T-cell signature cytokines distinguish pulmonary sarcoidosis from pulmonary tuberculosis.

Sarcoidosis is a systemic inflammatory disorder characterized by tissue infiltration due to mononuclear phagocytes and lymphocytes and associated noncaseating granuloma formation. Pulmonary sarcoidosis (PS) shares a number of clinical, radiological, and histopathological characteristics with that of pulmonary tuberculosis (PTB). Due to this, clinicians face issues in differentiating between PS and PTB in a substantial number of cases. There is a lack of any specific biomarker that can diagnose PS distinctively from PTB. We compared T-cell-based signature cytokines in patients with PS and PTB. In this study, we proposed a serum biomarker panel consisting of cytokines from cells: T helper (Th) 1 [interferon-gamma (IFN-γ); tumor necrosis factor-alpha (TNF-α)], Th9 [interleukin (IL)-9], Th17 [IL-17], and T regulatory (Treg) [IL-10; transforming growth factor-beta (TGF-ß)]. We performed the principal component analysis that demonstrated that our serum cytokine panel has a significant predictive ability to differentiate PS from PTB. Our results could aid clinicians to improve the diagnostic workflow for patients with PS in TB endemic settings where the diagnosis between PS and PTB is often ambiguous.

Non-coding RNA in idiopathic interstitial pneumonia and Covid-19 pulmonary fibrosis.

Pulmonary fibrosis is the key feature of majority of idiopathic interstitial pneumonias (IIPs) as well as many patients with post-COVID-19. The pathogenesis of pulmonary fibrosis is a complex molecular process that involves myriad of cells, proteins, genes, and regulatory elements. The non-coding RNA mainly miRNA, circRNA, and lncRNA are among the key regulators of many protein coding genes and pathways that are involved in pulmonary fibrosis. Identification and molecular mechanisms, by which these non-coding RNA molecules work, are crucial to understand the molecular basis of the disease. Additionally, elucidation of molecular mechanism could also help in deciphering a potential diagnostic/prognostic marker as well as therapeutic targets for IIPs and post-COVID-19 pulmonary fibrosis. In this review, we have provided the latest findings and discussed the role of these regulatory elements in the pathogenesis of pulmonary fibrosis associated with Idiopathic Interstitial Pneumonia and Covid-19.

Signature transcriptome analysis of stage specific atherosclerotic plaques of patients.

BACKGROUND: Inflammation plays an important role in all the stages of atherosclerotic plaque development. The current study aimed at assessing the altered expression of genes functioning in inflammation within the early stage (ES) and advanced stage (AS) atherosclerotic plaques obtained from patients undergoing coronary artery bypass grafting (CABG) surgery and identifying biomarker panel/s that may detect the status of plaque stages using peripheral blood samples. METHODS: A section of ES and AS plaques and normal left internal mammary arteries (LIMA) were obtained from 8 patients undergoing the CABG surgery. Total RNA isolated was analyzed for mRNA and miRNA expression profile by Affymetrix arrays. A significant number of mRNAs was found to be differentially expressed in ES and AS plaque tissues relative to LIMA. The pathway analysis of differentially expressed mRNAs in the two plaque stages was also performed using DAVID Bioinformatics Database. RESULTS: The mRNAs were found to be involved in critical inflammatory processes such as the toll-like receptor signaling pathway and cytokine-cytokine receptor interaction. Few miRNAs targeting these mRNAs were also altered in the two plaque conditions. QRT-PCR results showed a similar expression pattern of a few of the mRNAs and miRNAs in peripheral blood of the same patients relative to healthy controls. CONCLUSION: Changes in mRNA and miRNA expression associated with various inflammatory processes occur in different atherosclerotic stage plaques as well as peripheral blood. Detection of such variations in patients' blood can be used as a possible prognostic tool to detect and/or predict the risk and stage of atherosclerosis.

Primary Immunodeficiency Disorders Among North Indian Children.

