HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions.

INTRODUCTION: Spontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. METHODOLOGY: The study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10-12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12-15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. RESULTS: Membrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%). Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. CONCLUSION: Our data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA-C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

A comparative study on regulation of HLA-G expression in bad obstetric history and in head and neck squamous cell carcinoma from Northeast India.

The expression of immunomodulatory molecule HLA-G is tissue restricted with abundant expression in placenta, mediating immune tolerance to fetus. Tumors hijack HLA-G to establish nutrient supply and evade host immune response. 14 base pair Insertion/Deletion polymorphism (rs371194629) and + 3142 G/C SNP (rs1063320) of 3'UTR of HLA-G were investigated in conjunction with miR-148A, miR-152 and HLA-G expression in SAB (Spontaneous abortion) history placenta and HNSCC tumor in a hospital-based case control study. Higher frequency of G allele of rs1063320 was seen in study participants as reported in other global populations. Both miR-148A and miR-152 were downregulated in tumor tissue. Predominance of 14 base pair "IN" allele of rs371194629 was noted in SAB placental tissue (p =<0.0001) with lower expression of HLA-G levels. In conclusion, 14 base pair Insertion/Deletion in linkage with + 3142 G/C SNP was related to lower HLA-G protein expression in SAB tissue, contradictorily HLA-G protein level was manipulated by tumors by suppressing microRNAs.

Immune Modulation in HLA-G Expressing Head and Neck Squamous Cell Carcinoma in Relation to Human Papilloma Virus Positivity: A Study From Northeast India.

Background: Tumor specific ectopic expression of the immunomodulatory molecule, HLA-G is known to mediate immune tolerance and promote carcinogenesis. Viruses too employ strategies to evade immune surveillance. Considering the role of both HLA-G and HPV in tumor growth and progression, it is pertinent to investigate the relationship between HLA-G and HPV in context of immune modulation in HNSCC. Method: A hospital based case-control study was conducted in histopathologically confirmed HNSCC tissues. HLA-G isoform expression and HPV association studies were carried out and mRNA levels of HLA-G, markers of proliferation and differentiation (ki-67, keratin 18, cyclin D1), immune checkpoint molecules (IL-10, PD-1. TGF-ß), SOCS (SOCS1 and SOCS3) and pro-inflammatory cytokine IFN-γ were determined. Results: Higher expression of HLA-G was noted in HPV positive tumors (5.14 fold, p = 0.002). HLA-G7 was the most frequent isoform (29/80) found in HNSCC particularly in HPV positive tumors (13/16). In HPV negative tumors, all the checkpoint molecules were upregulated along with pro-inflammatory IFN-γ. In contrast, in HPV positive tumors, IFN-γ expression was higher (2.12 fold) but levels of IL-10, PD-1, TGF-ß, SOCS1 and SOCS3 were markedly lower (fold change of IL-10 = 0.37, PD1 = 0.41, TGF-ß = 0.17, SOCS1 = 0.055, SOCS3 = 0.027). HPV positive tumors were more proliferative and differentiated with higher expression of ki-67 and keratin18 (6.25 fold, p = 0.079 and 10.62 fold, p = 0.009). Decreased expression of cyclin D1 was noted in HPV positive tumors (6.94 fold, p = 0.006) than HPV negative tumors (17.69 fold). Also, HLA-G7 expressing HPV positive tumors showed lowest expression of cyclin D1. Interestingly, SOCS showed normal expression in HLA-G7 expressing HPV negative tumors (1.2 and 1.4 fold). IFN-γ was downregulated in HPV positive tumors without HLA-G7 (0.31 fold). Conclusion: Our data suggests that SOCS were downregulated irrespective of HLA-G positivity and IFN- γ expression appeared to be mediated by HLA-G. SOCS are reported to have anti-tumor activity and also SOCS and soluble HLA-G are known to interfere with cell cycle progression. Hence, through regulating HLA-G expression, HPV positive tumors could mediate immune suppression by manipulating SOCS, IFN-γ, IL-10 and cyclin D1 pathways which needs further exploration.

Integrative Approaches to Understand the Mastery in Manipulation of Host Cytokine Networks by Protozoan Parasites with Emphasis on Plasmodium and Leishmania Species.

Diseases by protozoan pathogens pose a significant public health concern, particularly in tropical and subtropical countries, where these are responsible for significant morbidity and mortality. Protozoan pathogens tend to establish chronic infections underscoring their competence at subversion of host immune processes, an important component of disease pathogenesis and of their virulence. Modulation of cytokine and chemokine levels, their crosstalks and downstream signaling pathways, and thereby influencing recruitment and activation of immune cells is crucial to immune evasion and subversion. Many protozoans are now known to secrete effector molecules that actively modulate host immune transcriptome and bring about alterations in host epigenome to alter cytokine levels and signaling. The complexity of multi-dimensional events during interaction of hosts and protozoan parasites ranges from microscopic molecular levels to macroscopic ecological and epidemiological levels that includes disrupting metabolic pathways, cell cycle (Toxoplasma and Theileria sp.), respiratory burst, and antigen presentation (Leishmania spp.) to manipulation of signaling hubs. This requires an integrative systems biology approach to combine the knowledge from all these levels to identify the complex mechanisms of protozoan evolution via immune escape during host-parasite coevolution. Considering the diversity of protozoan parasites, in this review, we have focused on Leishmania and Plasmodium infections. Along with the biological understanding, we further elucidate the current efforts in generating, integrating, and modeling of multi-dimensional data to explain the modulation of cytokine networks by these two protozoan parasites to achieve their persistence in host via immune escape during host-parasite coevolution.

Lower expression of GATA3 and T-bet correlates with downregulated IL-10 in severe falciparum malaria.

