Disruption of the PGE2 synthesis / response pathway restrains atherogenesis in programmed cell death-1 (Pd-1) deficient hyperlipidemic mice.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs limits their clinical utility. Combinatorial strategies enhance their antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E2, formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE2 synthesis /response pathway - global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4 - on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr-/-) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE2 biosynthesis, reflected by its major urinary metabolite, PGE2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4-/- mice was impaired. Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.

Impact of Social Restrictions During COVID-19 on the Aquatic Levels of Antimicrobials and Other Drugs in Delhi.

The relative contribution of factors responsible for the environmental exposure of active pharmaceutical ingredients (APIs) is of interest for appropriate remedial measures. This study was carried out to evaluate the post-lockdown levels of APIs in water resources, in comparison to our previously published study from 2016. The environmental levels of 28 drugs from different classes were analyzed in surface water (Yamuna River), aquifers, and leachate samples collected from 26 locations in Delhi-NCR using the previously validated liquid chromatography-mass spectrometry (LC-MS/MS) methods. In addition, the prevalence of antimicrobial resistance in coliforms isolated from targeted surface water samples was also studied. This study revealed that more than 90% of APIs, including antibiotics, decreased drastically in both surface water and aquifers compared to our previous data. Selected samples subjected to antimicrobial resistance (AMR) analysis revealed the presence of cephalosporin-resistant coliform bacteria. Tracing cephalosporins in the surface and drain water samples revealed the presence of ceftriaxone in the drain and water samples from Yamuna River. Higher levels of ceftriaxone in landfill leachate were also found, which were found to be associated with coliform resistance and indicate the un-segregated disposal of medical waste into landfills. Social restrictions enforced due to COVID-19 resulted in a drastic decrease in antimicrobials and other APIs in aquatic water resources. Increased ceftriaxone and cephalosporin resistance was seen in coliform from surface water and drain, indicating the possibility of hospital waste and treatment-related drugs entering Yamuna River. Enforcement of the regulations for the safe disposal of antibiotics at hospitals and preliminary disinfection of hospital sewage before its inflow into common drains might help minimize the spread of antibiotic resistance in the environment.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

PGF2α signaling drives fibrotic remodeling and fibroblast population dynamics in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-ß1-independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr-/-) line showed attenuated weight loss and gene dosage-dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Disruption of Prostaglandin F2α Receptor Signaling Attenuates Fibrotic Remodeling and Alters Fibroblast Population Dynamics in A Preclinical Murine Model of Idiopathic Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptfgr) are implicated as a TGFß1 independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER - SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen treated IER-SftpcI73T mice develop an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr null (FPr-/-) line showed attenuated weight loss and gene dosage dependent rescue of mortality compared to FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single cell RNA sequencing, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α/FPr dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Ocular surface sphingolipids associate with the refractory nature of vernal keratoconjunctivitis: newer insights in VKC pathogenesis.

BACKGROUND: The etiopathogenesis of vernal keratoconjunctivitis (VKC) is incompletely understood. Bioactive lipids play a key role in allergic disorders. This study focused on the sphingolipid metabolism on the ocular surface of VKC and to explore if it has a contributory role in the refractoriness of the disease. METHODS: Active VKC cases, (n=87) (classified as mild/moderate and severe/very severe based on the disease symptoms) and age-matched healthy controls (n=60) were recruited as part of a 2-year prospective study at a tertiary eye care centre in South India. Conjunctival imprint cytology was used to assess gene expression of enzymes of sphingolipids metabolism. Sphingolipids were estimated in the tears by LC-MS/MS analysis. In vitro study was done to assess IgE-induced alterations in sphingosine-1-phosphate (S1P) receptor expression and histone modification in cultured mast cells. RESULTS: Significantly altered gene expression of the sphingolipids enzymes and S1P receptor (SIP3R) were observed in conjunctival imprint cells of VKC cases. Pooled tears analysis revealed significantly lowered levels of S1P(d17:0), S1P(d20:1) (p<0.001) and S1P(d17:1) (p<0.01) specifically in severe/very severe VKC compared with both mild/moderate VKC and control. Cer(d18:/17:0) (p<0.001), ceramide-1-phosphate (C1P)(d18:1/8:0) (p<0.01) and C1P(d18:1/2.0 (p<0.05) were lowered in severe/very severe VKC compared with mild/moderate VKC. Cultured mast cells treated with IgE revealed significantly increased gene expression of S1P1 and 3 receptors and the protein expression of histone deacetylases (1, 6). CONCLUSION: Altered sphingolipid metabolism in the ocular surface results in low tear ceramide and sphingosine levels in severe/very severe VKC compared with the mild/moderate cases. The novel finding opens up fresh targets for intervention in these refractory cases.

