The novel ORFV protein ORFV113 activates LPA-p38 signaling

Viruses have evolved mechanisms to subvert critical cellular signaling pathways that regulate a wide range of cellular functions, including cell differentiation, proliferation and chemotaxis, and innate immune responses. Here, we describe a novel ORFV protein, ORFV113, that interacts with the G protein-coupled receptor Lysophosphatidic acid receptor 1 (LPA1). Consistent with its interaction with LPA1, ORFV113 enhances p38 kinase phosphorylation in ORFV infected cells in vitro and in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. Infection of cells with virus lacking ORFV113 (OV-IA82Δ113) significantly decreased p38 phosphorylation and viral plaque size. Infection of cells with ORFV in the presence of a p38 kinase inhibitor markedly diminished ORFV replication, highlighting importance of p38 signaling during ORFV infection. ORFV113 enhancement of p38 activation was prevented in cells in which LPA1 expression was knocked down and in cells treated with LPA1 inhibitor. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host. Notably, ORFV113 represents the first viral protein that modulates p38 signaling via interaction with LPA1 receptor.

Bifidobacterium bifidum Enhances the Intestinal Epithelial Tight Junction Barrier and Protects against Intestinal Inflammation by Targeting the Toll-like Receptor-2 Pathway in an NF-κB-Independent Manner

Defective intestinal tight junction (TJ) barrier is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). To date, there are no effective therapies that specifically target the intestinal TJ barrier. Among the various probiotic bacteria, Bifidobacterium, is one of the most widely studied to have beneficial effects on the intestinal TJ barrier. The main purpose of this study was to identify Bifidobacterium species that cause a sustained enhancement in the intestinal epithelial TJ barrier and can be used therapeutically to target the intestinal TJ barrier and to protect against or treat intestinal inflammation. Our results showed that Bifidobacterium bifidum caused a marked, sustained enhancement in the intestinal TJ barrier in Caco-2 monolayers. The Bifidobacterium bifidum effect on TJ barrier was strain-specific, and only the strain designated as BB1 caused a maximal enhancement in TJ barrier function. The mechanism of BB1 enhancement of intestinal TJ barrier required live bacterial cell/enterocyte interaction and was mediated by the BB1 attachment to Toll-like receptor-2 (TLR-2) at the apical membrane surface. The BB1 enhancement of the intestinal epithelial TJ barrier function was mediated by the activation of the p38 kinase pathway, but not the NF-κB signaling pathway. Moreover, the BB1 caused a marked enhancement in mouse intestinal TJ barrier in a TLR-2-dependent manner and protected against dextran sodium sulfate (DSS)-induced increase in mouse colonic permeability, and treated the DSS-induced colitis in a TJ barrier-dependent manner. These studies show that probiotic bacteria BB1 causes a strain-specific enhancement of the intestinal TJ barrier through a novel mechanism involving BB1 attachment to the enterocyte TLR-2 receptor complex and activation of p38 kinase pathway.

A phase 2 double-blind placebo-controlled 24-week treatment clinical study of the p38 alpha kinase inhibitor neflamapimod in mild Alzheimer’s disease

Background In preclinical studies, p38⍺ kinase is implicated in Alzheimer’s disease (AD) pathogenesis. In animal models, it mediates impaired synaptic dysfunction in the hippocampus, causing memory deficits, and is involved in amyloid-beta (Aβ) production and tau pathology. Methods The REVERSE-SD (synaptic dysfunction) study was a multi-center phase 2, randomized, double-blind, placebo-controlled trial of the p38⍺ kinase inhibitor neflamapimod; conducted December 29, 2017, to June 17, 2019; 464 participants screened, and 161 randomized to either 40 mg neflamapimod (78 study participants) or matching placebo (83 study participants), orally twice daily for 24 weeks. Study participants are as follows: CSF AD-biomarker confirmed, Clinical Dementia Rating (CDR)-global score 0.5 or 1.0, CDR-memory score ≥0.5, and Mini-Mental State Examination (MMSE) 20–28. The primary endpoint was the improvement in episodic memory, assessed by combined change in Z-scores of Hopkins Verbal Learning Test-Revised (HVLT-R) Total and Delayed Recall. Secondary endpoints included change in Wechsler Memory Scale-IV (WMS) Immediate and Delayed Recall composites, CDR-SB, MMSE, and CSF biomarkers [total and phosphorylated tau (T-tau and p-tau181), Aβ1-40, Aβ1-42, neurogranin, and neurofilament light chain]. Results At randomization, the mean age is 72, 50% female, 77% with CDR-global score 0.5, and mean MMSE score 23.8. The incidence of discontinuation for adverse events and serious adverse events (all considered unrelated) was 3% each. No significant differences between treatment groups were observed in the primary or secondary clinical endpoints. Significantly reduced CSF levels with neflamapimod treatment, relative to placebo, were evident for T-tau [difference (95% CI): −18.8 (−35.8, −1.8); P=0.031] and p-tau181 [−2.0 (−3.6, −0.5); P=0.012], with a trend for neurogranin [−21.0 (−43.6, 1.6); P=0.068]. In pre-specified pharmacokinetic-pharmacodynamic (PK-PD) analyses, subjects in the highest quartile of trough plasma neflamapimod levels demonstrated positive trends, compared with placebo, in HLVT-R and WMS. Conclusions and relevance A 24-week treatment with 40 mg neflamapimod twice daily did not improve episodic memory in patients with mild AD. However, neflamapimod treatment lowered CSF biomarkers of synaptic dysfunction. Combined with PK–PD findings, the results indicate that a longer duration study of neflamapimod at a higher dose level to assess effects on AD progression is warranted. Trial registration ClinicalTrials.gov identifier: NCT03402659. Registered on January 18, 2018

