Resmi Ravindran

NOD1 and NOD2 signalling links ER stress with inflammation

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Profiling Antibodies to Mycobacterium tuberculosis by Multiplex Microbead Suspension Arrays for Serodiagnosis of Tuberculosis

ABSTRACT Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests.

A recombinant attenuated Mycobacterium tuberculosis vaccine strain is safe in immunosuppressed simian immunodeficiency virus-infected infant macaques

ABSTRACT Many resource-poor countries are faced with concurrent epidemics of AIDS and tuberculosis (TB) caused by human immunodeficiency virus (HIV) and Mycobacterium tuberculosis, respectively. Dual infections with HIV and M. tuberculosis are especially severe in infants. There is, however, no effective HIV vaccine, and the only licensed TB vaccine, the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine, can cause disseminated mycobacterial disease in HIV-infected children. Thus, a pediatric vaccine to prevent HIV and M. tuberculosis infections is urgently needed. We hypothesized that a highly attenuated M. tuberculosis strain containing HIV antigens could be safely administered at birth and induce mucosal and systemic immune responses to protect against HIV and TB infections, and we rationalized that vaccine safety could be most rigorously assessed in immunocompromised hosts. Of three vaccine candidates tested, the recombinant attenuated M. tuberculosis strain mc26435 carrying a simian immunodeficiency virus (SIV) Gag expression plasmid and harboring attenuations of genes critical for replication (panCD and leuCD) and immune evasion (secA2), was found to be safe for oral or intradermal administration to non-SIV-infected and SIV-infected infant macaques. Safety was defined as the absence of clinical symptoms, a lack of histopathological changes indicative of M. tuberculosis infection, and a lack of mycobacterial dissemination. These data represent an important step in the development of novel TB vaccines and suggest that a combination recombinant attenuated M. tuberculosis-HIV vaccine could be a safe alternative to BCG for the pediatric population as a whole and, more importantly, for the extreme at-risk group of HIV-infected infants.

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

Plasma Antibody Profiles as Diagnostic Biomarkers for Tuberculosis

ABSTRACT Two billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), worldwide. Ten million to 20 million of the infected individuals develop disease per year. TB is a treatable disease, provided that it is diagnosed in a timely manner. The current TB diagnostic methods are subjective, inefficient, or not cost-effective. Antibody-based blood tests can be used efficiently and cost-effectively for TB diagnosis. A major challenge is that different TB patients generate antibodies against different antigens. Therefore, a multiplex immunoassay approach is needed. We have developed a multiplex panel of 28 M. tuberculosis antigen-coated microbeads. Plasma samples were obtained from over 300 pulmonary TB patients and healthy controls in a country where TB is endemic, Pakistan. Multiplex data were analyzed using computational tools by multivariate statistics, classification algorithms, and cluster analysis. The results of antibody profile-based detection, using 16 selected antigens, closely correlated with those of the sputum-based diagnostic methods (smear microscopy and culture) practiced in countries where TB is endemic. Multiplex microbead immunoassay had a sensitivity and specificity of approximately 90% and 80%, respectively. These antibody profiles could potentially be useful for the diagnosis of nonpulmonary TB, which accounts for approximately 20% of cases of disease. Since an automated, high-throughput version of this multiplex microbead immunoassay could analyze thousands of samples per day, it may be useful for the diagnosis of TB in millions of patients worldwide.

Multiplexed measurements of immunomodulator levels in peripheral blood of healthy subjects: Effects of analytical variables based on anticoagulants, age, and gender.

Multiplex microbead immunoassay (MMIA) is a powerful technology for a wide range of biomedical and clinical applications. It is important to study the normal concentration ranges of immunomodulators under different sample preparation conditions and age groups of subjects in order to more precisely determine their reference values for use in assessing alterations of their levels in disease. The aim of this study was to determine the plasma concentrations of immunomodulators (cytokines, chemokines, and growth factors) in the peripheral blood from healthy subjects by the use of a large multiplex panel, and to determine the effects of different anticoagulants, age, and gender on the immunomodulator levels. In addition, the assay precision for these biomarker analytes was determined. Plasma samples from 107 healthy subjects, aged 18 to 85 years, were collected in three different anticoagulants (sodium citrate, EDTA, Heparin); corresponding serum samples were also obtained. Multiplex microbead immunoassays were performed for measuring a total of 23 analytes including chemokines, cytokines, and growth factors (IL‐1β, IL‐1ra, IL‐2, IL‐6, IL‐7, IL‐8, IL‐12 p70, IL‐17, IFN‐γ, IP‐10, MCP‐1, PDGF‐BB, RANTES, TNF‐α, IL‐1a, IL‐16, HGF, MIG, TNF‐β, PDGF‐ABBB, EGF, Flt‐3 Ligand, VEGF). For these analytes, our results showed that the anticoagulant affected the concentration measurements and the coefficients of variation. However, the relative levels of the analytes (profiles) of samples collected in a particular anticoagulant are consistent. The analytes IL‐1β, IL‐7, Flt‐3 Ligand, and IL‐12p70 show the largest variation (up to fourfold) between the age groups. In addition, no statistically significant differences in the level of the analytes were found between the sexes.