Single-dose oral ivermectin in mild and moderate COVID-19 (RIVET-COV): A single-centre randomized, placebo-controlled trial.

INTRODUCTION: Ivermectin is an antiparasitic drug which has in-vitro efficacy in reducing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load. Hence, Ivermectin is under investigation as a repurposed agent for treating COVID-19. METHODS: In this pilot, double blind, randomized controlled trial, hospitalized patients with mild-to-moderate COVID-19 were assigned to a single oral administration of an elixir formulation of Ivermectin at either 24 mg or 12 mg dose, or placebo in a 1:1:1 ratio. The co-primary outcomes were conversion of RT-PCR to negative result and the decline of viral load at day 5 of enrolment. Safety outcomes included total and serious adverse events. The primary outcomes were assessed in patients who had positive RT-PCR at enrolment (modified intention-to-treat population). Safety outcomes were assessed in all patients who received the intervention (intention-to-treat population). RESULTS: Among the 157 patients randomized, 125 were included in modified intention-to-treat analysis. 40 patients each were assigned to Ivermectin 24 mg and 12 mg, and 45 patients to placebo. The RT-PCR negativity at day 5 was higher in the two Ivermectin arms but failed to attain statistical significance (Ivermectin 24 mg, 47.5%; 12 mg arm, 35.0%; and placebo arm, 31.1%; p-value = 0.30). The decline of viral load at day 5 was similar in each arm. No serious adverse events occurred. CONCLUSIONS: In patients with mild and moderate COVID-19, a single oral administration of Ivermectin did not significantly increase either the negativity of RT-PCR or decline in viral load at day 5 of enrolment compared with placebo.

Monocarbonyl curcuminoids as antituberculosis agents with their moderate in-vitro metabolic stability on human liver microsomes.

Tuberculosis, an airborne infectious disease, results in a high morbidity and mortality rate. The continuous emergence of TB resistance strains including MDR (multidrug-resistant tuberculosis), XDR (extensive drug-resistant tuberculosis), and especially TDR (totally drug-resistant tuberculosis) is a major public health threat and has intensified the need to develop new antitubercular agents. A natural product, curcumin, possesses diverse biological activities but suffers due to a lack of water solubility and bioavailability. To overcome these limitations, a series of 17 water-soluble monocarbonyl curcuminoids was synthesized and evaluated for antimycobacterial activity. All compounds exhibited good to moderate anti-TB activity with MIC99 in the range of 3.12-25.0 µM, out of which 7c and 7p were found the most potent compounds with MIC99 in the range of 3.12-6.25 µM. Furthermore, these compounds were observed to be nonhaemolytic, nontoxic, and stable under both physiological as well as reducing conditions. In-vitro metabolic stability data of the representative compound 7p with the human liver microsome revealed that these compounds possess a moderate metabolism with a half-life of 1.2 h and an intrinsic clearance of 1.12 ml/h/mg.

Effect of rutin on retinal VEGF, TNF-α, aldose reductase, and total antioxidant capacity in diabetic rats: molecular mechanism and ocular pharmacokinetics.

PURPOSE: The current study was conducted to explore the potential of rutin in preventing sight-threatening diabetic retinopathy. METHODS: Wistar albino rats (either sex) weighing 200-225 g were intraperitoneally injected with 45 mg/kg streptozotocin (pH 4.5). Rats having blood glucose ≥ 300 mg/dL were divided into two groups (n = 8; each group). Group I served as diabetic control and received normal saline p.o. Group II received rutin 50 mg/kg p.o. for 24 weeks. At the end of 24 weeks, retinal fundus and fluorescein imaging were done, rats were killed, and retinal biochemical assessments were conducted. Moreover, ocular pharmacokinetics of rutin was assessed in the normal rats after a single oral dose of 50 mg/kg. RESULTS: Rutin treatment significantly (p < 0.001) lowered retinal vascular endothelial growth factor, tumor necrosis factor-α, and aldose reductase. Rutin treatment significantly (p < 0.001) elevated the levels of total antioxidant capacity of the retinas. Fundus examination of rutin-treated group showed significantly lower tortuosity index and normal fluorescein angiography. Rutin was detected in the retina as well as in aqueous humor of normal rats. CONCLUSION: Rutin treatment significantly arrested the biochemical disturbances of diabetic retinopathy. The distribution of orally ingested rutin in ocular tissues further substantiate its site-specific action.

Hybridization of Fluoro-amodiaquine (FAQ) with Pyrimidines: Synthesis and Antimalarial Efficacy of FAQ-Pyrimidines.

