Bidyut Das

Non-Classical monocytes display inflammatory features: Validation in Sepsis and Systemic Lupus Erythematous

Given the importance of monocytes in pathogenesis of infectious and other inflammatory disorders, delineating functional and phenotypic characterization of monocyte subsets has emerged as a critical requirement. Although human monocytes have been subdivided into three different populations based on surface expression of CD14 and CD16, published reports suffer from contradictions with respect to subset phenotypes and function. This has been attributed to discrepancies in reliable gating strategies for flow cytometric characterization and purification protocols contributing to significant changes in receptor expression. By using a combination of multicolour flow cytometry and a high-dimensional automated clustering algorithm to confirm robustness of gating strategy and analysis of ex-vivo activation of whole blood with LPS we demonstrate the following: a. ‘Classical’ monocytes are phagocytic with no inflammatory attributes, b. ‘Non-classical’ subtype display ‘inflammatory’ characteristics on activation and display properties for antigen presentation and c. ‘Intermediate’ subtype that constitutes a very small percentage in circulation (under physiological conditions) appear to be transitional monocytes that display both phagocytic and inflammatory function. Analysis of blood from patients with Sepsis, a pathogen driven acute inflammatory disease and Systemic Lupus Erythmatosus (SLE), a chronic inflammatory disorder validated the broad conclusions drawn in the study.

Mannose binding lectin: a biomarker of systemic lupus erythematosus disease activity

IntroductionA role for mannose binding lectin (MBL) in autoimmune diseases has been demonstrated earlier and elevated level of MBL has been shown in systemic lupus erythematosus (SLE) patients. In the current study, we investigated MBL as a potential biomarker for disease activity in SLE.MethodsIn a case control study SLE patients (93 females) and 67 age, sex, ethnicity matched healthy controls were enrolled. Plasma MBL levels were quantified by enzyme linked immunosorbent assay (ELISA). Clinical, serological and other markers of disease activity (C3, C4 and anti-dsDNA) were measured by standard laboratory procedures.ResultsPlasma MBL levels were significantly high in SLE patients compared to healthy controls (P < 0.0001). MBL levels were variable in different clinical categories of SLE. Lower levels were associated with musculoskeletal and cutaneous manifestations (P = 0.002), while higher and intermediate MBL levels were significantly associated with nephritis in combination with other systemic manifestations (P = 0.01 and P = 0.04 respectively). Plasma MBL correlated with systemic lupus erythematosus disease activity index (SLEDAI) (P = 0.0003, r = 0.36), anti-dsDNA (P < 0.0001, r = 0.54), proteinuria (P < 0.0001, r = 0.42) and negatively correlated with C3 (P = 0.007, r = -0.27) and C4 (P = 0.01, r = -0.29).ConclusionsPlasma MBL is a promising marker in the assessment of SLE disease activity.

A role for tumour necrosis factor-alpha in acute lymphatic filariasis.

A spectrum of clinical manifestations is a feature of human lymphatic filariasis. The acute disease is characterized by periodic and self limiting episodes of adenolymphangitis, fever and associated constitutional symptoms, while the chronic disease includes long lasting manifestations such as lymphoedema and/or hydrocoele. The microfilariae carriers are generally free of clinical symptoms. In the present study circulating Tumour Necrosis Factor (TNF‐α) was measured in human bancroftian filariasis with different clinical manifestations. Significantly elevated levels were observed only in patients with acute disease and not in microfilariae carriers or in patients with chronic manifestations. A detailed analysis of the acute cases indicated an absence of correlation between TNF‐α levels and duration of the episodes. However, a significant positive correlation was observed between the severity of the disease and the TNF‐α levels. About 85% of the acute cases with severe manifestations showed raised levels of TNF‐α while only 6.5% of mild cases showed such levels. Manifestation of fever was also significantly associated with higher levels of TNF‐α—while 80% of acute cases with fever had significant levels only 24% of acute cases without fever had high levels of TNF‐α. Based on these observations we propose a mediatory role for TNF‐α in acute filariasis and the possible use of TNF‐α inhibitors for clinical management of the disease.

