Macrophage angiotensin AT2 receptor activation is protective against early phases of LPS-induced acute kidney injury.

Lipopolysaccharide (LPS)-induced acute kidney injury (AKI) is characterized by inflammation and infiltration of immune cells, mainly neutrophils and macrophages, and results in sudden renal dysfunction. Previously, we have reported the anti-inflammatory and renoprotective role of the angiotensin II type 2 receptor (AT2R), expressed on kidney tubular cells and immune cells, in LPS-induced AKI. Moreover, in vitro studies revealed macrophage AT2R activation shifts the cells to the anti-inflammatory M2 subtype. However, the protective role of the macrophage AT2R in a model of AKI is unknown. The present study addressed this question by adoptive transfer of bone marrow-derived macrophages (BMDMs) in systemic macrophage-depleted mice. We acquired significant systemic macrophage depletion by two doses of liposomal clodronate (CLD), and the mice were repopulated with BMDMs (CD11b+F4/80+, double positive) primed with AT2R agonist C21 (CLD + MacC21 + LPS) or vehicle (CLD + Mac + LPS) in vitro for 60 min, followed by LPS (5 mg/kg body wt ip) challenge. We observed a gradual increase in the CD11b+ cells at 2 and 24 h after the LPS challenge. However, kidney CD11b+ cells in the CLD + Mac + LPS group were elevated compared with the CLD + MacC21 + LPS group at 2 h after the LPS challenge. The level of inflammatory cytokine (tumor necrosis factor-α) was elevated at 2 h, which was reduced significantly in CLD + MacC21 + LPS-treated animals. Also, CLD + MacC21 + LPS-treated animals had elevated plasma and renal IL-10, indicating an anti-inflammatory role of C21-treated BMDMs. Renal functional injury in CLD + MacC21 + LPS-treated animals was partially improved. Collectively, the data demonstrate that BMDM AT2R stimulation results in anti-inflammation and partial renoprotection against early stages of LPS-induced AKI.NEW & NOTEWORTHY Endotoxin such as lipopolysaccharide (LPS) induces acute kidney injury (AKI), which is a risk factor for and often leads to chronic kidney diseases. The present study revealed that bone marrow-derived macrophage activation of the angiotensin II type 2 receptor (AT2R) contributes to the anti-inflammation and partial renoprotection against early stages of LPS-induced AKI. Since AT2R is an emerging anti-inflammatory and organ-protective target, this study advances our understanding of AT2R's anti-inflammatory mechanisms associated with renoprotection.

Angiotensin type 2 receptor activation limits kidney injury during the early phase and induces Treg cells during the late phase of renal ischemia.

Kidney infiltrating immune cells such as monocytes, neutrophils, and T cells play critical roles in renal ischemia-reperfusion (IR) injury and repair. Recently, the angiotensin II type 2 receptor (AT2R) has been implicated in protecting kidneys against injury and monocyte infiltration, particularly in chronic kidney disease. However, the role of AT2R in IR injury and repair phases and T cell modulation is unknown. To address this question, Sprague-Dawley rats were subjected to IR with or without AT2R agonist C21 treatment. IR caused early (2 h postreperfusion) renal functional injury (proteinuria, plasma urea, and creatinine) and enhanced immune cells (T cells and CD4 T cells) infiltration and levels of the proinflammatory cytokines monocyte chemoattractant protein-1, TNF-α, and IL-6. C21 treatment reversed these changes but increased the anti-inflammatory IL-10 level. On day 3, C21 treatment increased CD4+FoxP3+ (regulatory T cells) and CD4+IL-10+ cells and reduced kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in the kidney compared with the IR control, suggesting the involvement of AT2R in kidney repair. These data indicate that AT2R activation protects the kidney against IR injury and immune cell infiltration in the early phase and modulates CD4 T cells toward the regulatory T cell phenotype, which may have long-term beneficial effects on kidney function.NEW & NOTEWORTHY The angiotensin II type 2 receptor agonist C21 has been known to have a renoprotective role in various kidney pathologies. C21 treatment (before renal ischemia) attenuated postischemic kidney injury, kidney dysfunction, and immune cell infiltration during the injury phase. Also, C21 treatment modulated the kidney microenvironment by enhancing anti-inflammatory responses mainly mediated by IL-10. During the repair phase, C21 treatment enhanced IL-10-secreting CD4 T cells and FoxP3-secreting regulatory T cells in Sprague-Dawley rats.