To evade the possible toxicity associated with the formation of quinone-imine metabolite in amodiaquine (AQ), the para-hydroxyl group was replaced with a -F atom, and the resulting 4'-fluoro-amodiaquine (FAQ) was hybridized with substituted pyrimidines. The synthesized FAQ-pyrimidines displayed better in vitro potency than chloroquine (CQ) against the resistant P. falciparum strain (Dd2), exhibiting up to 47.3-fold better activity (IC50: 4.69 nM) than CQ (IC50: 222 nM) and 2.8-fold better potency than artesunate (IC50: 13.0 nM). Twelve compounds exhibited better antiplasmodial activity than CQ against the CQ-sensitive (NF54) strain. Two compounds were evaluated in vivo against a P. berghei-mouse malaria model. Mechanistic heme-binding studies, computational docking studies against Pf-DHFR and in vitro microsomal stability studies were performed for the representative molecules of the series to assess their antimalarial efficacy.

Molecular and Metabolic Markers of Fructose Induced Hepatic Insulin Resistance in Developing and Adult Rats are Distinct and Aegle marmelos is an Effective Modulator.

The time course of pathogenesis of fructose mediated hepatic insulin resistance (HepIR) is not well-delineated and we chronicle it here from post-weaning to adulthood stages. Weaned rats were provided for either 4 or 8 weeks, i.e., upto adolescence or adulthood, chow + drinking water, chow + fructose, 15% or chow + fructose, 15% + hydroalcoholic extract of leaves of Aegle marmelos (AM-HM, 500 mg/kg/d, po) and assessed for feed intake, fructose intake, body weight, fasting blood sugar, oral glucose tolerance test, HOMA-IR, insulin tolerance test and lipid profile. Activities of enzymes (glucose-6-phosphatase, hexokinase, phosphofructokinase, aldehyde dehydrogenase), hormones (leptin, ghrelin, insulin), insulin signaling molecules (Akt-PI3k, AMPK, JNK) hallmarks of inflammation (TNF-α), angiogenesis (VEGF), hypoxia (HIF-1), lipogenesis (mTOR) and regulatory nuclear transcription factors of de novo lipogenesis and hepatic insulin resistance gene (SREBP-1, FoxO1) that together govern the hepatic fructose metabolism, were also studied. The effect of fructose-rich environment on metabolic milieu of hepatocytes was confirmed using (human hepatocellular carcinoma) HepG2 cells. Using in vitro model, fructose uptake and glucose output from isolated murine hepatocytes were measured to establish the HepIR under fructose environment and delineate the effect of AM-HM. The leaves from the plant Aegle marmelos (L) Correa were extracted, fractionated and validated for rutin content using LC-MS/MS. The rutin content of extract was quantified and correlated with oral pharmacokinetic parameters in rat. The outcomes of the study suggest that the molecular and metabolic markers of fructose induced HepIR in developing and adult rats are distinct. Further, AM-HM exerts a multi-pronged attack by raising insulin secretion, augmenting insulin action, improving downstream signaling of insulin, reducing overall requirement of insulin and modulating hepatic expression of glucose transporter (Glut2). The butanol fraction of AM-HM holds promise for future development.

Un-segregated waste disposal: an alarming threat of antimicrobials in surface and ground water sources in Delhi.

Exposure of active pharmaceutical compounds (APCs) to the environment during human use is of potential importance in the emergence of drug resistance, changing soil microbiota and their residual effect on living organisms. Thus, this study aimed to assess the extent of exposure of APCs in the hydrologic cycle in and around New Delhi. This study analyzed the presence of 28 drugs from different classes in the surface water (river Yamuna) and aquifers collected from 48 places in Delhi (within the radius of 40 km). The collected water samples were quantified for APCs content using LC-MS/MS. This study revealed that aquifers are extensively affected in most areas based on the accumulation of APCs in water resources to the levels > 0.01 µg/L. Interestingly, a geographical plot of total APCs studied indicated clustering in aquifers with such high levels closer to an unscientific landfill. This 30-year-old un-segregated landfill is found to drain leachate into surface water that had high APCs. This study further revealed that apart from therapeutic usage, the main source of ecological exposure could be due to the disposal of unused and expired pharmaceutical compounds into landfills. For the first time, this study revealed the existence of antimicrobial agents and other APCs in the aquifers of Delhi with levels > 0.1 µg/L, which is a matter of serious concern in terms of multi-drug resistance and other environmental perils. This study warrants the enforcement of regulations for the disposal of unused/expired APCs in high-density population areas.