OBJECTIVES: To report the distribution pattern of various categories of primary immunodeficiency disorders (PIDs) in children from North India, frequency of warning signs and critical parameters for evaluation. METHODS: In this retrospective study, 528 children below 18 y of age after clinical assessment and presentation suggestive of PID were further screened by immunophenotyping for immune cell markers by flow cytometry. RESULTS: A total of 120 (23%) children were diagnosed with PID with median age at diagnosis being 2.5 y in males and 3.5 y in females and an average delay in diagnosis from onset of symptoms being approximately 5 y. Chronic lower respiratory tract infections, gastrointestinal symptoms like persistent diarrhea and failure to thrive were amongst the most common warning signs in these patients. PIDs were classified according to the International Union of Immunological Societies' (IUIS) criteria. The diagnosis of index study subjects included combined humoral and cellular immunodeficiency (29%), phagocytic defects (29%), followed by predominantly antibody deficiency (18%), innate immunity and dysregulation (17%) and other well-defined syndromes (7%). A family history of PID (23%), consanguineous marriage (8%) and previous sibling death (23%) were observed as major clinical predictors/clues for underlying PID. All children received prophylactic antibiotics and/or antifungals in addition to specific therapy for underlying immune deficiency. CONCLUSIONS: The field of PIDs in India remains largely unexplored and we are faced with various challenges in the diagnosis of PIDs due to lack of awareness as well as absence of equipped immunological laboratory support. The authors propose a methodical step-wise laboratory diagnostic approach that can facilitate early diagnosis and timely intervention of these mis/underdiagnosed disorders.

Rituximab modulates T- and B-lymphocyte subsets and urinary CD80 excretion in patients with steroid-dependent nephrotic syndrome.

BACKGROUND: Rituximab, a monoclonal antibody targeting B lymphocytes, effectively sustains remission in steroid-dependent nephrotic syndrome (SDNS). We studied its effects on lymphocyte subsets and urinary CD80 excretion (uCD80) in patients with SDNS. METHODS: Blood and urine samples were collected from 18 SDNS patients before rituximab, and after 1 month and 1 year or at first relapse. T and B lymphocytes and uCD80 were determined by flow cytometry and ELISA, respectively. RESULTS: Treatment was associated with reduction in counts of Th17, Th2, and memory T cells, and increased T-regulatory (Treg) cells. The Th17/Treg ratio declined from baseline (median 0.6) to 1 month (0.2, P = 0.006) and increased during relapse (0.3, P = 0.016). Ratios of Th1/Th2 cells at baseline, 1 month after rituximab, and during relapse were 7.7, 14.0 (P = 0.0102), and 8.7, respectively. uCD80 decreased 1 month following rituximab (45.5 vs. 23.0 ng/g creatinine; P = 0.0039). B lymphocytes recovered earlier in relapsers (60.0 vs.183.0 days; P < 0.001). Memory B cells were higher during relapse than remission (29.7 vs.18.0 cells/µL; P = 0.029). CONCLUSION: Rituximab-induced sustained remission and B-cell depletion was associated with reduced numbers of Th17 and Th2 lymphocytes, and increased Treg cells; these changes reversed during relapses. Recovery of B cells and memory B cells predicted the occurrence of a relapse.

Impact of energy devices on the post-operative systemic immune response in women undergoing total laparoscopic hysterectomy for benign disease of the uterus.