HLA-B*39:06 Efficiently Mediates Type 1 Diabetes in a Mouse Model Incorporating Reduced Thymic Insulin Expression.

Type 1 diabetes (T1D) is characterized by T cell-mediated destruction of the insulin-producing ß cells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with impaired immunological tolerance to insulin and increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06-transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared with littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR Vß family usage, confirming that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, it will facilitate the thymic escape of insulin-reactive HLA-B*39:06-restricted T cells, which participate in ß cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR Vß usage by CD8 T cells, demonstrating that some TCR Vß families have a preference for particular class I MHC alleles.

CD4+ TCRγδ+ FoxP3+ cells: An unidentified population of immunosuppressive cells towards disease progression leprosy patients.

This study, for the first time, reveals the role of M. leprae-specific CD4+ TCRγδ+ FoxP3+ cells in the progression and pathogenesis of leprosy. Co-culture with CD4+ CD25- cells suggested the immunosuppressive nature of CD4+ TCRγδ+ cells in dose-dependent manner. Isolation of CD4+ TCRγδ+ cells from leprosy patients and then culture in presence of M. leprae cell wall antigens (MLCwA) along with TGF ß, IPP and IL-2 suggested that these cells are M. leprae specific. TGF-ß-mediated SMAD3 signalling was turned out to be major factor towards the expression of FoxP3 in these cells. SMAD3 silencing during induction of these cells barely showed the induction of FoxP3. High density of SMAD3 binding at TGFßRII in CD4+ TCRγδ+ FoxP3+ furthermore suggested the TGF-ß-directed SMAD3 signalling in these cells. Taken together the above data, we can conclude that CD4+ TCRγδ+ FoxP3+ cells possess the potential to track the severity of the disease in leprosy patients.

Genetically modified human CD4(+) T cells can be evaluated in vivo without lethal graft-versus-host disease.

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD-scid IL2rγ(null) (NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4(+) T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA-DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA-DR4 and to be deficient for murine class II MHC molecules. CD4(+) T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3 months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.

FoxP3 provides competitive fitness to CD4⁺CD25⁺ T cells in leprosy patients via transcriptional regulation.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. FoxP3 have been shown to have important implications in various diseases. The present study describes the mechanism of action of FoxP3 in CD4⁺CD25⁺ T cells derived from leprosy patients. Increased molecular interactions of FoxP3 with histone deacetylases 7/9 in the nucleus of CD4⁺CD25⁺ T cells derived from borderline lepromatous leprosy/lepromatous leprosy (BL/LL) patients were found to be responsible for FoxP3-driven immune suppression activities during the progression of leprosy. Further, downregulation of CTLA-4 and CD25 genes in siFoxP3-treated PBMCs derived from BL/LL patients elucidated the transcription-activating nature of FoxP3. This observation was supported by direct binding of FoxP3 to the promoter region of the CTLA-4 and CD25 genes, and FoxP3's molecular interaction with histone acetyl transferases. The study also revealed that the increased expression of miR155 in CD4⁺CD25⁺ cells from BL/LL governs the competitive fitness of these cells. Again, reduced Annexin V & propidium iodide staining and Nur77 expression, and concomitantly increased Ki-67 positivity suggested that CD4⁺CD25⁺ cells derived from BL/LL patients are more competitively fit than those from borderline tuberculoid leprosy/tuberculoid leprosy and healthy controls. Taken together, the study shows the orchestration of FoxP3 leading to competitive fitness of Treg cells in leprosy.

Angiotensin II type 2 receptor activation preserves megalin in the kidney and prevents proteinuria in high salt diet fed rats.

Proteinuria is a risk factor for and consequence of kidney injury. Angiotensin II type 2 receptor (AT2R) is an emerging reno-protective target and is anti-proteinuric under pathological conditions, including high salt-fed obese animals. However, the mechanisms remain unknown, particularly whether the anti-proteinuric activity of AT2R is independent of its anti-hypertensive and anti-inflammatory effects. In the present study, obese Zucker rats were fed high sodium (4%) diet (HSD) for 48 h, a time in which blood pressure does not change. HSD caused proteinuria without affecting glomerular slit diaphragm proteins (nephrin and podocin), glomerular filtration rate, inflammatory and fibrotic markers (TNFα, IL-6, and TGF-ß), ruling out glomerular injury, inflammation and fibrosis but indicating tubular mechanisms of proteinuria. At cellular and molecular levels, we observed a glycogen synthase kinase (GSK)-3ß-mediated megalin phosphorylation, and its subsequent endocytosis and lysosomal degradation in HSD-fed rat kidneys. Megalin is a major proximal tubular endocytic protein transporter. The AT2R agonist C21 (0.3 mg/kg/day, i.p.) administration prevented proteinuria and rescued megalin surface expression potentially by activating Akt-mediated phosphorylation and inactivation of GSK-3ß in HSD-fed rat kidneys. Overall, AT2R has a direct anti-proteinuric activity, potentially via megalin regulation, and is suggested as a novel target to limit kidney injury.

