A protective role for type I interferon signaling following infection with Mycobacterium tuberculosis carrying the rifampicin drug resistance-conferring RpoB mutation H445Y.

Interleukin-1 (IL-1) signaling is essential for controlling virulent Mycobacterium tuberculosis (Mtb) infection since antagonism of this pathway leads to exacerbated pathology and increased susceptibility. In contrast, the triggering of type I interferon (IFN) signaling is associated with the progression of tuberculosis (TB) disease and linked with negative regulation of IL-1 signaling. However, mice lacking IL-1 signaling can control Mtb infection if infected with an Mtb strain carrying the rifampin-resistance conferring mutation H445Y in its RNA polymerase ß subunit (rpoB-H445Y Mtb). The mechanisms that govern protection in the absence of IL-1 signaling during rpoB-H445Y Mtb infection are unknown. In this study, we show that in the absence of IL-1 signaling, type I IFN signaling controls rpoB-H445Y Mtb replication, lung pathology, and excessive myeloid cell infiltration. Additionally, type I IFN is produced predominantly by monocytes and recruited macrophages and acts on LysM-expressing cells to drive protection through nitric oxide (NO) production to restrict intracellular rpoB-H445Y Mtb. These findings reveal an unexpected protective role for type I IFN signaling in compensating for deficiencies in IL-1 pathways during rpoB-H445Y Mtb infection.

NF-κB signaling deficiency in CD11c-expressing phagocytes mediates early inflammatory responses and enhances Mycobacterium tuberculosis control.

Early innate immune responses play an important role in determining the protective outcome of Mycobacterium tuberculosis (Mtb) infection. Nuclear factor kappa B (NF-κB) signaling in immune cells regulates the expression of key downstream effector molecules that mount early anti-mycobacterial responses. Using conditional knockout mice, we studied the effect of abrogation of NF-κB signaling in different myeloid cell types and its impact on Mtb infection. Our results show that absence of IKK2-mediated signaling in all myeloid cells resulted in increased susceptibility to Mtb infection. In contrast, absence of IKK2-mediated signaling specifically in CD11c+ myeloid cells induced early pro-inflammatory cytokine responses, enhanced the recruitment of myeloid cells and mediated early resistance to Mtb. Abrogation of IKK2 in MRP8-expressing neutrophils did not impact either disease pathology or Mtb control. Thus, we describe an early immunoregulatory role for NF-κB signaling in CD11c-expressing phagocytes, and a later protective role for NF-κB in LysM-expressing cells during Mtb infection.

Lung type 3 innate lymphoid cells respond early following Mycobacterium tuberculosis infection.

Tuberculosis is the leading cause of death due to an infectious disease worldwide. Innate lymphoid type 3 cells (ILC3s) mediate early protection during Mycobacterium tuberculosis (Mtb) infection. However, the early signaling mechanisms that govern ILC3 activation or recruitment within the lung during Mtb infection are unclear. scRNA-seq analysis of Mtb-infected mouse lung innate lymphoid cells (ILCs) has revealed the presence of different clusters of ILC populations, suggesting heterogeneity. Using mouse models, we show that during Mtb infection, interleukin-1 receptor (IL-1R) signaling on epithelial cells drives ILC3 expansion and regulates ILC3 accumulation in the lung. Furthermore, our data revealed that C-X-C motif chemokine receptor 5 (CXCR5) signaling plays a crucial role in ILC3 recruitment from periphery during Mtb infection. Our study thus establishes the early responses that drive ILC3 accumulation during Mtb infection and points to ILC3s as a potential vaccine target. IMPORTANCE: Tuberculosis is a leading cause of death due to a single infectious agent accounting for 1.6 million deaths each year. In our study, we determined the role of type 3 innate lymphoid cells in early immune events necessary for achieving protection during Mtb infection. Our study reveals distinct clusters of ILC2, ILC3, and ILC3/ILC1-like cells in Mtb infection. Moreover, our study reveal that IL-1R signaling on lung type 2 epithelial cells plays a key role in lung ILC3 accumulation during Mtb infection. CXCR5 on ILC3s is involved in ILC3 homing from periphery during Mtb infection. Thus, our study provides novel insights into the early immune mechanisms governed by innate lymphoid cells that can be targeted for potential vaccine-induced protection.

Deciphering the Patterns of Dual Primary Cases Registered at the Hospital-Based Cancer Registry: First Experience from Rural Cancer Center in North India.

