD25+ regulatory T cells (Tregs), proliferation of splenocytes, production of reactive oxygen species (ROS) by neutrophils and serum glucose levels. Changes in mRNA levels of two antioxidant genes in liver, viz, superoxide dismutase (SOD1) and catalase (CAT) were quantified with real-time PCR (QRT-PCR). Nicotinamide (50, 100 and 200 mg/kg) was supplemented p.o. to pregnant diabetic rats from days 6 through 20 of gestation. The highest dose enhanced expression of Tregs and increased splenocytes proliferation in both resting and lipopolysaccharide (LPS)-stimulated cells. Oxidative burst activity of neutrophils in response to phorbol myristate acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP) or E. coli activation was reduced. mRNA expressions of superoxide dismutase (SOD) and catalase (CAT) genes were upregulated by nicotinamide. In summary, nicotinamide boosted the immune system through stimulation of adaptive immune cells with enhancement of antioxidant defences and reduced production of ROS. Serum glucose level was normalised by nicotinamide (200 mg/kg). These findings provide evidence for usage of nicotinamide as a supplement or as adjunct to therapeutic agents in gestational diabetes and in pregnant individuals with weakened immune systems.

Immunomodulatory activity of polyphenols derived from Cassia auriculata flowers in aged rats.

The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats. Rats (24-26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and B cells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulated cells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, and CD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementation reduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation. Our results collectively show that polyphenols derived from CA boost T cell immunity by increasing the number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutrophils that could potentially harm multiple biological systems in aged individuals.