Cell-mediated immune response to epitopic MAP (multiple antigen peptide) construct of LcrV antigen of Yersinia pestis in murine model.

Yersinia pestis is the causative agent of plague. Cellular immunity seems to play an important role in defense against this disease. The subunit vaccine based on V (Lcr V) antigen has been proved to be immunogenic in animals and in humans. The multiple antigen peptide (MAP), incorporating all the relevant B and T cell epitopes is highly immunogenic in mice through intranasal route of immunization in PLGA particles containing CpG-ODN as an immunoadjuvant inducing humoral and mucosal immune response. In the present study, cell-mediated immune response using same MAP was studied in murine model. Primary and memory T cell responses were studied in outbred and inbred mice immunized intranasally with MAP in the presence of two immunoadjuvants (Murabutide and CpG-ODN). All the three compartments (Spleen, Lamina propria and Peyer's patches) of the lymphoid system showed increased lymphoproliferative response. Highest lymphoproliferative response was observed especially with CpG-ODN. Cytokine profile in the culture supernatant showed highest Th(1) and Th(17) levels. FACS analysis showed expansion of both CD4(+) and CD8(+) T cells producing gamma-interferon, perforin and granzyme-B with major contribution from CD4(+) T cells.

Induction of cell-mediated immune responses to peptide antigens of P. vivax in microparticles using intranasal immunization.

T-cells play a critical role in resistance to malaria, not only because they function as helper cells for an antibody response, but also because they serve as effector cells. Such cellular immunity is directly implicated in protection from sporozoites as well as from blood stage parasites. The aim of this study was to induce cell mediated immune responses to peptide antigens of Plasmodium vivax co-encapsulated with CpG oligodeoxynucleotide (ODN) in microparticles. In the present study, we have investigated the immunomodulatory effects of two CpG adjuvants, CpG 1826 and CpG 2006 to the five peptide antigens of Plasmodium vivax derived from circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen (Pvs24) in microparticle delivery. The T-cell proliferation response study of the cells collected from spleen, lamina propria and peyer's patches showed significantly high (p<0.001) stimulation index when primed with peptide antigens in microparticles co-encapsulating CpG ODN adjuvant as compared to peptide alone primed mice. The cytokine measurement profile of IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-10 in culture supernatants of cells primed with peptide antigens in microparticles co-encapsulating CpG ODN showed higher levels of IFN- gamma followed by TNF-alpha and IL-2, with relatively low levels of IL-4 and IL-10.

Significance of anti-HBc screening of blood donors and its association with occult hepatitis B virus infection: Implications for blood transfusion.

BACKGROUND & OBJECTIVES: Expansions of blood donor screening and improved laboratory detection of viral markers have remarkably reduced the risk for infection with transfusion-transmitted viruses. This study was aimed to evaluate the presence of anti-HBc and to determine the presence or absence of HBV DNA in the serum samples from HBsAg negative, anti-HBc positive blood donors in a tertiary care hospital blood bank from Delhi. METHODS: A total of 2175 HBsAg negative, first time volunteer blood donors were included in the study from blood bank, Lok Nayak Hospital, New Delhi. The blood specimens from all these subjects were evaluated for anti-HBV-core antigen (anti-HBc) serology, anti-HBV-surface antigen (anti-HBs) titres and HBeAg. The presence of HBV DNA was evaluated by testing, through polymerase chain reaction (PCR) techniques. RESULTS: Of the 2175 HBsAg negative voluntary blood donors, 413 (19.8%) were tested to be positive for anti-HBc alone. Of these, 153 (group-I) were anti-HBs negative whereas group-II comprises a total of 260 anti-HBs positive cases i.e. 89 out of 413 had anti-HBs titres of 10-99 IU/l and the remaining 171 had anti-HBs titres of 100-500 IU/l. HBV DNA was detected in 7.5 per cent anti-HBc positive samples irrespective of anti-HBs status. INTERPRETATION & CONCLUSION: Our results showed that 18.9 per cent of our donor population was anti-HBc reactive, and hence inclusion of anti-HBc testing will lead to a high discard rate. The presence of HBV DNA in fairly high percentage of anti-HBc positive samples highlighted the need for a stringent and better screening system to prevent occult HBV infection.