Low producer MBL genotypes are associated with susceptibility to systemic lupus erythematosus in Odisha, India

Variants of MBL gene have been associated with autoimmune disorders. The aim of this study was to explore whether common polymorphisms in MBL gene are associated with susceptibility to systemic lupus erythematosus (SLE) and its clinical manifestations in a cohort from eastern India. A total of 108 female SLE patients and 105 age, sex, and ethnically matched healthy controls were enrolled in the study. MBL2 codon and promoter polymorphisms were genotyped by AS-PCR and dARMS PCR, respectively. Plasma level of MBL was quantified by ELISA. Higher frequency of BB genotype and minor allele (B) was observed in patients of SLE compared to healthy controls (BB genotype: P = 0.0002; OR = 5.75, 95% CI = 2.09-15.76, B allele: P < 0.0001; OR = 2.78, 95% CI = 1.66-4.64). MBL codon 54, H-550L, Y-221X polymorphisms and combined MBL genotypes contributed to plasma MBL levels. Prevalence of MBL low producer genotype (LXA/LYB, LYB/LYB and LXB/LXB) was significantly higher in SLE patients compared to healthy control. (P = 0.005; OR = 3.09, 95% CI = 1.38-6.91). On analysis of clinical manifestations, MBL low producer genotype was significantly associated with autoimmune haemolytic anaemia (P = 0.006; OR = 13.06). Results of the present study indicate MBL2 variants as possible risk factors for development of SLE and clinical manifestation in eastern India.

TLR-9 promoter polymorphisms (T-1237C and T-1486C) are not associated with systemic lupus erythematosus: A case control study and meta-analysis

Toll like receptors (TLRs) are essential molecules implicated in both innate and adaptive immune response. Polymorphisms in TLR gene have been associated with various infectious diseases and autoimmune disorders. Role of TLR9 has been elegantly demonstrated in both human systemic lupus erythematosus (SLE) and mice model of lupus. In the present study we investigated association of TLR-9 promoter polymorphisms (T-1237C and T-1486C) with susceptibility/resistance to SLE in an Eastern Indian state which is endemic to parasitic diseases. 210 Female SLE patients who fulfilled the American College of Rheumatology criteria were enrolled along with matched healthy controls from Odisha, India. TLR-9 polymorphisms (T-1237C and T-1486C) were typed by polymerase chain reaction followed by restriction fragment length polymorphism. For meta-analysis, relevant literatures were searched from PubMed database and comprehensive meta-analysis V2 software was employed for analysis. Allele and genotype frequency of TLR-9 promoter polymorphisms (T-1237C and T-1486C) were comparable among SLE patients and controls. Further, meta-analysis of earlier reports and present study did not reveal a significant association of TLR-9 (T-1237C and T-1486C) polymorphisms with SLE. Data from the present study suggest that TLR-9 promoter polymorphisms are not associated with susceptibility to SLE in an area endemic to parasitic diseases.

Complement Receptor 1 Variants Confer Protection from Severe Malaria in Odisha, India

Background In Plasmodium falciparum infection, complement receptor-1 (CR1) on erythrocyte’s surface and ABO blood group play important roles in formation of rosettes which are presumed to be contributory in the pathogenesis of severe malaria. Although several studies have attempted to determine the association of CR1 polymorphisms with severe malaria, observations remain inconsistent. Therefore, a case control study and meta-analysis was performed to address this issue. Methods Common CR1 polymorphisms (intron 27 and exon 22) and blood group were typed in 353 cases of severe malaria (SM) [97 cerebral malaria (CM), 129 multi-organ dysfunction (MOD), 127 non-cerebral severe malaria (NCSM)], 141 un-complicated malaria and 100 healthy controls from an endemic region of Odisha, India. Relevant publications for meta-analysis were searched from the database. Results The homozygous polymorphisms of CR1 intron 27 and exon 22 (TT and GG) and alleles (T and G) that are associated with low expression of CR1 on red blood cells, conferred significant protection against CM, MOD and malaria deaths. Combined analysis showed significant association of blood group B/intron 27-AA/exon 22-AA with susceptibility to SM (CM and MOD). Meta-analysis revealed that the CR1 exon 22 low expression polymorphism is significantly associated with protection against severe malaria. Conclusions The results of the present study demonstrate that common CR1 variants significantly protect against severe malaria in an endemic area.