OBJECTIVE: Laparoscopic surgery is associated with reduced surgical stress response, lesser post- operative immune function, and consequent early recovery compared with conventional open surgery. There is a lack of evidence regarding the inflammatory stress response with the use of different energy devices. The present study was conducted to evaluate and compare the inflammatory response in total laparoscopic hysterectomy (TLH) using three different energy devices. MATERIAL AND METHODS: A prospective randomized controlled study was conducted in 60 women with abnormal uterine bleeding undergoing TLH. They were divided into three groups based on the energy devices used, namely integrated bipolar and ultrasonic energy (Thunderbeat), ultrasonic (Harmonic) and electrothermal bipolar vessel sealing system (Ligasure). Cytokines and chemokines were measured in all three groups at different time points. RESULTS: Serum levels of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) increased postsurgery in all three groups and gradually declined by 72 hours. The geometric mean serum (IL)-6 levels was highest with Ligasure at 24 hours as compared with the other groups. Levels of TNF-α, macrophage inflammatory protein (MIP-1) α, MIP-1 ß were also higher at 3 hours in the Ligasure group. When the differences between the groups were measured at different time points, there was a significantly greater increase in serum IL-6 levels in the Ligasure group at 24 hours (p=0.010). No significant difference was found in the post-operative course between the groups. CONCLUSION: A greater inflammatory response was seen after the use of Ligasure indicating greater tissue damage. However, this response was not correlated with any difference in postoperative recovery.

Efficacy and Safety of Mycobacterium indicus pranii as an adjunct therapy in Category II pulmonary tuberculosis in a randomized trial.

Prolonged treatment of tuberculosis (TB) often leads to poor compliance, default and relapse, converting primary TB patients into category II TB (Cat IITB) cases, many of whom may convert to multi-drug resistant TB (MDR-TB). We have evaluated the immunotherapeutic potential of Mycobacterium indicus pranii (MIP) as an adjunct to Anti-Tubercular Treatment (ATT) in Cat II pulmonary TB (PTB) patients in a prospective, randomized, double blind, placebo controlled, multicentric clinical trial. 890 sputum smear positive Cat II PTB patients were randomized to receive either six intra-dermal injections (2 + 4) of heat-killed MIP at a dose of 5 × 108 bacilli or placebo once in 2 weeks for 2 months. Sputum smear and culture examinations were performed at different time points. MIP was safe with no adverse effects. While sputum smear conversion did not show any statistically significant difference, significantly higher number of patients (67.1%) in the MIP group achieved sputum culture conversion at fourth week compared to the placebo (57%) group (p = 0.0002), suggesting a role of MIP in clearance of the bacilli. Since live bacteria are the major contributors for sustained incidence of TB, the potential of MIP in clearance of the bacilli has far reaching implications in controlling the spread of the disease.

Cedrus deodara: In vitro antileishmanial efficacy & immumomodulatory activity.

BACKGROUND & OBJECTIVES: The existing antileishmanial drugs for complete cure of visceral leishmaniasis (kala-azar) are limited. The available drugs are either toxic or less effective leading to disease relapse or conversion to post-kala-azar dermal leishmaniasis. Several herbal extracts have been shown to have antileishmanial activity, but a herbal drug may not always be safe. In the present study, the extract of Cedrus deodara leaves has been standardized and tested for immunomodulatory antileishmanial activities. METHODS: The extracts of C. deodara leaves with different solvents such as benzene, chloroform, ethyl acetate and methanol were made by soxhlation process. Solvents were removed under reduced pressure and temperature using rotary evaporator. The antileishmanial bioassay test was performed with in vitro maintained parasites. Immunomodulatory activity of different extracts was tested by flow cytometry. Standardization of the effective fraction was performed with Linalool as a marker compound through reverse-phase high-performance liquid chromatography. RESULTS: The extract with the use of benzene solvent showed strong antileishmanial activities within a dose 25-200 µg/ml culture with non-significant haemolytic activities and significant immunomodulant activities against the host cells. Linalool was found to be 1.29 per cent in the effective extract of C. deodara. INTERPRETATION & CONCLUSIONS: The antileishmanial activity of C. deodara, as assessed by bioassay testing on. LEISHMANIA DONOVANI: parasites and immunomodulatory effect of benzene extract of leaves on host cells indicated that it might be a potential new safe therapeutic target to cure the visceral leishmaniasis.

