Comprehending the crosstalk between Notch, Wnt and Hedgehog signaling pathways in oral squamous cell carcinoma - clinical implications.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a malignant oral cavity neoplasm that affects many people, especially in developing countries. Despite several advances that have been made in diagnosis and treatment, the morbidity and mortality rates due to OSCC remain high. Accumulating evidence indicates that aberrant activation of cellular signaling pathways, such as the Notch, Wnt and Hedgehog pathways, occurs during the development and metastasis of OSCC. In this review, we have articulated the roles of the Notch, Wnt and Hedgehog signaling pathways in OSCC and their crosstalk during tumor development and progression. We have also examined possible interactions and associations between these pathways and treatment regimens that could be employed to effectively tackle OSCC and/or prevent its recurrence. CONCLUSIONS: Activation of the Notch signaling pathway upregulates the expression of several genes, including c-Myc, ß-catenin, NF-κB and Shh. Associations between the Notch signaling pathway and other pathways have been shown to enhance OSCC tumor aggressiveness. Crosstalk between these pathways supports the maintenance of cancer stem cells (CSCs) and regulates OSCC cell motility. Thus, application of compounds that block these pathways may be a valid strategy to treat OSCC. Such compounds have already been employed in other types of cancer and could be repurposed for OSCC.

Downregulation of Notch4 - a prognostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma / Regulação descendente de Notch4 - um marcador prognóstico na distinção entre carcinoma verrucoso oral e carcinoma de células escamosas oral

Abstract Introduction: Oral verrucous carcinoma is a special form of well-differentiated squamous cell carcinoma which possesses specific clinical, morphologic and cytokinetic features that differ from other types of oral cancers and hence diagnosis requires immense experience in histopathology. Hence it is certainly important to distinguish such a lesion from other oral tumors as treatment strategies vary widely between them. Objective: In search of a critical diagnostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma, Notch4 receptor, one of the key regulatory molecules of the Notch signaling family has been aberrantly activated in the progression of several types of tumors. However its function in oral verrucous carcinoma remains unexplored. Thus the present study aims in determining the differential expression pattern of Notch4 in oral verrucous carcinoma and oral squamous cell carcinoma. Methods: Ten patients reported positive for oral cancer (5 patients with oral verrucous carcinoma and 5 patients with oral squamous cell carcinoma). Five normal tissue samples were also obtained and evaluated for clinicopathological parameters and immunohistochemistry, western blotting and real time polymerase chain reaction for Notch4 expression. Results: Our results reveal that the expression of Notch4 was considerably high in oral squamous cell carcinoma lesions compared to normal tissue, whereas in oral verrucous carcinoma, irrespective of the clinicopathological features, complete regulação descendente of Notch4 was observed. Conclusions: These preliminary findings strongly support the fact that Notch4 is downregulated in oral verrucous carcinoma and could be considered as a suitable prognostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma. This distinguishing marker can help in improving therapeutic options in patients diagnosed with oral verrucous carcinoma.