Plasma antibody profiles in non-human primate tuberculosis

Tuberculosis (TB) in non‐human primates (NHPs) is highly contagious, requiring efficient identification of animals infected with Mycobacterium tuberculosis. Tuberculin skin test is usually used but lacks desirable sensitivity/specificity and efficiency.

Exploratory Study on Plasma Immunomodulator and Antibody Profiles in Tuberculosis Patients

ABSTRACT Host immune responses to Mycobacterium tuberculosis are generally able to contain infection and maintain a delicate balance between protection and immunopathology. A shift in this balance appears to underlie active disease observed in about 10% of infected individuals. Effects of local inflammation, combined with anti-M. tuberculosis systemic immune responses, are directly detectable in peripheral circulation, without ex vivo stimulation of blood cells or biopsy of the affected organs. We studied plasma immunomodulator and antibody biomarkers in patients with active pulmonary tuberculosis (TB) by a combination of multiplex microbead immunoassays and computational tools for data analysis. Plasma profiles of 10 immunomodulators and antibodies against eight M. tuberculosis antigens (previously reported by us) were examined in active pulmonary TB patients in a country where TB is endemic, Pakistan. Multiplex analyses were performed on samples from apparently healthy individuals without active TB from the same community as the TB patients to establish the assay baselines for all analytes. Over 3,000 data points were collected from patients (n = 135) and controls (n = 37). The data were analyzed by multivariate and computer-assisted cluster analyses to reveal patterns of plasma immunomodulators and antibodies. This study shows plasma profiles that in most patients represented either strong antibody or strong immunomodulator biomarkers. Profiling of a combination of both immunomodulators and antibodies described here may be valuable for the analysis of host immune responses in active TB in countries where the disease is endemic.

Validation of multiplex microbead immunoassay for simultaneous serodetection of multiple infectious agents in laboratory mouse.

Multiplex methodologies enable simultaneous detection of antibodies against several infectious agents allowing sample conservation, cost effectiveness, and amenability to high-throughput/automation. We have previously described a multiplex microbead immunoassay for serodetection of ten, high-priority mouse infectious pathogens. Here, we present a validation of this multiplex diagnostic system using approximately four hundred serum samples from different groups of mice. Computer assisted multivariate analysis of the resulting high volume data (8000 data points) was performed. This computational approach enabled presentation of data in a variety of easily interpretable formats (e.g., correlation tables and heat maps). Importantly, this computer aided approach was instrumental for the evaluation of assay accuracy, sensitivity, specificity, and robustness during the study. Crucial pieces of information were obtained to make timely adjustments for assay refinement. This progressive approach to developing an implementation-ready clinical assay, facilitated by computational analysis, produced a highly efficient, accurate and dependable serodiagnostics system. This system has effectively replaced the current state-of-the-art methodology (ELISA) used in mouse colony health management at the University of California and the Jackson Laboratory. A pathway to develop multiplex serology tests for infectious disease diagnosis described here serves as a model for multiplex immunoassay design, clinical validation, refinement and implementation.

Abnormal mammary development in 129: STAT1-null mice is stroma-dependent

Female 129:Stat1-null mice (129S6/SvEvTac-Stat1tm1Rds homozygous) uniquely develop estrogen-receptor (ER)-positive mammary tumors. Herein we report that the mammary glands (MG) of these mice have altered growth and development with abnormal terminal end buds alongside defective branching morphogenesis and ductal elongation. We also find that the 129:Stat1-null mammary fat pad (MFP) fails to sustain the growth of 129S6/SvEv wild-type and Stat1-null epithelium. These abnormalities are partially reversed by elevated serum progesterone and prolactin whereas transplantation of wild-type bone marrow into 129:Stat1-null mice does not reverse the MG developmental defects. Medium conditioned by 129:Stat1-null epithelium-cleared MFP does not stimulate epithelial proliferation, whereas it is stimulated by medium conditioned by epithelium-cleared MFP from either wild-type or 129:Stat1-null females having elevated progesterone and prolactin. Microarrays and multiplexed cytokine assays reveal that the MG of 129:Stat1-null mice has lower levels of growth factors that have been implicated in normal MG growth and development. Transplanted 129:Stat1-null tumors and their isolated cells also grow slower in 129:Stat1-null MG compared to wild-type recipient MG. These studies demonstrate that growth of normal and neoplastic 129:Stat1-null epithelium is dependent on the hormonal milieu and on factors from the mammary stroma such as cytokines. While the individual or combined effects of these factors remains to be resolved, our data supports the role of STAT1 in maintaining a tumor-suppressive MG microenvironment.