Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

HLA-DR3 mediated CD4 T cell response against GAD65 in type 1 diabetes patients.

AIM: We planned this study to identify diabetogenic glutamic acid decarboxylase (GAD65) peptides possibly responsible for human leucocyte antigen (HLA)-DR3/DQ2-mediated activation of GAD65-specific CD4 T cells in type 1 diabetes (T1D). METHODS: Top 30 GAD65 peptides, found to strongly bind in silico with HLA-DR3/DQ2 molecules, were selected and grouped into four pools. The peptides were used to stimulate CD4 T cells of study subjects in 16-h peripheral blood mononuclear cell culture. CD4 T cells' stimulation in terms of interferon-gamma (IFN-γ), interleukin (IL)-17, tumor necrosis factor-alpha (TNF-α), and IL-10 expression was analyzed using flow cytometry. RESULTS: Although all four GAD65 peptide pools (PP1-4) resulted in significantly higher expression of IFN-γ by CD4 T cells (p = .003, p < .0001, p = .026, and p = .002, respectively), only pool 2 showed significant increase in IL-17 expression (p < .0001) in T1D patients vs healthy controls. Interpeptide group comparison for immunogenicity revealed significantly higher IFN-γ and IL-17 expressions and significantly lower IL-10 expression for PP2 compared to other groups (p < .0001, p = .02, and p = .04, respectively) in patients but not in controls. Further, group 2 peptides resulted in significant increase in CD4 T cells' expression of IFN-γ and IL-17 (p = .002 for both) and significant decrease in IL-10 (p = .04) in HLA-DRB1*03-DQA1*05-DQB1*02+ patients vs HLA-DRB1*03-DQA1*05-DQB1*02+ controls. The CD4 T cells' expression of IL-17 was significantly higher (p = .03) in recently diagnosed vs long-standing HLA-DRB1*03-DQA1*05-DQB1*02+ T1D patients. CONCLUSION: GAD65 peptides, particularly those belonging to PP2, induced CD4 T cells to express IFN-γ and IL-17 cytokines in T1D patients, suggesting that group 2 peptides possibly presented by HLA-DR3 molecule to CD4 T cells shift immune balance toward inflammatory phenotype in patients.

Near-Infrared Bioluminescence Imaging of Macrophage Sensors for Cancer Detection In Vivo.

Melanoma is an aggressive type of skin cancer with a poor prognosis after it gets metastasized. The early detection of malignant melanoma is critical for effective therapy. Because melanoma often resembles moles, routine skin check-up may help for timely identification of suspicious areas. Recently, it has been shown that the interplay of melanoma cells with the immune system can help develop efficient therapeutic strategies. Here, we leveraged engineered macrophages (BMC2) as cell-based sensors for metastatic melanoma. To perform dual-color bioluminescence imaging (BLI) in vivo, macrophages were engineered to express a green click beetle luciferase (CBG2) and a near-infrared fluorescent dye (DiR), and B16F10 melanoma cells were instead engineered to express a near-infrared click beetle luciferase (CBR2). Using real-time in vivo dual-color BLI and near-infrared fluorescence (FL) imaging, we could demonstrate that macrophages were able to sense and substantially accumulate in subcutaneous and metastatic melanoma tissues at 72 h after systemic injections. Together, we showed the potentiality to use optical imaging technologies to track circulating macrophages for the non-invasive detection of metastatic melanoma.

Differential HLA Association of GAD65 and IA2 Autoantibodies in North Indian Type 1 Diabetes Patients.

The human leucocyte antigen (HLA) association with type 1 diabetes (T1D) is well known but there are limited studies investigating the association between ß-cell autoantibodies and HLA genes. We evaluated the prevalence of GAD65 and IA-2 autoantibodies (GADA and IA2A) in 252 T1D patients from North India and investigated the genetic association of GADA and IA2A with HLA class I and class II genes/haplotypes. GADA and IA2A were detected in 50.79% and 15.87% of T1D patients, respectively, while only 8.73% had both GADA and IA2A. HLA-DRB1∗03 was observed to be significantly higher in GADA+ T1D patients as compared to GADA- (91.41% vs. 66.13%, Bonferroni-corrected P (P c) = 1.11 × 10-5; OR = 5.45; 95% CI: 2.67-11.08). Similarly, HLA-DQB1∗02 was found to be significantly increased in GADA+ patients (94.53%, P c = 2.19 × 10-5; OR = 6.27; 95% CI: 2.7-14.49) as compared to GADA- (73.39%). The frequencies of HLA-DRB1∗04 and DQB1∗03 were increased in IA2A+ patients (45.0% and 52.5%, respectively) as compared to that in IA2A- (25.94% and 33.96%, respectively). Further, the frequency of DRB1∗03-DQB1∗02 haplotype was found to be significantly increased in GADA+ T1D patients as compared to GADA- (60.55% vs. 41.94%, P = 3.94 × 10-5; OR = 2.13; 95%CI = 1.49-3.03). Similarly, HLA-DRB1∗04-DQB1∗03 haplotype was found to be significantly increased in IA2A+ T1D patients compared to IA2A- patients (22.5% vs. 12.97%; P = 0.041; OR = 1.95; 95%CI = 1.08-3.52). None of the HLA class I genes (HLA-A, B, and Cw) was found to be associated with GADA or IA2A in people with T1D. Our findings suggest that HLA-DRB1∗03/DQB1∗02 and HLA-DRB1∗04/DQB1∗03 might play an important role in the development of GADA and IA2A, respectively.