Resumo Introdução: O carcinoma verrucoso de cavidade oral é uma forma especial de carcinoma de células escamosas bem diferenciada que tem características clínicas, morfológicas e citocinéticas específicas que diferem de outros tipos de cânceres orais. Por essa razão, o diagnóstico requer grande experiência em histopatologia. Portanto, é certamente importante distingui-lo de outros tumores orais, pois as respectivas estratégias de tratamento variam muito. Objetivo: Em busca de um marcador de diagnóstico crítico na distinção entre o carcinoma verrucoso e o carcinoma de células escamosas de cavidade oral, o receptor Notch4, uma das principais moléculas reguladoras da família de sinalizadores Notch, foi ativado de maneira anormal na progressão de vários tipos de tumores. No entanto, sua função no carcinoma verrucoso permanece inexplorada. Assim, o presente estudo tem como objetivo determinar o padrão de expressão diferencial de Notch4 no carcinoma verrucoso e de células escamosas de cavidade oral. Método: Dez pacientes tiveram resultado positivo para câncer oral (cinco pacientes com carcinoma verrucoso e cinco pacientes com carcinoma de células escamosas) e cinco amostras normais foram também obtidas. Além da avaliação dos parâmetros clínico-patológicos, foram feitos análise imuno-histoquímica, Western Blot e reação de polimerase em cadeia em tempo real para a expressão de Notch4. Resultados: Nossos resultados revelam que a expressão de Notch4 foi consideravelmente alta em carcinomas de células escamosas em comparação com os tecidos normais, enquanto que no carcinoma verrucoso, independentemente das características clínico-patológicas, observou-se regulação descendente completa de Notch4. Conclusão: Esses achados preliminares apoiam fortemente o fato de que Notch4 estava regulado para baixo no carcinoma verrucoso oral e poderia ser considerado um marcador prognóstico adequado para distinguir entre carcinoma verrucoso e carcinoma de células escamosas de cavidade oral. Esse marcador distintivo pode ajudar a melhorar as opções terapêuticas em pacientes com diagnóstico de carcinoma verrucoso oral.

Downregulation of Notch4 - a prognostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma.

INTRODUCTION: Oral verrucous carcinoma is a special form of well-differentiated squamous cell carcinoma which possesses specific clinical, morphologic and cytokinetic features that differ from other types of oral cancers and hence diagnosis requires immense experience in histopathology. Hence it is certainly important to distinguish such a lesion from other oral tumors as treatment strategies vary widely between them. OBJECTIVE: In search of a critical diagnostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma, Notch4 receptor, one of the key regulatory molecules of the Notch signaling family has been aberrantly activated in the progression of several types of tumors. However its function in oral verrucous carcinoma remains unexplored. Thus the present study aims in determining the differential expression pattern of Notch4 in oral verrucous carcinoma and oral squamous cell carcinoma. METHODS: Ten patients reported positive for oral cancer (5 patients with oral verrucous carcinoma and 5 patients with oral squamous cell carcinoma). Five normal tissue samples were also obtained and evaluated for clinicopathological parameters and immunohistochemistry, western blotting and real time polymerase chain reaction for Notch4 expression. RESULTS: Our results reveal that the expression of Notch4 was considerably high in oral squamous cell carcinoma lesions compared to normal tissue, whereas in oral verrucous carcinoma, irrespective of the clinicopathological features, complete regulação descendente of Notch4 was observed. CONCLUSIONS: These preliminary findings strongly support the fact that Notch4 is downregulated in oral verrucous carcinoma and could be considered as a suitable prognostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma. This distinguishing marker can help in improving therapeutic options in patients diagnosed with oral verrucous carcinoma.

1, 25-dihydroxyvitamin D3 downregulates cytotoxic effector response in pulmonary tuberculosis.

1,25-dihydroxyvitaminD3 [1,25(OH)2D3] modulates both the innate and adaptive immunity in tuberculosis. We explored the effect of 1,25(OH)2D3 on cytolytic molecules like perforin, granulysin, and granzyme-B in T-cells and natural killer cells during M. tuberculosis (Mtb) infection. Peripheral blood mononuclear cells (PBMCs) from 45 healthy controls (HCs) and 45 pulmonary tuberculosis (PTB) patients were cultured with Mtb in the absence or presence of 1,25(OH)2D3 for 72 h. The percentage of perforin, granulysin, and granzyme-B positive cells were estimated by flow cytometry. 1,25(OH)2D3 significantly decreased the percentage of cytolytic molecules in total, CD4+, CD8+ and CD56+ cells in HCs and PTB patients (p < 0.05). Moreover, 1,25(OH)2D3 downregulates IFN-γ levels while upregulate the anti-inflammatory cytokine IL-10. Correlation revealed that the total percentage of cytolytic molecules were positively correlated with IFN-γ level, whereas negatively correlated with IL-10 level in both the study subjects (p < 0.05). This results suggests that 1,25(OH)2D3 downregulate the expression of cytolytic molecues and act as anti-inflammatory in adaptive immune response, which might help to reduce inflammation and tissue damage during the active stage of the disease.