Objectives The objective is to present the patterns of dual primary malignancies diagnosed at the Pathology Laboratory of Cancer Hospital with the support from hospital-based cancer registry (HBCR), Sangrur, Punjab, India for the years 2018 and 2019. Methods HBCR abstracts data from electronic medical records. Trained cancer registry staff abstracts cases in standard pro forma. Dual primary was coded as per the International Agency for Research on Cancer rule and was rechecked by the pathologist. Statistical Analysis Data about multiple primary was entered and documented in an Excel sheet. Time interval was calculated by subtracting the date of diagnosis for second primary and first primary. Results A total of 6,933 cases were registered, 45 cases are dual primary (26 females, 19 males) of which 64.4% are synchronous and 35.6% metachronous cases. Seventy-nine percent received cancer-directed treatment for synchronous and 87% for metachronous. The most common sites of the primary tumor were breast (33%), head and neck (22.2%), gynecological sites (11%), prostate (9%), esophagus (4%), and remaining other tumors (20.8%). Most common sites for second malignancies were gastrointestinal (GI) tract (31%), gynecological sites (18%), head and neck (16%), hematological malignancies (7%), soft tissue sarcoma (4%), breast (2%), and other sites (22%). Conclusion More than 70% of cases of primary tumors were in breast, head and neck, gynecological, and prostate. Of these, more than 60% of the second malignancy was found in the GI tract, gynecological, and head and neck sites. Around two-thirds of dual tumors are synchronous. Breast cancer cases have higher incidence of second malignancy. Regular follow-up is necessary to assess the survival of the second primary.

The feasibility of using of a comprehensive unit-based safety program (CUSP) for improving antimicrobial stewardship at a tertiary care hospital in national capital region of India: A prospective quasi experimental study.

PURPOSE: To examine the feasibility of Comprehensive Unit-based Safety Program (CUSP) as a strategy tool to improve antibiotic stewardship in low and middle income countries (LMIC) in resource limited setting. The primary outcome measure is identification of inappropriate prescriptions. The secondary outcome parameters are App adoption trends and antimicrobial prescription pattern and practices. MATERIAL AND METHODS: A prospective quasi-experimental design was used to operationalizing the CUSP intervention. The project considered the data of 482 patients from two mixed Medical ICUs admitted during June 2019 to April 2020. The information was collected on antimicrobials prescription pattern and practices for identification of inappropriate use as well as app adoption trend with respect to Electronic Medical Record (EMR) Orders Placed, Clinical Notes and Checklist Filled. The intervention in the study comprised of development of an antibiotic monitoring stewardship (AMS) data collection app for ease of use and for Clinical Decision Support System (CDSS) to identify the cases of inappropriate use of antibiotics. RESULTS: Data of patients was reviewed to create algorithms for empirical and directed antibiotic therapy as well as to create a CDSS app. Out of 793 prescriptions initially during July-September 2019, 19 (2.4%) were inappropriate antimicrobial prescription. The continuous monitoring of antimicrobial prescription helped in reducing the irrational use and bring it to level zero at the end. CONCLUSION: It requires commitment from the management, and seamless communication within Clinical, Microbiology, Pharmacology and data management teams to create and run a successful CUSP program towards Antimicrobial Resistance. Tools such as the CDSS can smoothen the process.

Mycobacterium tuberculosis infection drives a type I IFN signature in lung lymphocytes.

Mycobacterium tuberculosis (Mtb) infects 25% of the world's population and causes tuberculosis (TB), which is a leading cause of death globally. A clear understanding of the dynamics of immune response at the cellular level is crucial to design better strategies to control TB. We use the single-cell RNA sequencing approach on lung lymphocytes derived from healthy and Mtb-infected mice. Our results show the enrichment of the type I IFN signature among the lymphoid cell clusters, as well as heat shock responses in natural killer (NK) cells from Mtb-infected mice lungs. We identify Ly6A as a lymphoid cell activation marker and validate its upregulation in activated lymphoid cells following infection. The cross-analysis of the type I IFN signature in human TB-infected peripheral blood samples further validates our results. These findings contribute toward understanding and characterizing the transcriptional parameters at a single-cell depth in a highly relevant and reproducible mouse model of TB.

IRF8 and BATF3 interaction enhances the cDC1 specific Pfkfb3 gene expression.