Diverse human leukocyte antigen association of type 1 diabetes in north India.

BACKGROUND: Type 1 diabetes (T1D) is a complex disease, with involvement of various susceptibility genes. Human leukocyte antigen (HLA) on chromosome 6p21 is major susceptibility region. This study examined genetic association of HLA genes with T1D. METHODS: The study recruited 259 T1D patients and 706 controls from north India. PCR-SSP and LiPA were used to type HLA Class I and II alleles. RESULTS: At HLA Class I locus, HLA-A*02, A*26, B*08 and B*50 were significantly increased in patients vs controls (39.8% vs 28.9% [Bonferroni-corrected P {Pc } = 0.032], 24.7% vs 9.6% [Pc = 4.83 × 10-8 ], 37.2% vs 15.7% [Pc = 1.92 × 10-9 ], and 19.4% vs 5.5% [Pc = 4.62 × 10-9 ], respectively). Similarly, in Class II region, DRB1*03 showed a strong positive association with T1D (78.7% vs 17.5% in controls; P = 1.02 × 10-9 ). Association of DRB1*04 with T1D (28.3% vs 15.5% in controls; Pc = 3.86 × 10-4 ) was not independent of DRB1*03. Negative associations were found between T1D and DRB1*07, *11, *13, and *15 (13.8% vs 26.1% in controls [Pc = 0.00175], 3.9% vs 16.9% in controls [Pc = 6.55× 10-6 ], 5.5% vs 21.6% in controls [Pc = 2.51 × 10-7 ], and 16.9% vs 43.9% in controls [Pc = 9.94× 10-10 ], respectively). Compared with controls, patients had significantly higher haplotype frequencies of A*26-B*08-DRB1*03-DQA1*05-DQB1*02 (10.43% vs 1.96%; P = 7.62 × 10-11 ), A*02-B*50-DRB1*03-DQA1*05-DQB1*02 (6.1% vs 0.71%; P = 2.19 × 10-10 ), A*24-B*08-DRB1*03-DQA1*05-DQB1*02 (4.72% vs 0.8%; P = 5.4 × 10-7 ), A*02-B*08-DRB1*03-DQA1*05-DQB1*02 (2.36% vs 0.18%; P = 3.6 × 10-5 ), and A*33-B*58-DRB1*03-DQA1*05-DQB1*02 (4.33% vs 1.25%; P = 0.00019). CONCLUSIONS: In north India, T1D is independently associated only with HLA-DRB1*03 haplotypes, and is negatively associated with DRB1*07, *11, *13, and *15.

Telomere length variation and expression analysis of shelterin complex genes during gallbladder carcinogenesis.

BACKGROUND: Telomeres, which are bound with shelterin protein complex, play an important role in maintaining genomic stability and its dysfunction may lead to carcinogenesis. Here, we aimed to analyze whether shelterin complex gene expression and telomere length variation, play any role in gallbladder carcinogenesis. METHODS: Telomere length analysis was carried out by monochrome multiplex qPCR, whereas expression analysis of shelterin genes was carried out using RT-qPCR. Statistical analysis was carried out using SigmaPlot 11 software. RESULTS: We found significantly reduced telomere length in tumor tissues, and this reduction was seen in both, tumors with or without gallstones in comparison to adjacent non tumor and gallstone (chronic calculous cholecystitis: Inflamed) tissues. Inflamed tissues showed increased telomere length as compared to both adjacent non tumor and tumor tissues. Expression analysis of five shelterin genes showed significant downregulation of TERF1, POT1, and TINF2 genes in inflamed tissues as compared to non tumor and tumor tissues. POT1 was also found to be significantly upregulated in tumor tissues and specifically in tumor tissues with gallstones compared to inflamed tissues. CONCLUSION: This study, thus, suggests that, gallstone does not affect telomere length and even after having increased telomere length, decreased expression of some shelterin genes in inflamed tissue might cause telomeres to cap improperly, possibly leading to telomere dysfunction and further, gallbladder carcinogenesis. Also, increased expression of POT1 in tumor tissues with gallstones could act as a diagnostic marker in patients with gallstones.