Hypoxia induced EMT: A review on the mechanism of tumor progression and metastasis in OSCC.

Hypoxia, a condition of low oxygen tension in tissues, has emerged as a crucial factor in tumor pathophysiology. Hypoxic microenvironment gives rise to altered cellular metabolism and triggers varied molecular responses. These responses promote tumor progression and confer radiation resistance and chemo resistance to tumors. The predominant molecules that are associated with hypoxia research are the hypoxia inducible factors (HIFs). HIFs are known to regulate a large group of genes that are involved in cell survival, proliferation, motility, metabolism, pH regulation, extracellular matrix function, inflammatory cell recruitment and angiogenesis by inducing the expression of their downstream target genes. The process of epithelial to mesenchymal transition (EMT) has been associated with metastasis in cancer. Reports also suggest that hypoxia triggers EMT in several types of cancer including breast cancer, prostate cancer and oral cancer. Oral cancer is a predominant cancer in Central and South East Asia. However, in the recent times, the incidence rates of oral cancer have been increasing in Northern and Eastern Europe as well. This review articulates the role of hypoxia and the associated factors like HIFs in inducing EMT in oral cancer (OSCC).

Clinical Applications of Induced Pluripotent Stem Cells - Stato Attuale.

In an adult human body, somatic stem cells are present in small amounts in almost all organs with the function of general maintenance and prevention of premature aging. But, these stem cells are not pluripotent and are unable to regenerate large cellular loss caused by infarctions or fractures especially in cells with limited replicative ability such as neurons and cardiomyocytes. These limitations gave rise to the idea of inducing pluripotency to adult somatic cells and thereby restoring their regeneration, replication and plasticity. Though many trials and research were focused on inducing pluripotency, a solid breakthrough was achieved by Yamanaka in 2006. Yamanaka's research identified 4 genes (OCT-4, SOX-2, KLF-4 and c-MYC) as the key requisite for inducing pluripotency in any somatic cells (iPSCs). Our study, reviews the major methods used for inducing pluripotency, differentiation into specific cell types and their application in both cell regeneration and disease modelling. We have also highlighted the current status of iPSCs in clinical applications by analysing the registered clinical trials. We believe that this review will assist the researchers to decide the parameters such as induction method and focus their efforts towards clinical application of iPSCs.

Regulatory role of Cdx-2 and Taq I polymorphism of vitamin D receptor gene on chemokine expression in pulmonary tuberculosis.

Vitamin D receptor (VDR) gene variants have been shown to be regulating the immune response in tuberculosis. We studied the regulatory role of VDR promoter Cdx-2 and 3'UTR TaqI gene variants on chemokine levels from culture filtrate antigen (CFA) stimulated with or without 1,25(OH)2D3 treated peripheral blood mononuclear cells of 50 pulmonary tuberculosis patients (PTB) and 51 normal healthy controls (HCs). In CFA with 1,25(OH)2D3 treated cultures, the MIP-1α, MIP-1ß, RANTES levels were significantly decreased in Cdx-2 AA genotype compared to GG genotype, while a significantly increased MIG level was observed in Cdx-2 AA genotype (p<0.05). In TaqI polymorphism, tt genotype significantly decreased MIP-1ß and RANTES levels compared to TT genotype. Moreover, a significantly increased level of IP-10 and MIG was observed in TaqI tt genotype compared with TT genotype (p<0.05). The results suggests that the 1,25(OH)2D3 may alter the chemokine response through the VDR polymorphic variants during infection.