Dendritic cells (DCs) play central role in innate as well as adaptive immune responses regulated by diverse DC subtypes that vary in terms of surface markers, transcriptional profile and functional responses. Generation of DC diversity from progenitor stage is tightly regulated by complex molecular inter-play between transcription factors. We earlier demonstrated that Batf3 and Id2 expression have a synergistic effect on the Irf8 directed classical cDC1 development. In present study, Bi-molecular fluorescence complementation assay suggested that IRF8 interacts with BATF3, and ID2 may aid cDC1 development independently. Genome wide recruitment analysis of IRF8 and BATF3 from different DC subtypes led to identification of the overlapping regions of occupancy by these two transcription factors. Further analysis of overlapping peaks of IRF8 and BATF3 occupancy in promoter region within the cDC1 subtype specific transcriptional pattern identified a metabolically important Pfkfb3 gene. Among various immune cell types; splenic cDC1 subtype displayed enhanced expression of Pfkfb3. Analysis of Irf8-/-, Irf8R294C and Batf3DCKO DC confirmed direct regulation of Pfkfb3 enhanced expression specifically in cDC1 subtype. Further we show that inhibition of PFKFB3 enzymatic activity by a chemical agent PFK15 led to reduction in cDC1 subtype in both in vitro FLDC cultures as well as in vivo mouse spleens. Together, our study identified the direct regulation of cDC1 specific enhanced expression of Pfkfb3 in glycolysis and cDC1 biology.

Mutation in Irf8 Gene (Irf8R294C ) Impairs Type I IFN-Mediated Antiviral Immune Response by Murine pDCs.

Plasmacytoid dendritic cells (pDCs) are the key producers of type I interferons (IFNs), thus playing a central role in initiating antiviral immune response. Besides robust type I IFN production, pDCs also act as antigen presenting cells post immunogenic stimulation. Transcription factor Irf8 is indispensable for the development of both pDC and cDC1 subset. However, the mechanism underlying the differential regulation by IRF8 in cDC1- and pDC-specific genomic architecture of developmental pathways still remains to be fully elucidated. Previous studies indicated that the Irf8R294C mutation specifically abrogates development of cDC1 without affecting that of pDC. In the present study using RNA-seq based approach, we have found that though the point mutation Irf8R294C did not affect pDC development, it led to defective type I IFN production, thus resulting in inefficient antiviral response. This observation unraveled the distinctive roles of IRF8 in these two subpopulations-regulating the development of cDC1 whereas modulating the functionality of pDCs without affecting development. We have reported here that Irf8R294C mutation also caused defect in production of ISGs as well as defective upregulation of costimulatory molecules in pDCs in response to NDV infection (or CpG stimulation). Through in vivo studies, we demonstrated that abrogation of type I IFN production was concomitant with reduced upregulation of costimulatory molecules in pDCs and increased NDV burden in IRF8R294C mice in comparison with wild type, indicating inefficient viral clearance. Further, we have also shown that Irf8R294C mutation abolished the activation of type I IFN promoter by IRF8, justifying the low level of type I IFN production. Taken together, our study signifies that the single point mutation in Irf8, Irf8R294C severely compromised type I IFN-mediated immune response by murine pDCs, thereby causing impairment in antiviral immunity.

Determinants of completion of cancer directed treatment: an experience from a rural cancer centre, Sangrur, Punjab state, India.

In low and middle-income countries, access to cancer diagnosis and treatment is suboptimal. Further, compliance to cancer treatment is a major issue due to various reasons including financial barriers, lack of family support and fear of treatment. This article discusses the determinants of treatment completion in cancer patients of a government-run hospital, in a rural part of Punjab in India. The Sangrur hospital-based cancer registry data for the year 2018 have been used. We have registered 2,969 cancer cases, out of which 2,528 (85%) cases were eligible for the analysis. Of the total 2,528 cases, 1,362 (54%) cases completed the cancer directed treatment and 1,166 (46%) did not. The data have been collected from the electronic medical record (EMR) department and entered into CanReg5 software. The bivariate and multivariate binary logistic regression analysis was performed to see the effect of variables on the treatment completion. The results indicate that the elderly age group (>60 years) (odds ratio (OR): 0.52, (95% confidence interval (CI): 0.31-0.86)), distance from hospital (OR: 0.67, (95% CI: 0.50-0.89)) and access to government health schemes (OR: 0.13, (95% CI: 0.10-0.19)] have direct correlation with the treatment completion. The educated patients (OR: 1.49, (95% CI: 1.13-1.96)) and patients who received curative treatment (OR: 2.7, (95% CI: 1.88-3.88)) have shown 58% and 84% compliance to treatment completion, respectively. The other variables like the clinical extent of disease, religion, gender and income do not have any significant effect on the treatment completion. Determinants like age (young), education, distance from the hospital, curative treatment and availability of government health schemes for financial support have shown positive effects on treatment completion. These factors have to be considered by the cancer hospitals, health departments and policymakers while planning for cancer care or control in India.