Bacteraemia in adult patients presenting with malaria in India

Severe falciparum malaria is a major health problem in Odisha, India, contributing to high mortality. Multi organ dysfunction is a predominant manifestation of severe disease in Odisha, unlike in Africa, where cerebral malaria and anaemia are common. There are several studies implicating bacteraemia with severe malaria in African children while there are no reports in adults in India. This study has addressed this issue by enrolling 67 P. falciparum infected adult patients categorized into severe and uncomplicated malaria. Blood culture failed to confirm bacteraemia in any sample with the exception of one case of uncomplicated malaria. Study is inconclusive with regard to use of antibiotics in adult patients.

Host metabolic responses to Plasmodium falciparum infections evaluated by 1H NMR metabolomics

The human malarial parasite Plasmodium falciparum causes the most severe forms of malarial infections, which include cerebral malaria and various organ dysfunctions amongst adults in India. So far no dependable clinical descriptor is available that can distinguish cerebral malaria from other symptomatically similar diseases such as sepsis and encephalitis. This study aims at evaluating the differential metabolic features of plasma samples from P. falciparum patients with varying severities, and patients suffering from symptomatically similar diseases. 1H Nuclear Magnetic Resonance (NMR) based metabolic profiling of the plasma of the infected individuals and the control population was performed. The differences in the plasma profiles were evaluated through multivariate statistical analyses. The results suggest malaria-specific elevation of plasma lipoproteins. Such an increase was absent in control populations. In addition, cerebral malaria patients exhibited a decrease in plasma glycoproteins; such a reduction was not observed in malarial patients without cerebral symptoms. The data presented here indicates that the metabolism and/or transport of the plasma lipids is specifically perturbed by malarial infections. The differential perturbation of the plasma glycoprotein levels in cerebral malaria patients may have important implications in the diagnosis of cerebral malaria.

MBL-2 polymorphisms (codon 54 and Y-221X) and low MBL levels are associated with susceptibility to multi organ dysfunction in P. falciparum malaria in Odisha, India

Background: Mannose binding lectin, a plasma protein protects host from virus, bacteria, and parasites. Deficiency in MBL levels has been associated with susceptibility to various infectious diseases including P. falciparum malaria. Common MBL polymorphisms in promoter and coding regions are associated with decrease in plasma MBL levels or production of deformed MBL, respectively. In the present study, we hypothesized that MBL2 variants and plasma MBL levels could be associated with different clinical phenotypes of severe P. falciparum malaria. Methods: A hospital based study was conducted in eastern Odisha, India which is endemic to P. falciparum malaria. Common MBL-2 polymorphisms (codon 54, H-550L, and Y-221X) were typed in 336 cases of severe malaria (SM) [94 cerebral malaria (CM), 120 multi-organ dysfunction (MOD), 122 non-cerebral severe malaria (NCSM)] and 131 un-complicated malaria patients (UM). Plasma MBL levels were quantified by ELISA. Results: Severe malaria patients displayed lower plasma levels of MBL compared to uncomplicated falciparum malaria. Furthermore, on categorization of severe malaria patients into various subtypes, plasma MBL levels were very low in MOD patients compared to other categories. Higher frequency of AB genotype and allele B was observed in MOD compared to UM (AB genotype: P = 0.006; B allele: P = 0.008). In addition, prevalence of YX genotype of MBL Y-221X polymorphism was also statistically more frequent in MOD case than UM (P = 0.009). Conclusions: The observations of the present study reveal that MBL-2 polymorphisms (codon 54 and Y-221X) and lower plasma MBL levels are associated with increased susceptibility to multi organ dysfunctions in P. falciparum malaria.