Effect of vitamin D3 on chemokine levels and regulatory T-cells in pulmonary tuberculosis.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] the active form of vitamin D3 acts as an immunomodulator in various immune cells. The present study is aimed to study the effect of 1,25(OH)2D3 on chemokine levels and regulatory T-cells in 51 healthy controls (HCs) and 50 pulmonary tuberculosis (PTB) patients. Peripheral blood mononuclear cells were cultured with culture filtrate antigen (CFA) of Mycobacterium tuberculosis in the presence or absence of 1,25(OH)2D3 at 10(-7) M concentration for 72 h and the percentage positive regulatory T-cell subsets were studied using flow cytometry. The chemokine levels were estimated in the culture supernatants by ELISA. 1,25(OH)2D3 significantly upregulated the frequency of regulatory T-cell subsets while suppressed the production of chemokine levels in CFA stimulated cultures of HCs and PTB patients (p<0.05). Correlation analysis revealed a significant negative correlation between CD4+Foxp3+ regulatory T-cells and MCP-1, MIP-1ß and IP-10 in CFA stimulated with 1,25(OH)2D3 treated cells (p<0.05). The results suggested that 1,25(OH)2D3 upregulated regulatory T-cells and act as anti-inflammatory by downregulating chemokine levels which could be beneficial to protect the host from inflammation and tissue damage during infection.

Genotoxicity evaluation of metformin and glimepiride by micronucleus assay in exfoliated urothelial cells of type 2 diabetes mellitus patients.

Micronucleus (MN) assay was performed on the exfoliated urothelial cells to detect the genotoxic effects of the anti-hyperglycemic drugs, metformin and glimepiride in T2DM patients and to use it as a biomarker for DNA damage by assessing the frequency of micronuclei in the exfoliated urothelial cells. A total of 201 subjects (147 T2DM patients & 54 Normal cases) were selected from diverse age groups (25-75 years) and the mean MN frequency was examined per 1000 cells in all the subjects. Relative to the control group (5.02 ± 1.01), an increased MN frequency was observed in females (26.15 ± 2.15) when compared to males (23.08 ± 2.09) in T2DM patients. Further analysis showed that there was a profound increase in the number of MN in the patients using metformin alone (23.02 ± 4.44), or combination of metformin & glimepiride (24.98 ± 2.87) than to the subjects using glimepiride alone (17.52 ± 3.28). It has been proven by this simple, reliable and non-invasive method that metformin has a potential role in causing genotoxicity and that the MN observed in exfoliated urothelial cells could be used as a reliable biomarker in monitoring the genotoxic risk of the anti-hyperglycemic drugs.

Effect of 1,25-dihydroxy vitamin D3 on cathelicidin expression in patients with and without cavitary tuberculosis.

1,25-dihydroxy vitamin D3 (1,25(OH)2D3) is a potent immuno-modulator which induces LL-37, the active peptide of cathelicidin, and restricts the growth of Mycobacterium tuberculosis (Mtb) in human macrophages. In the present study, we investigated the effect of 1,25(OH)2D3 on cathelicidin antimicrobial peptide (CAMP) expression in healthy controls (HCs) and pulmonary tuberculosis (PTB) patients. Peripheral blood mononuclear cells (PBMCs) from 50 HCs and 35 PTB patients were cultured for 72 h either with Mtb alone or Mtb with 1,25(OH)2D3 at 10(-7) M concentration. 1,25(OH)2D3 significantly up regulated the macrophage phagocytosis, CD14, CAMP gene expression and hCAP18 protein in HCs and PTB patients (p < 0.05). A significant positive correlation was observed between macrophage phagocytosis and CAMP gene expression in both the study groups (p < 0.05). Moreover, 1,25(OH)2D3 up regulated CAMP gene expression was more prominent in PTB patients without lung cavity (less severe form of disease) as compared to patients with cavitary TB (severe form of disease) (p < 0.05). The present study suggests that vitamin D may be used as an adjunct to anti-TB treatment and may be useful for a quicker recovery from less severe forms of TB disease.

1,25-Dihydroxy vitamin D3 downregulates pro-inflammatory cytokine response in pulmonary tuberculosis.