Increased level of soluble adenosine deaminase in bone marrow of visceral leishmaniasis patients: an inverse relation with parasite load.

Adenosine deaminase (ADA) which degrades adenosine to inosine, is known to be pro-inflammatory molecule in many diseases. Adenosine suppresses the functioning of the immune system and thus promotes dissemination of the parasite. In our previous finding, the level of soluble ADA in serum of visceral leishmaniasis (VL) was found to be increased as compared to healthy controls. However, it cannot be fairly interpreted unless their level is demonstrated at the disease site, where the parasite resides. We designed this study to correlate the level of soluble ADA (sADA) with parasitic load at the disease site i.e. bone marrow (BM). We found increased levels of sADA in BM as compared to the unaffected BM. Furthermore, a significant inverse correlation is observed between the parasite load and level of sADA at the disease site.

Differential expression of HLA-G and ILT-2 receptor in human tuberculosis: Localized versus disseminated disease.

Human leukocyte antigen-G (HLA-G) is an anti-inflammatory and immunosuppressive molecule that can modulate immune cell activation. The role of HLA-G in tuberculosis, an immune-mediated and chronic bacterial disease remains to be elucidated. We investigated the expression profile of soluble and membrane bound HLA-G in pulmonary TB (PTB), TB pleural effusion (TB-PE, localized disease) and Miliary TB (disseminated form). The expression of HLA-G receptor, ILT-2 was also determined on the immune cells. We observed that the plasma sHLA-G levels were significantly increased in Miliary TB than in TB-PE patients. In contrast, immunophenotyping revealed that the percent frequency of CD3(+) T cells expressing HLA-G was significantly reduced in Miliary TB as compared to TB-PE, whereas frequency of CD14(+) monocytes expressing HLA-G was significantly higher in TB-PE patients. Strikingly in the TB-PE cases, comparison of disease site, i.e. pleural effusion with peripheral blood showed increased expression of both soluble and surface HLA-G, whereas ILT-2 expressing cells were reduced at the local disease site. Furthermore, we demonstrated that in TB-PE cases, HLA-G expression on CD3(+) T cells was influenced by broad spectrum MMP inhibitor. Thus, differential expression of HLA-G could potentially be a useful biomarker to distinguish different states of TB disease.

Evidence of a novel allergenic protein Narcin in the bulbs of Narcissus tazetta.

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.

Recruitment of Th1 effector cells in human tuberculosis: hierarchy of chemokine receptor(s) and their ligands.

Selective recruitment of IFN-γ biased Th1 effector cells at the pathologic site(s) determines the local immunity of tuberculosis (TB). We observed the enrichment of CXCR3, CCR5 and CD11a(high) T cells in the peripheral blood, pleural fluid and bronchoalveolar lavage of TB pleural effusion (TB-PE) and miliary tuberculosis (MTB) patients respectively. CXCR3(+)CCR5(+) T cells were significantly high at the local disease site(s) in both the forms of TB and their frequency was highest among activated lymphocytes in TB-PE. Interestingly, all CCR5(+) cells were invariably positive for CXCR3 but all CXCR3(+) cells did not co-express CCR5 in pleural fluid whereas the situation was reverse in bronchoalveolar lavage. These CXCR3(+)CCR5(+) cells dominantly produced IFN-γ in response to Mycobacterium tuberculosis antigen. In vitro chemotaxis assay indicates dominant role of RANTES and IP-10 in the selective recruitment of CXCR3(+)CCR5(+)cells at the tubercular pathologic sites.

Inhibiting the programmed death 1 pathway rescues Mycobacterium tuberculosis-specific interferon γ-producing T cells from apoptosis in patients with pulmonary tuberculosis.

