M. G. R. Rajan

G1-4A, a Polysaccharide from Tinospora cordifolia Inhibits the Survival of Mycobacterium tuberculosis by Modulating Host Immune Responses in TLR4 Dependent Manner

Rapid emergence of drug resistance in Mycobacterium tuberculosis (MTB) is a major health concern and demands the development of novel adjunct immunotherapeutic agents capable of modulating the host immune responses in order to control the pathogen. In the present study, we sought to investigate the immunomodulatory effects of G1-4A, a polysaccharide derived from the Indian medicinal plant Tinospora cordifolia, in in-vitro and aerosol mouse models of MTB infection. G1-4A treatment of MTB infected RAW264.7 macrophages significantly induced the surface expression of MHC-II and CD-86 molecules, secretion of proinflammatory cytokines (TNF-α, IL-β, IL-6, IL-12, IFN-γ) and nitric oxide leading to reduced intracellular survival of both drug sensitive (H37Rv) as well as multi drug resistant strains (Beijing and LAM) of MTB, which was partially attributed to G1-4A induced NO production in TLR4-MyD88 dependent manner. Similarly, bacillary burden was significantly reduced in the lungs of MTB infected BALB/c mice treated with G1-4A, with simultaneous up-regulation of the expression of TNF-α, INF-γ and NOS2 in the mouse lung along with increased levels of Th1 cytokines like IFN-γ, IL-12 and decreased levels of Th2 cytokine like IL-4 in the serum. Furthermore, combination of G1-4A with Isoniazid (INH) exhibited better protection against MTB compared to that due to INH or G1-4A alone, suggesting its potential as adjunct therapy. Our results demonstrate that modulation of host immune responses by G1-4A might improve the therapeutic efficacy of existing anti-tubercular drugs and provide an attractive strategy for the development of alternative therapies to control tuberculosis.

Activation of murine macrophages by G1-4A, a polysaccharide from Tinospora cordifolia, in TLR4/MyD88 dependent manner

Abstract Macrophages are centrally placed in the innate immune system and their activation is crucial to the generation of appropriate immune response in the event of any pathogenic invasion, tumorigenesis or other human diseases. Many plant derived polysaccharides are known to activate macrophages. In the present study, effects of G1‐4A, a polysaccharide derived from Tinospora cordifolia, on the activation of macrophages were investigated. Our data demonstrated the up regulation of expression of TNF‐&agr;, IL‐&bgr;, IL‐6, IL‐12, IL‐10 and IFN‐&ggr; in RAW 264.7 cell line and peritoneal macrophages after G‐14A treatment. Nitric oxide levels were also enhanced along with up‐regulation of NOS2 expression in murine macrophages post G1‐4A treatment. Further, G1‐4A treatment up‐regulated the surface expression of MHC‐II and CD‐86 in macrophages. Using siRNA against TLR4, MyD88 and anti‐TLR4 blocking antibodies, we established that G1‐4A activated macrophages by classical pathway in TLR4‐MyD88 dependent manner. Additionally, G1‐4A treatment activated p38, ERK and JNK MAPKs in macrophages. Using pharmaceutical inhibitors of above MAPKs we concluded that G1‐4A activates the macrophages by activation of p38, ERK and JNK MAPKs in RAW264.7 macrophages. Thus our data suggests the activation of macrophages by classical pathway after treatment of G1‐4A. HighlightsG1‐4A treatment activates murine macrophages through classical pathway.G1‐4A mediated macrophage activation is TLR4/MyD88 dependent.ERK, JNK and p38 MAPK were involved in G1‐4A mediated macrophage activation.

A multi-analyte immunoassay for thyroid related analytes

ABSTRACT We describe the development and validation of multianalyte immunoassays (MAIA) for three analytes, viz., thyroxine (T4), thyroid stimulating hormone (TSH), and thyroglobulin (Tg) essential for assessment of thyroid function but having widely varying molecular weights. Using polycarbonate (PC) track-etched membranes (TEM) as an immobilization support and 125I as the tracer, both competitive assay for T4 and non-competitive assay for TSH and Tg were performed on the same TEM. MAIA was found to be highly sensitive and precise with clinically useful working range and correlated very well with individual analyte immunoassays. While we have demonstrated this assay format with radiotracer, it can be used with non-isotopic tracers equally well.