1,25-Dihydroxy vitamin D3 [1,25(OH)2D3] is a potent immunomodulator and regulates various immune responses to Mycobacterium tuberculosis (Mtb). The present study aimed to understand the effect of 1,25(OH)2D3 on pro-inflammatory cytokine response to Mtb antigen. Peripheral blood mononuclear cells from 42 healthy controls (HCs) and 42 pulmonary tuberculosis (PTB) patients were cultured with culture filtrate antigen (CFA) of Mtb with and without 1,25(OH)2D3 at 10(-7)M concentration for 72 h. The levels of IL-1α, IL-1ß, TNF-α, TNF-ß, IL-17 and IL-23 were estimated in the culture supernatants by ELISA. 1,25(OH)2D3 significantly suppressed all the CFA induced pro-inflammatory cytokines (p<0.05) studied except IL-1ß in both HCs and PTB patients. Among the PTB patients, the observed suppression was visible both in patients with and without cavitary tuberculosis. The present study results suggest that 1,25(OH)2D3 downregulates the production of pro-inflammatory cytokines and may control the exacerbated inflammatory response that may protect the host from excessive tissue damage at the site of infection.

Efficiency of the intestinal bacteria in the degradation of the toxic pesticide, chlorpyrifos.

Chlorpyrifos (CP) is the most commonly used pesticide throughout the world. Its widespread use in agriculture and its potential toxicity to humans from ingestion of CP contaminated food have raised concerns about its risk to health. Human intestinal microflora has the ability to degrade pesticides, but the exact mechanisms involved and the metabolite end-products formed are not well understood. The primary objective of this work was to analyse the in vitro degradation of CP by five model intestinal bacteria namely Lactobacillus lactis, L. fermentum, L. plantarum, Escherichia coli and Enterococcus faecalis. Plate assay results revealed that L. lactis, E. coli and L. fermentum could grow with high concentrations of CP (>1,400 µg/mL), whereas E. faecalis and L. plantarum could grow with concentrations as low as 400 and 100 µg/mL, respectively. The best three CP degraders were therefore used in further experiments. The degradation of CP-induced organophosphorous phosphatase (OPP) production and that OPP concentration were higher in the supernatant (extracellular) rather than inside the cells by factor of up to 28. L. fermentum degraded 70 % CP with 3,5,6-trichloro-2-pyridinol (TCP) detected as the end product. L.lactis degraded up to 61 % CP with chlorpyrifos oxon detected as the end product, whereas E.coli degraded a lesser concentration (16 %) to chlorpyrifos-oxon and diethylphosphate.

Effect of vitamin D3 on chemokine expression in pulmonary tuberculosis.

1,25 Dihydroxy vitamin D(3) (vitamin D(3)) is an immunomodulator and its deficiency has been associated with susceptibility to tuberculosis. We have studied the immunoregulatory role of vitamin D(3) on various chemokine expression in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 21 pulmonary tuberculosis (PTB) patients and 24 healthy controls (HCs) were cultured for 48 h with culture filtrate antigen (CFA) of Mycobacterium tuberculosis with or without vitamin D(3) at a concentration 1 × 10(-7)M. The relative mRNA expression of monocyte chemoattractant protein-1 (MCP-1, CCL2), macrophage inflammatory protein-1α (MIP-1α, CCL3), macrophage inflammatory protein-1ß (MIP-1ß, CCL4), and regulated upon-activation, normal T cell-expressed and secreted (RANTES, CCL5) and IFN-γ inducible protein-10 (IP-10, CXCL10) chemokines were estimated from 48 h old macrophages using real-time polymerase chain reaction (RT-PCR). The culture supernatants were used to estimate the various chemokines including monokine induced by IFN-γ (MIG, CXCL9) levels using cytometric bead array. In HCs, vitamin D(3) significantly suppressed the MCP-1 mRNA expression of CFA stimulated cells (p=0.0027), while no such effect was observed in PTB patients. Vitamin D(3) showed no significant effect on MIP-1α, MIP-1ß and RANTES in both the study groups. The CFA induced IP-10 mRNA and protein expression was significantly suppressed by vitamin D(3) in both the study groups (p<0.05). A similar suppressive effect of vitamin D(3) was observed with MIG protein in healthy controls (p=0.0029) and a trend towards a suppression was observed in PTB patients. The suppressive effect of vitamin D(3) is more prominent in CXC chemokines rather than CC chemokines. This suggests that vitamin D(3) may down regulate the recruitment and activation of T-cells through CXC chemokines at the site of infection and may act as a potential anti-inflammatory agent.