Genotype-phenotype relationship of CCL5 in pulmonary tuberculosis infection in Sahariya tribe: A pilot study.

BACKGROUND & OBJECTIVES: Sahariya, a primitive tribe of Central India, has shown significantly increased incidence of pulmonary tuberculosis (PTB). Our previous study on Sahariya showed a significant association of -403G>A single nucleotide polymorphism (SNP) of CCL5 with susceptibility to PTB. Hence, this study was aimed to analyze a genotype-phenotype relationship of this disease-associated SNP to develop a potential diagnostic marker for TB in this tribe. METHODS: The present study was carried out on 70 plasma samples from Sahariya tribe, wherein the plasma CCL5 level was determined using a commercially available ELISA kit. RESULTS: The level of CCL5 decreased significantly in patients who were on therapy/completed their therapy [inactive TB patient/inactive PTB (IPTB)], particularly with AA genotype of -403G>A (P=0.046). The level, with AA genotype, was also found to gradually decrease in sputum 3+ and 1+/2+ than in sputum-negative samples. Similarly, the CCL5 level was found to be higher in sputum-positive/active TB patients than in IPTB group and healthy controls. INTERPRETATION & CONCLUSIONS: Our results suggested that the CCL5 level was influenced collectively not only by the genotypes of -403G>A SNP and bacillary load but also by the treatment. Thus, CCL5 may be considered for the development of a diagnostic marker and also as an indicator of recovery.

Dysfunction of subtelomeric methylation and telomere length in gallstone disease and gallbladder cancer patients of North Central India.

BACKGROUND: Telomeres play an important role in cancer progression. Recently it has been shown that subtelomeric methylation negatively regulates telomere length in various diseases, including cancers. Here, we evaluated the influence of subtelomeric methylation in telomere dysfunction in gallbladder cancer (GBC), and whether this dysfunction is affected by the presence of gallstones. METHODS: Relative telomere length and subtelomeric methylation levels were assessed using monochrome multiplex quantitative polymerase chain reaction and bisulfite sequencing, respectively, in different gallbladder tissue types including different grades of GBC, gallstones and adjacent non-tumor. RESULTS: We found telomere length to shorten significantly in overall GBC, but specifically in early grade cancer. We also found D4Z4 and DNF92 subtelomeric sequences to be hypermethylated and hypomethylated, respectively, in GBC; however, their methylation levels differed significantly, only in early grade cancer. We could not find any specific correlation between subtelomeric methylation and telomere length in GBC. Interestingly, telomere length and subtelomeric methylation differed significantly in GBC without gallstones but not in GBC with gallstones. CONCLUSIONS: This study, thus, suggests that telomere dysfunction and changes in methylation levels may occur earlier in the progression of GBC, while the presence of gallstones may have no influence on telomere length as well as on methylation levels.

Distribution of HLA-A, B and DRB1 alleles in Sahariya tribe of North Central India: an association with pulmonary tuberculosis.

Sahariya, a primitive tribe, native of North Central India, is characterized by a significantly increased incidence of pulmonary tuberculosis (PTB) as compared to other tribes from the same region. Host genetic factors are known to influence susceptibility to PTB at the population level. Since an association of immune regulatory genes, particularly HLA, with PTB susceptibility has already been reported in several studies, we investigated a similar association of HLA alleles with PTB pathogenesis in the Sahariya tribe. A total of 210 cases and 178 healthy individuals from Sahariya tribe were genotyped for HLA class I and II alleles using the PCR based SSP and Reverse-SSO methods. The study showed a significantly increased allelic frequency of HLA-DRB1(∗)15 (p=0.02) in the patients as compared to healthy controls. However, the allelic frequency of HLA-DRB1(∗)16 was significantly reduced in patients than in controls (p=0.01). Three locus haplotype analysis of HLA-A, B and DR revealed a significantly increased frequency of A(∗)24-B(∗)40-DRB1(∗)15 haplotype among patients than controls (p=0.005), while A(∗)02-B(∗)40-DRB1(∗)16 and A(∗)02-B40-DRB1(∗)03 (p=0.001 and 0.02, respectively) were significantly reduced in the patients. Our findings confirm a positive association of HLA-DRB1(∗)15 in Sahariya tribe similar to the one already shown in other Indian ethnic groups. On the other hand, HLA-A(∗)24-B(∗)40-DRB1(∗)15 haplotype was found to be specific to Sahariya tribe with a strong predisposition to PTB. Further, the protection offered by DRB1(∗)16 allele and associated haplotypes towards PTB in this tribe appears to be a novel finding that warrants further investigation with regard to resistance to PTB.