Emerging Role of Angiotensin AT2 Receptor in Anti-Inflammation: An Update.

The hyperactive RAS and inflammation are closely associated. The angiotensin-II/AT1R axis of the RAS has been explored extensively for its role in inflammation and a plethora of pathological conditions. Understanding the role of AT2R in inflammation is an emerging area of research. The AT2R is expressed on a variety of immune and non-immune cells, which upon activation triggers the release of a host of cytokines and has multiple effects that coalesce to anti-inflammation and prevents maladaptive repair. The anti-inflammatory outcomes of AT2R activation are linked to its well-established signaling pathways involving formation of nitric oxide and activation of phosphatases. Collectively, these effects promote cell survival and tissue function. The consideration of AT2R as a therapeutic target requires further investigations.

Development and Characterization of a Preclinical Model for the Evaluation of CD205-Mediated Antigen Delivery Therapeutics in Type 1 Diabetes.

Dendritic cells (DCs) are crucial for the production of adaptive immune responses to disease-causing microbes. However, in the steady state (i.e., in the absence of an infection or when Ags are experimentally delivered without a DC-activating adjuvant), DCs present Ags to T cells in a tolerogenic manner and are important for the establishment of peripheral tolerance. Delivery of islet Ags to DCs using Ag-linked Abs to the DC endocytic receptor CD205 has shown promise in the NOD mouse model of type 1 diabetes (T1D). It is important to note, however, that all myeloid DCs express CD205 in humans, whereas in mice, only one of the classical DC subsets does (classical DC1; CD8α+ in spleen). Thus, the evaluation of CD205-targeted treatments in mice will likely not accurately predict the results observed in humans. To overcome this challenge, we have developed and characterized a novel NOD mouse model in which all myeloid DCs transgenically express human CD205 (hCD205). This NOD.hCD205 strain displays a similar T1D incidence profile to standard NOD mice. The presence of the transgene does not alter DC development, phenotype, or function. Importantly, the DCs are able to process and present Ags delivered via hCD205. Because Ags taken up via hCD205 can be presented on both class I and class II MHC, both CD4+ and CD8+ T cells can be modulated. As both T cell subsets are important for T1D pathogenesis, NOD.hCD205 mice represent a unique, patient-relevant tool for the development and optimization of DC-directed T1D therapies.

An HLA-Transgenic Mouse Model of Type 1 Diabetes That Incorporates the Reduced but Not Abolished Thymic Insulin Expression Seen in Patients.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. Multiple genetic loci contribute to disease susceptibility in humans, with the most responsible locus being the major histocompatibility complex (MHC). Certain MHC alleles are predisposing, including the common HLA-A(∗)02:01. After the MHC, the locus conferring the strongest susceptibility to T1D is the regulatory region of the insulin gene, and alleles associated with reduced thymic insulin expression are predisposing. Mice express two insulin genes, Ins1 and Ins2. While both are expressed in beta cells, only Ins2 is expressed in the thymus. We have developed an HLA-A(∗)02:01-transgenic NOD-based T1D model that is heterozygous for a functional Ins2 gene. These mice exhibit reduced thymic insulin expression and accelerated disease in both genders. Immune cell populations are not grossly altered, and the mice exhibit typical signs of islet autoimmunity, including CD8 T cell responses to beta cell peptides also targeted in HLA-A(∗)02:01-positive type 1 diabetes patients. This model should find utility as a tool to uncover the mechanisms underlying the association between reduced thymic insulin expression and T1D in humans and aid in preclinical studies to evaluate insulin-targeted immunotherapies for the disease.

Preparation of peptide microspheres using tumor antigen-derived peptides.

Due to its distinct biological attributes, poly(D,L lactide-co glycolide) (PLGA) is one of the most preferred methods for DNA/protein/peptide encapsulation for therapeutics. Importantly, PLGA acts as an adjuvant for weakly immunogenic antigens and mimics booster responses after a single dose of administration, thereby serving as a single-shot vaccine delivery vehicle. Efficient delivery of antigens to antigen-presenting cells (APC) has been made possible by the use of a PLGA particle-based vaccine delivery system. Also, the plasma half-life of the PLGA-encapsulated vaccine increases as it is protected from degradation, prior to its further release. PLGAs are reported to be catabolized into individual nontoxic units once inside the host and further degraded via normal metabolic pathways. In this chapter, we have described the preparation and characterization of tumor peptide encapsulated PLGA microparticles as a model for controlled-release peptide delivery system.