Cloning and sequencing of Indian water buffalo (Bubalus bubalis) interleukin-3 cDNA.

Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

Toll-like receptor and TIRAP gene polymorphisms in pulmonary tuberculosis patients of South India.

Toll-like receptors (TLRs) are pattern recognition receptors and play an important role in innate immunity. Changes in TLRs and signaling molecules that result from polymorphisms are often associated with susceptibility to various infectious diseases. In the present study, we investigated whether variants in the TLR-1 1805T/G (Ile602Ser), TLR-2 2258G/A (Arg753Gln), TLR-4 896A/G (Asp299Gly), TLR-4 1196C/T (Thr399Ile), TLR-6 745C/T (Ser249Pro), TIRAP 975C/T (Ser180Leu) genes and TLR-9 promoter region polymorphisms at positions -1237C/T and -1486C/T are associated with susceptibility or resistance to pulmonary tuberculosis (PTB). Genotyping of TLR and TIRAP gene polymorphisms was performed by polymerase chain reaction followed by restriction fragment length polymorphism method in 212 healthy control subjects (HCs) and 206 PTB patients. The allele and genotype frequencies of various TLR genes were not different between the HCs and PTB patients. However, the study is underpowered to detect minor associations. The frequency of T allele of TIRAP 975C/T (Ser180Leu) polymorphism was significantly increased among PTB patients as compared to HCs [p = 0.026; Odds ratio (OR) 1.49, 95% Confidence interval (CI) 1.049-2.22]. A trend towards an increased frequency of TT genotype of TIRAP 975C/T was also observed in PTB patients [p = 0.078, OR 3.10 95% CI (0.96-10.05)]. The present study suggests that T allele of TIRAP 975C/T polymorphism may be associated with susceptibility to pulmonary TB in south Indian population. Further study on the regulatory role of this polymorphism may be helpful to understand the innate immunity in TB.

Interferon gamma gene +874A/T polymorphism and intracellular interferon gamma expression in pulmonary tuberculosis.

We investigated whether IFN-gamma gene +874(A/T) polymorphism influences intracellular interferon gamma expression in T-cell subsets of normal healthy subjects (NHS) and pulmonary tuberculosis patients (PTB). Peripheral blood mononuclear cells were stimulated with live Mycobacterium tuberculosis (MTB) and the intracellular IFN-gamma expression was studied using flow cytometry. Genotyping of IFN-gamma gene +874(A/T) was done using allele specific polymerase chain reaction. Significantly increased IFN-gamma expressing CD3+CD4+ and CD3+CD8+ T cells were observed in NHS with AA genotype compared to TT genotype in unstimulated (p=0.0308 and p=0.0157) and MTB stimulated (p=0.0494 and p=0.0287) cultures and this difference was not observed in PTB patients. The present study suggests that the variant genotypes of IFN-gamma (+874) may be associated with altered expression of IFN-gamma at the intracellular level and play an immunoregulatory role at the site of M. tuberculosis infection.

Plasma 1,25 dihydroxy vitamin D3 level and expression of vitamin d receptor and cathelicidin in pulmonary tuberculosis.

INTRODUCTION: Vitamin D(3), which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D(3) levels and increased risk of tuberculosis have been reported. STUDY SUBJECTS AND METHODS: Plasma 1,25 dihydroxy vitamin D(3) levels (1,25(OH)(2) D(3)) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D(3). VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. RESULTS: 1,25(OH)(2) D(3) were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)(2) D(3) levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D(3)-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). CONCLUSIONS: The present study suggests that PTB patients may have increased 1,25(OH)(2) D(3) levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)(2) D(3) might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.

CD209 gene polymorphisms in South Indian HIV and HIV-TB patients.