BACKGROUND: Overexpression of programmed death 1 (PD-1) receptor is thought to inhibit the effector T-cell response in human tuberculosis. However, the precise mechanism of such inhibition remains unclear. The present study addresses the role of PD-1 in dampening host T-cell function among patients with pulmonary tuberculosis. METHODS: Expression of PD-1 and its ligands (PD-L1/L2) on T cells, B cells, and monocytes was evaluated by flow cytometry (FACS). In vitro stimulation of peripheral blood mononuclear cells in the presence of Mycobacterium tuberculosis antigens was performed with and without blocking PD-1, and intracellular cytokine production was measured by FACS. RESULTS: We showed higher frequencies of T cells, monocytes, and B cells expressing PD-1 and its ligand(s) among patients with pulmonary tuberculosis. Infections with live M. tuberculosis upregulated PD-L1 expression on monocytes. In vitro PD-1 blocking rescued M. tuberculosis-specific interferon γ (IFN-γ)-producing T cells from undergoing apoptosis. The number of PD-1-expressing T cells decreased significantly during therapy and inversely correlated with IFN-γ-dominant T-cell response against M. tuberculosis. CONCLUSIONS: Manipulation of PD-1 signaling may restore the host T-cell response and thus may have therapeutic potential. PD-1 also may serve as a biomarker to monitor host immunity among patients with tuberculosis during therapy and vaccine studies.

Structural basis of the binding of fatty acids to peptidoglycan recognition protein, PGRP-S through second binding site.

Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5) to 10(-8) M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN-γ by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.

Impaired expression of CD26 compromises T-cell recruitment in human visceral leishmaniasis.

An inefficient Th1 response, coupled with skewed Th2 cytokine production, has been implicated to increase susceptibility to visceral leishmaniasis (VL) infection. The expression of the dipeptidyl peptidase Cd26 by polarized Th1 activates a chemokine cascade that recruits Th1 recruitment to the pathologic site. Here, we studied 42 patients with confirmed VL (mean age 24.80 ± 16.26 years; range 3-70 years; 25 males and 17 females), 30 endemic controls, and 10 nonendemic controls. We observed a decrease in constitutive and antigen-induced expression of CD26 on the T cells of VL patients. Soluble CD26 (sCD26) levels in serum and BM were also found to be significantly lower in VL patients. Following successful therapy, increased sCD26 expression was observed. Tuberculosis pleural effusion derived CCR5(+) CXCR3(+) effector T cells showed enhanced chemokine-driven migration in the presence of posttreatment BM aspirate containing high levels of sCD26. Moreover, T-cell migration could be inhibited by blocking RANTES, IP-10, and CD26 signaling from the posttreatment aspirate with Ab. Our results indicate that, in VL patients, impaired expression and secretion of CD26 compromises chemokine activation and thus T-cell recruitment, eventually resulting in a deficient state of local immunity at pathologic sites.

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Regulatory T cells suppress T cell activation at the pathologic site of human visceral leishmaniasis.

Suppression of T cell response is thought to be involved in the pathogenesis of visceral leishmaniasis (VL). Regulatory T cell (Treg) mediated immune-suppression is reported in animal models of Leishmania infection. However, their precise role among human patients still requires pathologic validation. The present study is aimed at understanding the frequency dynamics and function of Treg cells in the blood and bone marrow (BM) of VL patients. The study included 42 parasitologically confirmed patients, 17 healthy contact and 9 normal bone marrow specimens (NBM). We show i) the selective accumulation of Treg cells at one of the disease inflicted site(s), the BM, ii) their in vitro expansion in response to LD antigen and iii) persistence after successful chemotherapy. Results indicate that the Treg cells isolated from BM produces IL-10 and may inhibit T cell activation in IL-10 dependent manner. Moreover, we observed significantly higher levels of IL-10 among drug unresponsive patients, suggesting their critical role in suppression of immunity among VL patients. Our results suggest that IL-10 plays an important role in suppression of host immunity in human VL and possibly determines the efficacy of chemotherapy.

Clinical significance of sperm DNA damage threshold value in the assessment of male infertility.

Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.