Pramod Upadhyay

Immunogenicity and Protective Efficacy of “Mycobacterium w” against Mycobacterium tuberculosis in Mice Immunized with Live versus Heat-Killed M. w by the Aerosol or Parenteral Route

ABSTRACT As the disease caused by Mycobacterium tuberculosis continues to be a burden, there is a concerted effort to find new vaccines to combat this problem. One of the important vaccine strategies is whole bacterial vaccines. This approach relies on multiple antigens and built-in adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M. tuberculosis have been considered as alternatives to M. bovis for vaccine use. One such strain, “Mycobacterium w”, had been evaluated for its immunomodulatory properties in leprosy. A vaccine against leprosy based on killed M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae antigens. M. w shares antigens not only with M. leprae but also with M. tuberculosis, and initial studies have shown that vaccination with killed M. w induces protection against tuberculosis in Mycobacterium bovis BCG responder, as well as BCG nonresponder, strains of mice. Hence, we further studied the protective potential of M. w and the underlying immune responses in the mouse model of tuberculosis. We analyzed the protective efficacy of M. w immunization in both live and killed forms through the parenteral route and by aerosol immunization, compared with that of BCG. Our findings provide evidence that M. w has potential protective efficacy against M. tuberculosis. M. w activates macrophage activity, as well as lymphocytes. M. w immunization by both the parenteral route and aerosol adminstration gives higher protection than BCG given by the parenteral route in the mouse model of tuberculosis.

Needle-free, non-adjuvanted skin immunization by electroporation-enhanced transdermal delivery of diphtheria toxoid and a candidate peptide vaccine against hepatitis B virus

To explore the feasibility of non-adjuvanted, needle-free skin immunization, we examined the immune responses of mice immunized by three routes with two antigens (Ag) differing in molecular size and lipophilicity. Diphtheria toxoid (DT) or a myristylated peptide (MYR) administered either by transdermal electroporation (EP) or intradermally (i.d.) with a needle elicited markedly different responses. The EP route elicited higher responses to MYR than i.d. immunization, but lower responses to DT. Intraperitoneal (i.p.) immunization evoked responses against MYR that were higher than those evoked by EP. It was inferred that EP is a promising technique for non-adjuvanted skin immunization, specially with low molecular weight, weakly immunogenic Ag.

Combined chemo-immunotherapy as a prospective strategy to combat cancer: a nanoparticle based approach.

The prime objective of this study was to develop a combined chemo-immunotherapeutic formulation which could directly kill cancer cells as well as activate the immunosuppressed tumor microenvironment to mount a robust antitumor immune response. Paclitaxel (PTX) and SP-LPS (nontoxic derivative of lipopolysaccharide) were selected as anticancer drug and immunostimulant respectively. Poly(lactic-co-glycolic acid) (PLGA) based PTX and SP-LPS containing nanoparticles (TLNP) were prepared by the double-emulsion method (w/o/w) and characterized in terms of size, zeta potential and transmission electron microscopy (TEM). The release behavior of PTX and SP-LPS from the TLNP exhibited a biphasic pattern characterized by an initial burst followed by slow continuous release. In vitro anticancer activity of TLNP was found to be higher compared to PTX when studied in a tumor cell-splenocyte coculture system. TLNP activated murine monocytes induced the secretion of various proinflammatory cytokines. After iv administration of TLNP in tumor bearing C57BL/6 mice, the amount of PTX in the tumor mass was found to be higher in TLNP treated mice as compared to commercial Taxol group at all time points studied. In vitro studies suggest that nanoparticles containing PTX and SP-LPS have both direct cytotoxicity and immunostimulatory activity. Hence this might have potential as a chemo-immunotherapeutic formulation against cancer with advantage over present day chemotherapy with Taxol, in terms of tumor targeting, less toxicity and immunostimulation.

Nanoparticle mediated co-delivery of paclitaxel and a TLR-4 agonist results in tumor regression and enhanced immune response in the tumor microenvironment of a mouse model.

Inefficiency of cancer chemotherapy to improve life expectancy in majority of patients raises serious concern and warrants development of novel therapeutic strategies. Immunotherapy in combination with chemotherapy has shown promising outcomes in recent years. Herein, we report better tumor regression and enhancement of antitumor immune response at the tumor microenvironment by co-delivery of paclitaxel and a TLR4 agonist through a PLGA based nanoparticle preparation (TLNP). Particle characterization showed high encapsulation of both components and retention of their biological activities. In vivo tumor regression studies demonstrated clear benefit of TLNP over the paclitaxel. The mean tumor volume of the TLNP treated animals was found to be 40% less than that of the Paclitaxel treated animals. Flow cytometric analysis of tumor infiltrating immune cells indicated activation of antigen presenting cells and T-cells providing evidence of Th1 immune response. In vivo results are promising and could pave way for novel chemo-immunotherapeutic treatment modality.