Regorafenib enhances antitumor immunity via inhibition of p38 kinase/Creb1/Klf4 axis in tumor-associated macrophages

BackgroundRegorafenib and other multikinase inhibitors may enhance antitumor efficacy of anti-program cell death-1 (anti-PD1) therapy in hepatocellular carcinoma (HCC). Its immunomodulatory effects, besides anti-angiogenesis, were not clearly defined.MethodsIn vivo antitumor efficacy was tested in multiple syngeneic liver cancer models. Murine bone marrow–derived macrophages (BMDMs) were tested in vitro for modulation of polarization by regorafenib and activation of cocultured T cells. Markers of M1/M2 polarization were measured by quantitative reverse transcription PCR (RT-PCR), arginase activity, flow cytometry, and ELISA. Knockdown of p38 kinase and downstream Creb1/Klf4 signaling on macrophage polarization were confirmed by using knockdown of the upstream MAPK14 kinase, chemical p38 kinase inhibitor, and chromatin immunoprecipitation.ResultsRegorafenib (5 mg/kg/day, corresponding to about half of human clinical dosage) inhibited tumor growth and angiogenesis in vivo similarly to DC-101 (anti-VEGFR2 antibody) but produced higher T cell activation and M1 macrophage polarization, increased the ratio of M1/M2 polarized BMDMs and proliferation/activation of cocultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream Creb1/Klf4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells. Synergistic antitumor efficacy between regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.ConclusionRegorafenib may enhance antitumor immunity through modulation of macrophage polarization, independent of its anti-angiogenic effects. Optimization of regorafenib dosage for rational design of combination therapy regimen may improve the therapeutic index in the clinic.

NG2+/Nestin+ mesenchymal stem cells dictate DTC dormancy in the bone marrow through TGFβ2

In the bone marrow (BM) microenvironment, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell (HSC) quiescence1,2. Importantly, the BM can also harbour disseminated tumour cells (DTCs) from multiple cancers, which, like HSCs, can remain dormant3. The BM signals are so growth-restrictive that dormant BM DTCs can persist for years to decades only to awaken and fuel lethal metastasis3–10. The mechanisms and niche components regulating DTC dormancy remain largely unknown. Here, we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can instruct breast cancer (BC) DTCs to enter dormancy. NG2+/Nestin+ MSCs produce TGFβ2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27-CDK inhibitor induction. Importantly, genetic depletion of the NG2+/Nestin+ MSCs or conditional knock-out of TGFβ2 in the NG2+/Nestin+ MSCs led to awakening and bone metastatic expansion of otherwise dormant p27+/Ki67− DTCs. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy. Given that aged NG2+/Nestin+ MSCs can lose homeostatic control of HSC dormancy, our results suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche may result in a break from dormancy and BC bone relapse.

Synthesis, characterization, antitubercular and anti-inflammatory activity of new pyrazolo[3,4-d]pyrimidines

Aims and Objective: Copious proinflammatory cytokines including TNF-α and IL-1β are involved in progression of inflammation in human body. Inhibition of signaling mediated by proinflammatory cytokines offer effective in the treatment of inflammatory diseases. The treatment of dreadful infectious disease mycobacterium tuberculosis still remains a challenge owing to resistance to multiple drugs hence an urgent need for newer drugs. Pyrazolo[3,4-d]pyrimidines have been disclosed to possess numerous pharmacological activities including anti-inflammatory, antimicrobial and antitubercular activities. Here in we report the synthesis of pyrazolo[3,4-d]pyrimidines for anti-inflammatory and antitubercular activities. Materials and Methods: The targeted compounds having pyrazolo[3,4-d]pyrimidines 8a-m were synthesized in three step reactions with the formation of key intermediate 5-amino-4-cyno-1-phenyl pyrazole which upon cyclization resulted in 4amino pyrazolo[3,4-d]pyrimidine for subsequent benzoylation with substituted benzoyl chlorides to form 8a-m. Antiinflammatory activity of 8a-m was assessed at 25 mg/Kg dose and minimum inhibitory concentration against gram positive, gram negative and mycobacteria was also performed. Binding interactions were also measured in binding pocket of p38 kinase. Results: Four compounds 8a, 8b, 8e and 8i significant anti-inflammatory activity in rat paw edema model induced by carrageenan and among all 8b was potent with 80.6% activity. Numerous compounds exhibited potent activity against fungal strains than bacterial strains, compound 8k was most potent against gram negative bacteria Klebsiella pneumoniae. Compounds 8d, 8e and 8f exhibited antitubercular activity with MIC value of 6.25 μg/mL Conclusion: Substituted N-benzoylated amino pyrazolo[3,4-d]pyrimidines endowed significant and potent anti-inflammatory and antimicrobial activities. Molecular docking studies also revealed favorable interactions in active site of p38 kinase.