Vitamin D: The unexplored immunomodulator

In the year 1922, Elmer McCollum demonstrated cure of rickets in dogs by modified cod liver oil and named it as vitamin D. Since then it was believed that the major functions of vitamin D were to maintain calcium metabolism and bone homeostasis. Subsequently, it was increasingly recognised that vitamin D played many other roles in multiple systems which included a regulatory role for the immune system. A basic understanding of its mechanism of action provides insight into its capabilities as an immunomodulator. It is a steroid hormone the requirement of which in humans is mostly fulfilled by synthesis in the skin, utilizing UV light and to a limited degree from dietary sources. After several stages of hydroxylation of the precursor molecules, 1,25 dihydroxycholecalciferol (D3), the active biological molecule is ultimately produced in the kidney. Vitamin D interacts with its receptor, the VDR, and makes a heterodimer complex with retinoid X receptor (RXR), which gets translocated to the nucleus. Vitamin D responsive element (VDRE) present in the promoter region is targeted by vitamin D-VDR-RXR heterodimer which induces expression of vitamin D responsive genes. VDR receptors are widely distributed, including in the cells of the immune system, where the immune regulatory functions are demonstrated. Vitamin D has various effects on both B cells and T cells. It hinders B cell proliferation, differentiation and production of antibodies. It also suppresses T cell proliferation and directs the immune response toward Th2 from Th1. Furthermore, it effects T cell maturation, decreases interleukin (IL)-17 phenotype and induces T regulatory cell proliferation. Vitamin D has also significant effect on the innate immune system. It reduces the production of inflammatory molecules by monocytes and decreases the differentiation and maturation of dendritic cells. Peripheral blood mononuclear cells (PBMCs) derived from rheumatoid arthritis (RA) patients treated with 1,25-OH vitamin D showed suppression of IL-17, tumor necrosis factor (TNF)-a and IL-6 production, and up-regulation of osteoprotegerin (OPG)/receptor activator of nuclear factor jB ligand (RANKL) ratio, which was associated with an anti-inflammatory immune response. Furthermore, treatment of systemic lupus erythematosus (SLE) patients with 1,25-OH vitamin D have been shown to activate and increase the number of T regulatory cells, suppress IL-17 and decrease production of interferon (IFN)-a. Vitamin D exerts its effect through the VDR, but common mutations in VDR gene are believed to hamper vitamin D signalling. VDR polymorphisms such as FokI, BsmI and TaqI have been associated with autoimmune disorders. A recent meta-analysis showed association of FokI variant with susceptibility to RA in a European population and a GATA haplotype with atherosclerotic phenotype. Furthermore, FokI and TaqI polymorphisms conferred bad prognosis and increased risk for organ damage in SLE, while BsmI was associated with increased susceptibility to SLE in Asians. Further analyses indicated that TaqI, BsmI and FokI polymorphisms were associated with susceptibility to multiple sclerosis. The efficacy of vitamin D as an immune modulator has been increasingly demonstrated by in vitro studies, animal models and in epidemiological observations of several autoimmune diseases. In experimental models vitamin D has been shown to modulate outcome in CIA (collagen induced arthritis), lupus mice and EAE (experimental autoimmune encephalitis), the experimental equivalents for RA, SLE and multiple sclerosis, respectively. Despite the bounty of nature in the form of abundant sunshine which can boost vitamin D synthesis in the body, there are reports of widespread deficiency/insufficiency of vitamin D across vast populations of the world. What are the consequences? Besides poor musculosketal health it perhaps triggers many other problems, including autoimmune diseases in genetically susceptible individuals. The direct cause– effect relationship in autoimmune diseases has not been established by robust longitudinal studies but associational observations abound. The association between vitamin D deficiency and autoimmune disorders such as RA, SLE, multiple sclerosis and inflammatory bowel disease (IBD) has been widely reported. A recent meta-analysis and earlier reports have shown lower serum vitamin D in RA