RelB suppresses type I Interferon signaling in dendritic cells.

Type I Interferon (IFN) signaling plays a critical role in dendritic cell (DC) development and functions. Inhibition of hyper type I IFN signaling promotes cDC2 subtype development. Relb is essential to development of cDC2 subtype and here we analyzed its effect on type I IFN signaling in DCs. We show that Relb suppresses the homeostatic type I IFN signaling in cDC2 cultures. TLR stimulation of FL-DCs led to RelB induction coinciding with fall in IFN signatures; conforming with the observation Relb expression reduced TLR stimulated IFN induction along with decrease in ISGs. Towards understanding mechanism, we show that effects of RelB are mediated by increased levels of IκBα. We demonstrate that RelB dampened antiviral responses by lowering ISG levels and the defect in cDC2 development in RelB null mice can be rescued in Ifnar1-/- background. Overall, we propose a novel role of RelB as a negative regulator of the type I IFN signaling pathway; fine tuning development of cDC2 subtype.

Cutting Edge: ACVRL1 Signaling Augments CD8α+ Dendritic Cell Development.

Dendritic cells (DCs) are a collection of different subtypes, each of which is characterized by specific surface markers, gene-expression patterns, and distinct functions. Members of the IFN regulatory factor family play critical roles in DC development and functions. Recently, Irf8 was shown to activate TGF-ß signaling, which led to exacerbated neuroinflammation in the experimental autoimmune encephalomyelitis mouse model. We analyzed the effect of Irf8 on TGF-ß/bone morphogenetic protein pathway-specific genes in DCs and identified Acvrl1, a type I TGF-ß superfamily receptor, as a gene strongly induced by Irf8 expression. Among various DC subtypes, Acvrl1 is differentially expressed in CD8α(+) DCs. ACVRL1 signaling augmented Irf8-directed classical CD8α(+) DC development. Irf8 expression is essential for plasmacytoid DC and CD8α(+) DC development, and this study demonstrates that ACVRL1 signaling plays a pivotal role whereby it suppresses plasmacytoid DC development while enhancing that of CD8α(+) DCs, thus contributing to DC diversity development.

New class of methyl tetrazole based hybrid of (Z)-5-benzylidene-2-(piperazin-1-yl)thiazol-4(%H)-one as potent antitubercular agents.

In search of potential therapeutics for tuberculosis, we describe here the synthesis and in vitro antitubercular activity of a novel series of thiazolone piperazine tetrazole derivatives. Among all the synthesized derivatives, four compounds (10, 14, 20 and 33) exhibited more potent activity (MIC=3.08, 3.01, 2.62 and 2.51 µM) than ethambutol (MIC=9.78 µM) and pyrazinamide (MIC=101.53 µM) against Mycobacterium tuberculosis. Furthermore, they displayed no toxicity against Vero cells (C1008) and mouse bone marrow derived macrophages (MBMDMϕ). These investigated analogues have emerged as possible lead molecule to enlarge the scope of the study.

Investigation of Ugi-4CC derived 1H-tetrazol-5-yl-(aryl) methyl piperazinyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid: synthesis, biology and 3D-QSAR analysis.

Novel series of 7-piperazinylquinolones with tetrazole derivatives were synthesized and evaluated for their antibacterial activity against various strains of Staphylococcus aureus. All the synthesized compounds showed significant in vitro antibacterial activity against Gram-positive bacteria whereas some compounds displayed moderate activity against Gram-negative bacteria. Among all the synthesized compounds, compounds (6a-c, 6e-g, 6i-k, 6m, 6'f and 6'm) were found to be more effective with MIC ranging from (0.78-3.12 µg/mL) against S. aureus (ATCC-29213) than the control; ciprofloxacin (MIC = 25 µg/mL). Moreover, these analogues displayed no toxicity up to MIC = 0.39 µg/mL against mammalian cell line L-929. Furthermore, to correlate the biological activities of synthesized compounds with their 3D conformation, we attempted 3D-QSAR study.

Design and synthesis of a new class of 4-aminoquinolinyl- and 9-anilinoacridinyl Schiff base hydrazones as potent antimalarial agents.

A series of novel 4-aminoquinolinyl and 9-anilinoacridinyl Schiff base hydrazones have been synthesized and evaluated for their antimalarial activity. All compounds were evaluated in vitro for their antimalarial activity against chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 strain of Plasmodium falciparum and for cytotoxicity toward Vero cells. Compounds 17, 20, and 21 displayed good activity against the 3D7 strain with IC50 values ranging from 19.69 to 25.38 nm. Moreover, compounds 16, 17, 21, 24, 32, and 33 exhibited excellent activities (21.64-54.26 nm) against K1 strain and several compounds displayed ß-hematin inhibitory activity, suggesting that they act on the heme crystallization process such as CQ. Compounds were also found to be non-toxic with good selectivity index.

