Rangaiah Shashidharamurthy

Another View of T Cell Antigen Recognition: Cooperative Engagement of Glycolipid Antigens by Va14Ja18 Natural TCR

Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor β-chain repertoire and how α-galactosylceramide (α-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-αGalCer- and αGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional αβ TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.

Variations in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution

Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA.

Lipocalin 2 Deficiency Dysregulates Iron Homeostasis and Exacerbates Endotoxin-Induced Sepsis

Various states of inflammation, including sepsis, are associated with hypoferremia, which limits iron availability to pathogens and reduces iron-mediated oxidative stress. Lipocalin 2 (Lcn2; siderocalin, 24p3) plays a central role in iron transport. Accordingly, Lcn2-deficient (Lcn2KO) mice exhibit elevated intracellular labile iron. In this study, we report that LPS induced systemic Lcn2 by 150-fold in wild-type mice at 24 h. Relative to wild-type littermates, Lcn2KO mice were markedly more sensitive to endotoxemia, exhibiting elevated indices of organ damage (transaminasemia, lactate dehydrogenase) and increased mortality. Such exacerbated endotoxemia was associated with substantially increased caspase-3 cleavage and concomitantly elevated immune cell apoptosis. Furthermore, cells from Lcn2KO mice were hyperresponsive to LPS ex vivo, exhibiting elevated cytokine secretion. Additionally, Lcn2KO mice exhibited delayed LPS-induced hypoferremia despite normal hepatic hepcidin expression and displayed decreased levels of the tissue redox state indicators cysteine and glutathione in liver and plasma. Desferroxamine, an iron chelator, significantly protects Lcn2KO mice from LPS-induced toxicity, including mortality, suggesting that Lcn2 may act as an antioxidant in vivo by regulating iron homeostasis. Thus, Lcn2-mediated regulation of labile iron protects the host against sepsis. Its small size and simple structure may make Lcn2 a deployable treatment for sepsis.

Dynamics of the interaction of human IgG subtype immune complexes with cells expressing R and H allelic forms of a low-affinity Fc gamma receptor CD32A.

CD32A, the major phagocytic FcγR in humans, exhibits a polymorphism in the ligand binding domain. Individuals homozygous for the R allelic form of CD32A (CD32AR allele) are more susceptible to bacterial infections and autoimmune diseases as compared with H allelic CD32A (CD32AH) homozygous and CD32AR/H heterozygous individuals. To understand the mechanisms behind this differential susceptibility, we have investigated the dynamics of the interaction of these allelic forms of CD32A when they are simultaneously exposed to immune complexes (IC). Binding studies using Ig fusion proteins of CD32A alleles showed that the R allele has significantly lower binding not only to human IgG2, but also to IgG1 and IgG3 subtypes. Competition assays using purified molecules demonstrated that CD32AH-Ig outcompetes CD32AR-Ig for IC binding when both alleles simultaneously compete for the same ligand. CD32AH-Ig blocked the IC binding mediated by both the allelic forms of cell surface CD32A, whereas CD32AR-Ig blocked only CD32AR and was unable to cross-block IC binding mediated by CD32AH. Two-dimensional affinity measurements also demonstrated that CD32AR has significantly lower affinity toward all three subtypes as compared with CD32AH. Our data suggest that the lower binding of CD32AR not only to IgG2 but also to IgG1 and IgG3 might be responsible for the lack of clearance of IC leading to increased susceptibility to bacterial infections and autoimmune diseases. Our data further suggests that in humans, inflammatory cells from CD32AR/H heterozygous individuals may predominantly use the H allele to mediate Ab-coated target cell binding during phagocytosis and Ab-dependent cellular cytotoxicity, resulting in a phenotype similar to CD32AH homozygous individuals.

Differential Role of Lipocalin 2 During Immune Complex–Mediated Acute and Chronic Inflammation in Mice

OBJECTIVE Lipocalin 2 (LCN-2) is an innate immune protein that is expressed by a variety of cells and is highly up-regulated during several pathologic conditions, including immune complex (IC)-mediated inflammatory/autoimmune disorders. However, the function of LCN-2 during IC-mediated inflammation is largely unknown. Therefore, this study was undertaken to investigate the role of LCN-2 in IC-mediated diseases. METHODS The up-regulation of LCN-2 was determined by enzyme-linked immunosorbent assay in 3 different mouse models of IC-mediated autoimmune disease: systemic lupus erythematosus, collagen-induced arthritis, and serum-transfer arthritis. The in vivo role of LCN-2 during IC-mediated inflammation was investigated using LCN-2-knockout mice and their wild-type littermates. RESULTS LCN-2 levels were significantly elevated in all 3 of the autoimmune disease models. Further, in an acute skin inflammation model, LCN-2-knockout mice exhibited a 50% reduction in inflammation, with histopathologic analysis revealing notably reduced immune cell infiltration as compared to wild-type mice. Administration of recombinant LCN-2 to LCN-2-knockout mice restored inflammation to levels observed in wild-type mice. Neutralization of LCN-2 using a monoclonal antibody significantly reduced inflammation in wild-type mice. In contrast, LCN-2-knockout mice developed more severe serum-induced arthritis compared to wild-type mice. Histologic analysis revealed extensive tissue and bone destruction, with significantly reduced neutrophil infiltration but considerably more macrophage migration, in LCN-2-knockout mice compared to wild-type mice. CONCLUSION These results demonstrate that LCN-2 may regulate immune cell recruitment to the site of inflammation, a process essential for the controlled initiation, perpetuation, and resolution of inflammatory processes. Thus, LCN-2 may present a promising target in the treatment of IC-mediated inflammatory/autoimmune diseases.