Genetic polymorphisms of CCL2, CCL5, CCR2 and CCR5 genes in Sahariya tribe of North Central India: an association study with pulmonary tuberculosis.

The association of genetic variants of chemokines, CCL2 [MCP-1 (monocyte chemoattractant protein-1)], CCL5 [RANTES (regulated on activation, normal T-cell expressed and secreted)] and their receptors CCR2 and CCR5, respectively, earlier reported to be associated with susceptibility to pulmonary tuberculosis (PTB) in certain ethnic populations, were explored in Sahariya tribe, a primitive tribe of North Central India having a high prevalence of TB. We genotyped 215 cases and 215 controls of Sahariya tribe for polymorphisms in CCL2 (-2518A/G, -362G/C) by PCR-RFLP method and in CCR2 (V64I), CCL5 (-403G/A, -28C/G) and CCR5 (-59029G/A) by ARMS-PCR method. The frequencies of 'AA' genotype and 'A' allele of -403G/A were found significantly higher in cases than in controls (OR, 2.616 [95%CI, 1.302-5.320] and OR, 1.348 [95%CI, 0.980-1.853], respectively). Conversely, the frequencies of 'AA' genotype and 'A' allele of V64I were significantly (p=0.05 and 0.04, respectively) higher in controls than in cases. Also, the "AA" genotype of V64I was found to provide significant (p=0.05) protection against high bacillary load (3+). Likewise, the comparison of frequencies of different combinations of these polymorphisms further strengthens the association of -403G/A with susceptibility and V64I with resistance to TB in Sahariya tribe. However, no significant association of other polymorphisms with either resistance or susceptibility to TB was found. Thus, our findings support the association of -403G/A and V64I polymorphisms with genetic susceptibility and resistance to TB, respectively , alone or in combination with other polymorphisms in Sahariya tribe.

Coding and non-coding polymorphisms in VDR gene and susceptibility to pulmonary tuberculosis in tribes, castes and Muslims of Central India.

Vitamin D receptor (VDR) plays an important role in activating immune response against various infectious agents. This study was aimed to investigate the association between VDR gene polymorphisms and different clinical forms of pulmonary tuberculosis (TB) in different population groups. Four common polymorphisms (TaqI, ApaI, BsmI and FokI) of VDR gene were studied in clinically diagnosed TB patients and healthy controls from Sahariya tribe (n=377), Bhil tribe (n=95), Chhattisgarh tribe (n=33), general population from North-Central (NC) (n=1021) and South-Eastern (SE) region (n=646) and Muslims (n=217). Genotyping was carried out using PCR-RFLP method and re-confirmed by direct sequencing. The haplotype analysis was performed on Haploview 4.1 and statistical analysis was done using SPSS 13.0 software. We found that bb genotype of BsmI polymorphism conferred significant risk to smear positive and multiple drug resistant (MDR) TB in tribes [OR (CI)=3.7 (1.5-9.2), p=0.002], SE population [OR (CI)=2.1 (1.4-3.3), p=0.0004] and Muslims [OR (CI)=6.7 (1.1-39), p=0.01]. The subjects with FF genotype of FokI polymorphism appeared less likely (p=0.004) to develop MDR TB in NC population, whereas, those with Ff [OR (CI)=2.5 (1.3-5.0), p=0.004] and ff [OR (CI)=3.4 (1.2-9.3), p=0.01] genotypes were at high risk of MDR and smear positive disease, respectively. Similarly, tt genotype of TaqI polymorphism was found associated with high risk of smear positive TB in NC [OR (CI)=3.6 (0.9-14.2), p=0.05] as well as in SE [OR (CI)=4.7 (1.8-12.3), p=0.00003] population. Interestingly, tt genotype appeared strongly associated [OR (CI)=8.9 (2.7-29), p=0.00001] with high bacillary load outcome. In conclusion, genetic polymorphisms in VDR gene, alone or in combination (haplotypes) are associated with different clinical outcomes in pulmonary TB.