Discovery of a new class of natural product-inspired quinazolinone hybrid as potent antileishmanial agents.

The high potential of quinazolinone containing natural products and their derivatives in medicinal chemistry led us to discover four novel series of 53 compounds of quinazolinone based on the concept of molecular hybridization. Most of the synthesized analogues exhibited potent leishmanicidal activity against intracellular amastigotes (IC50 from 0.65 ± 0.2 to 7.76 ± 2.1 µM) as compared to miltefosine (IC50 = 8.4 ± 2.1 µM) and nontoxic toward the J-774A.1 cell line and Vero cells. Moreover, activation of Th1 type and suppression of Th2 type immune responses and induction in nitric oxide generation proved that 8a and 8g induce murine macrophages to prevent survival of parasites. Compounds 8a and 8g exhibited significant in vivo inhibition of parasite 73.15 ± 12.69% and 80.93 ± 10.50% against Leishmania donovani /hamster model. Our results indicate that compounds 8a, 8g, and 9f represent a new structural lead for this serious and neglected disease.

Synthesis and biological evaluation of a new class of 4-aminoquinoline-rhodanine hybrid as potent anti-infective agents.

Synthesis of novel 4-aminoquinoline-rhodanine hybrid using inexpensive starting materials via easy to operate methodology, and their biological activity is reported. All the compounds were screened for their in vitro antimalarial activity against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum, and their cytotoxicity toward VERO cell line. Compounds 9, 19, 21 and 23 exhibited excellent antimalarial activity with IC50 value ranging from 13.2 to 45.5 nM against chloroquine-resistant (K1) strain. Biochemical studies revealed that inhibition of hemozoin formation is the primary mechanism of action of these analogs for their antimalarial activity. Additionally, some derivatives (14, 18 and 26) of this series also exhibited the antimycobacterial activity against H37Rv strain of Mycobacterium tuberculosis with MIC value of 6.25 µM.

Discovery of triazine mimetics as potent antileishmanial agents.

The World Health Organization has classified the leishmaniasis as a major tropical disease. The discovery of new compounds for leishmaniasis is therefore a pressing concern for the anti-infective research program. We have synthesized 19 compounds of triazine dimers as novel antileishmanial agents. Most of the synthesized derivatives exhibited better activity against intracellular amastigotes (IC50 ranging from 0.77 to 10.32 µM) than the control, pentamidine (IC50 = 13.68 µM), and are not toxic to Vero cells. Compounds 14 and 15 showed significant in vivo inhibition of 74.41% and 62.64%, respectively, in L. donovani/hamster model. Moreover, expansion of Th1-type and suppression of Th2-type immune responses proved that compound 14 stimulates mouse macrophages to prevent the progression of leishmania parasite. The molecular docking studies involving PTR1 protein PDB further validated the concepts involved in the design of these compounds. Among the investigated analogues, compound 14 has emerged as the potential one to enlarge the scope of the study.

Cyanuric chloride catalyzed mild protocol for synthesis of biologically active dihydro/spiro quinazolinones and quinazolinone-glycoconjugates.

We have developed an efficient cyanuric chloride (2,4,6-trichloro-1,3,5-triazine, TCT) catalyzed approach for the synthesis of 2,3-dihydroquinazolin-4(1H)-one (3a-3x), 2-spiroquinazolinone (5, 7), and glycoconjugates of 2,3-dihydroquinazolin-4(1H)-one (10a, 10b) derivatives. The reaction allows rapid cyclization (8-20 min) with 10 mol % cyanuric chloride to give skeletal complexity in good to excellent yield. We believe that this novel procedure may open the door for the easy generation of new and bioactive quinazolinones.

Synthesis and antibacterial evaluation of novel 8-fluoro Norfloxacin derivatives as potential probes for methicillin and vancomycin-resistant Staphylococcus aureus.

A series of novel 8-fluoro Norfloxacin derivatives and the hybrids of its piperazinyl derivatives incorporated with 1,3,5-triazine and pyrimidine were synthesized. All the above compounds were evaluated for their antibacterial activity against Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus and methicillin & vancomycin-resistant S. aureus. Among all, compounds having Morpholine, N-methyl/phenyl/benzyl/pyrimidinyl piperazines and n-butylamine substitution at C-7 position, have shown increased potency in comparison to norfloxacin and ciprofloxacin.