Dendritic-cell-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), a pattern recognition receptor, is associated with immune functions and is also exploited by HIV-1 and Mycobacterium tuberculosis as a part of their immune evasion strategy. In the present study we investigated whether variants in the DC-SIGN encoding CD209 gene are associated with susceptibility to or protection against HIV-1 infection as well as development of tuberculosis (TB) among HIV-1 infected south Indian patients. CD209 gene variants in the promoter region (-336 and -139), in the intron and 3'-untranslated regions (In2+11 and 2281) were studied using polymerase chain reaction-based genotyping methods in 131 HIV patients without TB (HIV+TB-) and 107 HIV patients with TB (HIV+TB+), 107 HIV negative pulmonary TB patients (HIV-PTB+) and 157 healthy controls. Results revealed a decreased frequency of -336 G/G genotype among all HIV patients compared to healthy controls and -336 G/G genotype was not observed among HIV+TB- individuals (p=0.005; odds ratio (OR) 0 (95% confidence intervals (CI) 0-0.46); Peto's odds ratio 0.149 (95% CI 0.045-0.50)). Among HIV+ patients, those with TB had a significantly increased frequency of -336 G/G genotype (p=0.003; OR undefined; Peto's odds ratio 9.8 (95% CI 2.2-44.3)) compared to those without TB. Other polymorphisms were not significantly different between the various study groups. The results suggest that -336 G/G genotype while associated with protection against HIV-1 infection the same genotype is also associated with susceptibility to HIV-TB among south Indians.

Cytokine gene polymorphisms and cytokine levels in pulmonary tuberculosis.

Polymorphisms in the cytokine genes are known to influence cytokine levels and may be associated with outcome of infections. We investigated the polymorphisms in the cytokine genes namely IFN-gamma (+874 and +5644), IL-2 (-330 and +160), IL-4 (VNTR), IL-6 (-174), IL-10 (-1082 and -819) and IL-12B (+1188) in 188 normal healthy subjects (NHS) and 166 pulmonary tuberculosis patients (PTB) using polymerase chain reaction-based methods. To study the influence of cytokine gene polymorphisms on cytokine levels, phytohaemagglutinin and culture filtrate antigen of Mycobacterium tuberculosis-induced cytokine levels were measured by ELISA from 72-h-old peripheral blood mononuclear cell culture supernatants. Significantly decreased frequency of TT genotype of IL-2 -330 polymorphism (p=0.024, odds ratio (OR) 0.53, 95% CI 0.31-0.92) was observed in patients compared to NHS. The genotype frequencies of other polymorphisms were not different between patients and NHS. IL-12p40 levels were significantly decreased among NHS with AA genotype of IL-12B gene polymorphism compared to NHS with AC genotype (p<0.05). Increased levels of IL-12p40 were observed among patients with CC genotype of IL-12B gene compared to patients with other genotypes (p<0.01). The present study suggests that the TT genotype of IL-2 -330 polymorphism may be associated with the protection to PTB in south India. Further, +1188 polymorphism of IL-12B gene either alone or in combination with closely linked genes may regulate IL-12p40 production and may play a major role on acquired immunity to tuberculosis.

Regulatory region polymorphisms of vitamin D receptor gene in pulmonary tuberculosis patients and normal healthy subjects of south India.

Vitamin D receptor (VDR) gene variants are associated with differential susceptibility or resistance to tuberculosis in different ethnic groups. We investigated the polymorphisms in the 5' regulatory region of VDR gene in 206 normal healthy subjects and 166 patients with pulmonary tuberculosis from south India. Cdx-2 polymorphism was studied by polymerase chain reaction (PCR) with allele-specific primers, while genotyping of A1012G was done by PCR-based restriction fragment length polymorphism. A significantly decreased frequency of Cdx-2 G allele (P = 0.016) and G/G genotype (P = 0.010) and an increased frequency of A-A haplotype (A allele of Cdx-2 and A allele of A1012G) (P = 0.015) were observed in patients compared to controls. The study suggests that Cdx-2 G/G genotype may be associated with protection and A-A haplotype with susceptibility to tuberculosis.