Molecular linkages between diabetes and Alzheimer's disease: current scenario and future prospects.

After the revolutionary Rotterdam study that suggested there was an increased risk of developing Alzheimer's disease (AD) in patients with type-2 diabetes mellitus (T2DM), a number of studies have provided direct evidence for the linkage between AD and T2DM. In recent years, AD is considered as a neuroendocrine disorder, also referred as type-3 diabetes. There is a growing list of evidence to suggest that, in addition to impaired insulin signaling, there are a number of additional factors that may act as mechanistic links between AD and T2DM. These factors mainly include hypercholesterolemia, dyslipidemia, hypercystinemia, inflammation, impaired insulin signaling and impaired central nervous response to the adipose tissue-derived hormone leptin. Increased cholesterol plays a crucial role in the abnormal metabolism of the amyloid precursor protein, leading to the accumulation of ß-amyloid. In addition to impaired insulin signaling, diabetes has been found to accelerate the appearance of cerebrovascular inflammation and ß-amyloid peptide (Aß) deposition. Increased oxidative stress and production of advanced glycation end products are other probable marker linkages. However, the details of many of these molecular links still require extensive investigation. It is possible that a number of common molecular linkages exist between T2DM and AD. Understanding and analyzing the various molecular linkages between AD and T2DM may shed light on new tools that can be used for the early diagnosis and treatment of AD and also accelerate the identification of T2DM patients who are at high risk of AD.

B and T cell epitope mapping and study the humoral and cell mediated immune response to B-T constructs of YscF antigen of Yersinia pestis.

YscF antigen, a type III secretion protein has recently been shown partial protection in murine model. Five peptides of YscF antigen were predicted using DNASTAR and T-cell prediction software. Peptides were synthesised and authenticated using competitive, direct binding immunoassay with anti YscF/peptide sera raised in mice. Peptide P1 and P2 were found to be B cell epitope while P3 was minor B cell epitope. P4 peptide was a pure T cell epitope based on lymphoproliferative response, cytokines profile and T-bet expression. Furthermore, with an intention to enhance immunogenicity, three B-T constructs were designed between the above epitopes. Conjugate B1T1 and B2T1 showed higher serum IgG/IgA titre, respectively, as well as high secretory IgA plus secretory component (Sc) both in lung and intestinal washes. Also, these conjugates showed high T-cell proliferation in addition to higher Th1 type cytokines (IFN-γ and IL-2) in cells obtained from spleen, lamina propria and Peyer's patches. B3T1 stimulated cells showed moderate levels of IFN-γ and IL-2 but higher levels of IL-4. This study demonstrates superior immunogen of B1T1 and B2T1 of YscF antigen to be exploited as vaccine candidate for plague.

Multiple antigen peptide consisting of B- and T-cell epitopes of F1 antigen of Y. pestis showed enhanced humoral and mucosal immune response in different strains of mice.

Yersinia pestis is a causative agent of plague. F1 and V antigen based vaccines have shown remarkable protection in experimental animals. In order to develop epitope based immunogen, three B and one T-cell epitopes of F1 antigen with palmitate residue at amino terminal were assembled on a lysine backbone as multiple antigen peptide (MAP or F1-MAP). MAP was characterized by SDS-PAGE, immunoblot and immunoreactivity with anti F1 sera. MAP was entrapped in PLGA (polylactide-co-glycolide) microparticles and humoral, mucosal immune responses were studied after intranasal immunization with/without CpG ODN 1826 (CpG)/murabutide in different strains of mice. Serum and mucosal washes were measured for MAP specific IgG, IgA, sIgA and IgG subclasses in three strains of mice. F1-MAP showed high serum antibody and mucosal IgG and IgA peak antibody titers. MAP with CpG showed significantly high (p<0.001) peak antibody titer ranging from 102,400 to 204,800 for IgG and 6400 to 12,800 for IgA. High mucosal sIgA and its secretary component detection confirmed generation of mucosal response in intestinal and lung washes. MAP antisera also showed significant immunoreactivity with individual peptides. Moreover, antibody specific activity (IgG, IgA and sIgA) positively correlates with peak antibody titers. Predominantly IgG2a/IgG2b subclass was observed with CpG formulation but in other formulation a mixed IgG1 and IgG2a response was observed. The present study highlights the importance of multiple antigen peptide approach of F1-antigen with CpG as an alternative approach for subunit vaccine.