Immune Complex-Mediated Enhancement of Secondary Antibody Responses

Immunologic memory is a hallmark of the vertebrate immune system. The first antigenic exposure leads to a slow and modest immune response, whereas repeated exposure, even many years later, leads to a rapid and exaggerated response that is two to three orders of magnitude greater than the primary. In the case of humoral immunity, the increased efficacy of recall responses is due to the production of amplified levels of Ag-specific Ab, as well as the accelerated kinetics of their production. Current thinking suggests that this is due to selective activation of long-lived, Ag-specific memory B cells. A downside of restricting secondary responses solely to memory cells is that the repertoire of the memory B cell pool remains static while pathogens continue to evolve. In this study, we propose that during secondary responses, naive Ag-specific B cells participate alongside memory cells. We show that immune complexes formed in vivo between the Ag and pre-existing Abs from the primary response activate these naive B cells, inducing them to respond with accelerated kinetics and increased magnitude. Thus, the continued recruitment of new B cell clones after each antigenic exposure enables the immune system to stay abreast of rapidly changing pathogens.

Inhibition of expression of anti-apoptotic protein Bcl-2 and induction of cell death in radioresistant human prostate adenocarcinoma cell line (PC-3) by methyl jasmonate

Hormone refractory human prostate cancer cell lines are known to be radioresistant, a feature attributed to their ability to induce anti-apoptotic proteins of the Bcl-2 family when exposed to radiation. We investigated whether pro-apoptotic compounds such as methyl jasmonate, a plant stress hormone, can counteract the radiation-induced anti-apoptotic mechanism in a human prostate cancer cell line PC-3. Significant (p<0.05) increase in cytotoxicity was observed in the combined treatment groups compared to single treatments with methyl jasmonate or gamma-radiation. Treatment of irradiated PC-3 cells with methyl jasmonate resulted in suppression of anti-apoptotic Bcl-2 protein and elevation of caspase-3 activity. Our results showed increased apoptosis in the combined treatment group as compared to the irradiated group or the untreated control. In summary, methyl jasmonate suppressed the radiation-induced Bcl-2 expression and enhanced the radiation sensitivity of human prostate cancer cells.

Antibody Effector Functions Mediated by Fcγ-Receptors Are Compromised during Persistent Viral Infection

T cell dysfunction is well documented during chronic viral infections but little is known about functional abnormalities in humoral immunity. Here we report that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit a severe defect in Fcγ-receptor (FcγR)-mediated antibody effector functions. Using transgenic mice expressing human CD20, we found that chronic LCMV infection impaired the depletion of B cells with rituximab, an anti-CD20 antibody widely used for the treatment of B cell lymphomas. In addition, FcγR-dependent activation of dendritic cells by agonistic anti-CD40 antibody was compromised in chronically infected mice. These defects were due to viral antigen-antibody complexes and not the chronic infection per se, because FcγR-mediated effector functions were normal in persistently infected mice that lacked LCMV-specific antibodies. Our findings have implications for the therapeutic use of antibodies and suggest that high levels of pre-existing immune complexes could limit the effectiveness of antibody therapy in humans.

A novel microparticulate vaccine for melanoma cancer using transdermal delivery

In this study, we formulated a microparticulate melanoma cancer vaccine via the transdermal route. The vaccine was delivered using microneedle-based Dermaroller® which is available for cosmetic purposes. Unlike subcutaneous injections, administration using microneedles is painless and in general can increase the permeability of many compounds ranging in size from small molecules to proteins and microparticles that do not normally penetrate the skin. The vaccine microparticles were taken up by the antigen presenting cells which demonstrated a strong IgG titre level of 930 ug/mL in serum samples. The formulation increased the immunogenicity of the vaccine by incorporating the antigen into an albumin matrix having a size range of around 0.63–1.4 µm which acted as a synthetic adjuvant. The animals were vaccinated with 1 prime and 4 booster doses administered every 14 days over 8 weeks duration, followed by challenge with live tumour cells which showed protection after transdermal vaccination.

Oral microparticulate vaccine for melanoma using M-cell targeting.

Cancer vaccines are limited in their use, because of their inability to mount a robust anti-tumor immune response. Thus, targeting M-cells in the small intestine, which are responsible for entry of many pathogens, will be an attractive way to elicit a strong immune response toward particulate antigens. Therefore, in the present investigation, we demonstrated that efficient oral vaccination against melanoma antigens could be accomplished by incorporating the antigens in an albumin-based microparticle with a ligand AAL (Aleuria aurantia lectin) targeted specifically to M-cells. The oral microparticulate vaccine effectively protected the mice from subcutaneous challenge with tumor cells in prophylactic settings. The animals were vaccinated with antigen microparticles having a size range of around 1–1.25 µm where one prime and four booster doses were administered every 14 days over 10 weeks of duration, followed by challenge with live tumor cells, which showed complete tumor protection after oral vaccination. With the inclusion of ligand in the microparticles, we observed significantly higher IgG titers (1565 μg/mL) as compared to the microparticle formulations without AAL (872 μg/mL). This data suggests that ligand loaded microparticles may have the potential to target antigens to M-cells for an efficient oral vaccination.