External quality assessment of RIA for thyroid related hormones

An external quality assessment was conducted for RIA of thyroid related hormones. Thirtyfive laboratories (35 for T4, 34 for T3 and 23 for TSH) from different parts of country participated in the programme. Twentyfour samples (16 pools: 5 simple and 11 manipulated pools) in 8 batches, 3 per batch per month were sent for analysis of T4, T3 and TSH. Some of the samples were repeated 3 times at different occasions to assess the imprecision of the laboratory. The overall mean percent CV obtained for T4, T3 and TSH were 22.7, 36.32 and 52.38 respectively. The recovery for added T4 was 86.73% while that for T3 was 117.4%. A large variation was obtained for recovery of TSH. For T4 estimations, 13 laboratories had a desirable performance i. e. bias less than ±10.0% and variability of bias (VB) and imprecision (IP) less than 15.0%. None of the laboratories had a desirable performance for T3 or TSH. The number of laboratories with acceptable performance i. e. bias between ±10.0–15.0%, VB and IP between 20.0–25.0% for T4, T3 and TSH were 4, 3 and 0 respectively. The number of laboratories which required attention (bias between ±15.0–20.0%; VB and IP between 20.0–25.0%) were 5,7 and 1 respectively. The unacceptable results with larger bias, VB and IP for T4, T3 and TSH were 6, 18 and 17 respectively. Our results are in general agreement that the performance of T4 assay is better than T3 and both in turn are much better than TSH.Quantitation of circulating thyroid hormones (TH) viz. tri-iodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH), which form the largest percentage of hormones estimated in a clinical laboratory is mainly done by radio-immunoassay (RIA) procedures. The reliability and reproducibility of these assays are generally monitored by using internal quality control (IQC) samples in every assay batch. Thus, the IQC provides information whether the assay results are satisfactory and can be released. However, external quality assessment (EQA) is a procedure whereby an external agency undertakes evaluation of the quality of an analytical service by providing samples for analysis to individual laboratories performing the assay. The data gathered is analysed collectively. EQA therefore provides a means by which performance of a laboratory is assessed in relation to other laboratories and matching the assay unbiased by removing systematic error, if present. This is important since RIA procedure involves several steps (collection and storage of samples, quality of the reagents, procedure followed for performance of an assay, counting equipment used and the mode of the data analysis) and therefore prone to systematic errors. We therefore undertook the EQA programme for assessment of thyroid related hormones as a joint collaborative project of Bhabha Atomic Research Centre, India and International Atomic Energy Agency.

Quantitative analysis of SPECT imaging parameters in patients with resting perfusion defects on myocardial perfusion scintigraphy

Background: Functional status/contractile behaviour of hibernating myocardium was analyzed objectively by analyzing the available quantitative parameters obtained on gated SPECT myocardial perfusion imaging (MPI) using Emory cardiac toolbox (ECTB) software. Materials and Methods: In this retrospective study, 70 patients with perfusion defects on 99Tc-Sestamibi MPI (12 females, 58 males) who also underwent 18F-FDG Cardiac PET study for assessment of hibernating myocardium were included for analysis. Patients were divided in three categories based on summed rest score (SRS) obtained from ECTB software, depicting the extent of perfusion defects. In a study population matched for extent of perfusion defects, quantitative parameters obtained from ECTB software such as left ventricular ejection fraction (LVEF), left ventricular end diastolic volume (EDV), left ventricular end systolic volume (ESV) and left ventricular stroke volume (SV) were compared between patients showing evidence of hibernating myocardium and patients showing no evidence of hibernating myocardium. Student ‘t’ test was applied on the given observations and a P-value <0.05 was considered as a significant difference between the means in two categories. Results: There was no significant difference in LVEF, EDV, ESV and SV measurements between those who demonstrate hibernating myocardium and those who show no evidence of hibernating myocardium across all the categories of patients. Few trends were evident in the present study in LVEF, EDV and ESV measurements i.e., fall in mean LVEF with increasing SRS and rise in mean EDV and ESV with increasing SRS. Conclusions: The findings were consistent with the nature of hibernating myocardium i.e., non-contractile and dysfunctional. The fall in the LVEF was suggestive of deteriorating myocardial function with increasing extent of perfusion defects. The increasing left ventricular EDV and ESV with increasing extent of perfusion defects was suggestive of rising incidence of gross morphological LV cavity dilatation or “Dilated ischemic cardiomyopathy” in these patients.

External quality assessment for the RIA of thyroid related hormones: Second phase

An external quality assessment was conducted to assess the performance of various laboratories for RIA of thhyroid related hormones in two phases. In the first phase thirty five laboratories participated. At the end of first phase a meeting cum workshop was organised to discuss the results of first phase, difficultires faced by the participants and pinpoint the short oming. A second phase was then initiated with an objective of improvement in the performance, if any, where twelve samples from four pools were distributed to twenty four laboratories who participated for the second phase. The overall return of the results increased significantly from 71.8% (1586/2208) for the first phase to 92.4% (732/792) for the second phase. The inter laboratory %CV for T3, T4 and TSH were lower during the second phase (30.6%, 19.0% and 31.6% respectively) as compared to those observed during first phase (36.3%, 22.7% and 52.8% respectively). Similarly, there was an improvement in reproducibility of ALTM as %CV for T3, T4 and TSH decreased from 6.0%, 9.8% and 13.4% respectively to 4.5%, 4.6% and 8.5% respectively. The individual performances of the participating laboratories viz. bias, variability of bias and imprecision also showed a trend towards improvement as percent laboratories having desirable or acceptable results for T3, T4 and TSH increased from 10.7%, 60.7% and 0.0% respectively to 20.8%, 66.7% and 22.2% respectively. External quality assessment thus appears to be beneficial in assessing the performane of a laboratory in comfparison with other laboratories and indeed helps in improving the performance.