CD4+CD25+ T regs with acetylated FoxP3 are associated with immune suppression in human leprosy.

Leprosy is a chronic human disease that results from infection of Mycobacterium leprae. T reg cells have been shown to have important implications in various diseases. However, in leprosy, it is still unclear whether T regs can mediate immune suppression during progression of the disease. In the present study, we have proposed the putative mechanism leading to high proportion of T reg cells and investigated its significance in human leprosy. High levels of TGF-ß followed by adaptation of FoxP3(+) naive and memory (CD4(+)CD45RA(+)/RO(+)) T cells were observed as the principal underlying factors leading to higher generation of T reg cells during disease progression. Furthermore, TGF-ß was found to be associated with increased phosphorylation-mediated-nuclear-import of SMAD3 and NFAT towards BL/LL pole to facilitate FoxP3 expression in these cells, the same as justified after using nuclear inhibitors of SMAD3 (SIS3) and NFAT (cyclosporin A) in CD4(+)CD25(+) cells in the presence of TGF-ß and IL-2. Interestingly, low ubiquitination of FoxP3 in T reg cells of BL/LL patients was revealed to be a major driving force in conferring stability to FoxP3 which in turn is linked to suppressive potential of T regs. The present study has also pinpointed the presence of CD4(+)CD25(+)IL-10(+) sub class of T regs (Tr1) in leprosy.

IL-10 production from dendritic cells is associated with DC SIGN in human leprosy.

The defective antigen presenting ability of antigen presenting cells (APCs) modulates host cytokines and co-stimulatory signals that may lead to severity of leprosy. In the present study, we sought to evaluate the phenotypic features of APCs along with whether DC SIGN (DC-specific intercellular adhesion molecule-grabbing nonintegrin) influences IL-10 production while moving from tuberculoid (BT/TT) to lepromatous (BL/LL) pole in leprosy pathogenesis. The study revealed an increased expression of DC SIGN on CD11c⁺ cells from BL/LL patients and an impaired form of CD83 (∼50 kDa). However, the cells after treatment with GM-CSF+IL-4+ManLAM showed an increased expression of similar form of CD83 on DCs. Upon treatment with ManLAM, DCs were found to show increased nuclear presence of NF-κB, thus leading to higher IL-10 production. High IL-10 production from ManLAM treated PBMCs further suggested the role of DC SIGN in subverting the DCs function towards BL/LL pole of leprosy. Anti-DC SIGN treatment resulting in restricted nuclear ingression of NF-κB as well as its acetylation along with enhanced T cell proliferation validated our findings. In conclusion, Mycobacterium leprae component triggers DC SIGN on DCs to induce production of IL-10 by modulating intracellular signalling pathway at the level of transcription factor NF-κB towards BL/LL pole of disease.

Evaluation of CD4+/CD8+ T-cell expression and IFN-γ, perforin secretion for B-T constructs of F1 and V antigens of Yersinia pestis.

Yersinia pestis is a facultative bacterium that can survive and proliferate inside host macrophages and cause bubonic, pneumonic and systemic infection. Understanding the immune response generated by epitopes recognized by CD4+ and CD8+ T cells is important for the development of safe and effective vaccines designed to promote protective cellular immunity. Apart from humoral response, CD4+ T cells have shown to have a major role in combating the pneumonic form of the disease. In the present study, the secretion of IFN-γ and IL-4 by splenocytes, stimulated by different constructs of B and T cell epitopes of F1 and V antigens, was measured by ELISpot assay. We also measured perforin and IFN-γ expression as a function of cell mediated immunity by flow cytometry. Three B-T constructs of F1 and seven B-T constructs of V antigens produced a high number of IFN-γ secreting cells as compared to native antigen and a low number of IL-4 secreting cells. B-T conjugates of F1 and V antigens showed significantly high (p<0.001) percentage of CD4+ IFN-γ(+) cells as compared to CD8+ IFN-γ(+) cells. Thus, the study highlights the importance of Th1 cytokine and existence of high proportion of CD4+ T cells probably contributing protection in the host. This study proposes a new perspective for the development of vaccination strategies for Y. pestis that trigger T cell immune response.