Activation of Anti-Tumor Immune Response and Reduction of Regulatory T Cells with Mycobacterium indicus pranii (MIP) Therapy in Tumor Bearing Mice

Background Role of immune system in protecting the host from cancer is well established. Growing cancer however subverts immune response towards Th2 type and escape from antitumor mechanism of the host. Activation of both innate and Th1 type response is crucial for host antitumor activity. In our previous study it was found, that Mycobacterium indicus pranii (MIP) also known as M. w induces Th1 type response and activates macrophages in animal model of tuberculosis. Hence, we studied the immunotherapeutic potential of MIP in mouse tumor model and the underlying mechanisms for its antitumor activity. Methodology and Principal Findings Tumors were implanted by injecting B16F10 melanoma cells subcutaneously into C57BL/6 mice. Using the optimized dose and treatment regimes, anti-tumor efficacy of heat killed MIP was evaluated. In MIP treated group, tumor appeared in only 50–60% of mice, tumor growth was delayed and tumor volume was less as compared to control. MIP mediated immune activation was analysed in the tumor microenvironment, tumor draining lymph node and spleen. Induction of Th1 response and higher infiltration of immune cells in the tumor microenvironment was observed in MIP treated mice. A large fraction of these immune cells were in activated state as confirmed by phenotypic and functional analysis. Interestingly, percentage of Treg cells in the tumor milieu of treated mice was less. We also evaluated efficacy of MIP along with chemotherapy and found a better response as compared to chemotherapy alone. Conclusion MIP therapy is effective in protecting mice from tumor. It activates the immune cells, increases their infiltration in tumor, and abrogates tumor mediated immune suppression.

A novel ultrasound-enhanced latex agglutination test for the detection of antibodies against Mycobacterium tuberculosis in serum

A novel ultrasound-enhanced latex agglutination test is described for the detection of serum antibodies against Mycobacterium tuberculosis. The use of ultrasound to detect the low level of antibodies in serum enhances the sensitivity of the test. The technique is based on the fact that suspended latex particles become concentrated in an ultrasonic standing wave field, thereby increasing the rate of particle-particle collisions compared to the standard agglutination test procedure. Reactions were performed on piezo-ceramic wafers, which were connected to an oscillator at a frequency of 100 kHz. This method improved sensitivity significantly so that even 20 times diluted serum samples exhibited agglutination, which was clearly visible to the naked eye.

Anticancer and Immunostimulatory Activity by Conjugate of Paclitaxel and Non-toxic Derivative of LPS for Combined Chemo-immunotherapy

ABSTRACTPurposeCancer is a multifactorial syndrome; hence, multidimensional therapy with a chemo-immunotherapeutic conjugate could be more effective in curing the disease.MethodsWe used SP-LPS, a bio-polymer having potent immunostimulatory activity, for conjugation with paclitaxel to make a chemo-immunotherapeutic conjugate. Its physicochemical characterization was done by HPLC, NMR and IR spectra. Stability was measured at different pH, temperature and in tissue homogenates. Chemotherapeutic and immunostimulatory activity was evaluated in vitro and also in tumor microenvironment.ResultsThe conjugate self assembled into nanoparticulate structure, probably due to micelle formation. Stability was pH and temperature dependent. The conjugate exhibited chemotherapeutic and immunotherapeutic activity in vitro. In vivo antitumor activity was significantly higher and a higher percentage of activated immune cells were found in the tumor microenvironment of the conjugate-treated mice as compared to Taxol®-treated group.ConclusionsThis conjugate is a potential chemo-immunotherapeutic compound for the treatment of cancer with advantages over present day chemotherapy with Taxol in terms of higher anticancer activity, less toxicity and ease of delivery.

Adjuvant properties of thermal component of hyperthermia enhanced transdermal immunization: effect on dendritic cells.

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.

Real time monitoring of lymphocyte proliferation by an impedance method

Impedance methods are routinely used to estimate the concentration of viable bacteria in a culture. We have adapted an impedance method to monitor the growth of lymphocytes and used it to monitor lymphocyte proliferation in real time. In this method lymphocytes were cultured in modified micro-well strips with transparent indium-titanium oxide coated electrodes at the bottom. The assay was totally automated and did not involve handling of radioactive chemicals. As the method uses a non destructive method of recording the proliferation, the cells could be used for other studies after the proliferation assay. Due to the real time monitoring of proliferation, results could be obtained within 30 h and additional information such as the rate of proliferation and the limiting rate at time-->0 could also be calculated instantly.

Animal Models Correlating Immune Cells for the Development of NAFLD/NASH

This review mainly elaborates on the animal models available for understanding the pathogenesis of the second hit of non-alcoholic fatty liver disease (NAFLD) involving immune system. This is known to be a step forward from simple steatosis caused during the first hit, which leads to the stage of inflammation followed by more serious liver conditions like non-alcoholic steatohepatitis (NASH) and cirrhosis. Immune-deficient animal models serve as an important tool for understanding the role of a specific cell type or a cytokine in the progression of NAFLD. These animal models can be used in combination with the already available animal models of NAFLD, including dietary models, as well as genetically modified mouse models. Advancements in molecular biological techniques enabled researchers to produce several new animal models for the study of NAFLD, including knockin, generalized knockout, and tissue-specific knockout mice. Development of NASH/NAFLD in various animal models having compromised immune system is discussed in this review.