Interleukin (IL)-10, a non-redundant anti-inflammatory cytokine is produced by different cells and its production involves activation of cell-specific transcriptional regulatory machinery in response to specific pathogen. We have previously demonstrated downregulated levels of IL-10 in severe falciparum malaria. The present study investigated transcriptional regulation of IL-10 in severe malaria. Comparative expression analysis of cell-specific signalling proteins and transcription factors for IL-10 production during the stage of active infection and with resolution of parasitaemia was performed. Interestingly, T-bet and GATA3, the Th1 and Th2 transcription factors, respectively, were downregulated in severe malaria with fold change values of 0.59 and 0.86. Increase in the levels of both the factors with resolution of parasitaemia implicated a role for parasite in depressed levels of these factors. Further support for probable parasite manipulation of GATA3 was obtained from negative correlation of GATA3 with parasitaemia. In addition, a role for interferon-α in suppressing IL-10 transcription was evident from its negative correlation with GATA3 and IL-10 levels. In summary, IL-10 transcription in Th1 and Th2 is defective and appears to have major contribution to low levels in severe malaria.

Negative regulation of natural killer cell in tumor tissue and peripheral blood of oral squamous cell carcinoma.

Natural killer (NK) cells are the key lymphocytes in solid tumors. Its activity is regulated by both germline encoded receptors and cytokine microenvironment. We conducted a case-control study to investigate the activation status of NK cell in oral squamous cell carcinoma (OSCC). NK cell activation was assessed in context of NK cell cytotoxicity and transcript expression of NK cell receptors (NKp46 and KIRs) and NK cell associated cytokines (IL-1ß, IL-2, IL-10, IL-12ß, IL-15, IL-18, IL-21, IFN-γ, TNF-α and TGF-ß). The results revealed possible mechanisms involved in reduced NK cell activation in peripheral circulation: quantitative deficiency of NK cell number and lowered cytotoxicity together with qualitative NK impairments caused by--(1) decreased expression of NK activating receptor NKp46, (2) increased expression of NK suppressive cytokines--IL-10 and TGF-ß and (3) induction of FOXP3(+)CTLA4(+) suppressor cells. On the other hand, in the tumor tissue, escape of NK immune surveillance appeared to be modulated by upregulation of TGF-ß and IL-10 together with downregulation of NK cell activating cytokines (IL-2, IL-12ß, IL-15, IL-18, IL-21 and IFN-γ) and NK receptors (NKp46 and KIRs). In addition, our study supported the earlier contention that TNF-α and IL-1ß expression levels may be used as markers of malignant transformation in oral leukoplakia. In conclusion, the study provided an insight into the negative regulation of NK cell in tumor tissue and peripheral blood of OSCC patients, which can be exploited to boost the current NK cell and cytokine based immunotherapy for the treatment of oral cancer.

Heightened inflammation in severe malaria is associated with decreased IL-10 expression levels and neutrophils.

Dysregulation of the cytokine network in severe malaria owing to variations in factors like parasite load, strains and host factors is well documented but the key cytokines that are dysregulated remain poorly elucidated. Longitudinal changes in cytokine levels in an individual with parasitemia and disease resolution is likely to identify the key cytokines. We have analyzed the mRNA expression of cytokines over a 7-d period in severe (SM) and uncomplicated (UM) Plasmodium falciparum malaria. We found up-regulated expression of TNF-α, IL-1ß, IFN-γ and TGF-ß in SM, with decreased expression of IL-10 on d 0. Further, we observed a negative correlation of IL-10 expression with parasitemia and pro-inflammatory cytokines, suggesting IL-10 to be the key cytokine in tilting the balance to an inflammatory response. Longitudinal analysis revealed that the key cytokines associated with disease were TNF-α, IL-1ß, IFN-γ, IL-12α, RANTES and TGF-ß, while TNF-α, IL-10 and TGF-ß discriminated between SM and UM. A higher neutrophil count in SM and its positive association with parasite density and IL-1ß and IL-8 provides support for neutrophils in inflammation in malaria. Our findings suggest subversion of anti-inflammatory response in SM by parasite factors towards an exaggerated pro-inflammatory response with involvement of neutrophils, the classical inflammatory cells.

Association of killer cell immunoglobulin-like receptor gene 2DL1 and its HLA-C2 ligand with family history of cancer in oral squamous cell carcinoma.

Killer cell immunoglobulin-like receptors (KIR) are involved in regulating natural killer cell activation through recognition of their human leukocyte antigen (HLA) class I ligands. We conducted a case-control study with 169 oral squamous cell carcinoma (OSCC) patients and 177 healthy participants to study the genomic diversity of KIR and HLA loci and KIR gene expression in context of family history of cancer (FHC) in OSCC. Polymerase chain reaction (PCR) sequence-specific priming approach was used to type 16 KIR genes in individuals. SSP-real-time PCR was used for HLA class I ligand genotyping and real-time quantitative reverse transcriptase PCR was used to determine the expression of KIR gene. KIR2DL1(+)-HLA-C2(+) genotype was higher and positively associated with OSCC. Notably, all KIR2DL1(+)-HLA-C2(+) genotypes occurred exclusively in patients with FHC, showing a strong positive association of KIR2DL1(+)-HLA-C2(+) genotype with FHC. In addition, all younger age group patients (<55 years) with FHC were positive for KIR2DL1(+)-HLA-C2(+) genotype suggesting association of the genotype with early onset of disease. RNA transcript abundance of inhibitory KIR2DL1 in FHC patients, particularly of lower age groups (<45 and 45-54 years), supports the contention. Further, KIR2DL3(+)-HLA-C(+) genotype was negatively associated with OSCC. Our findings suggest KIR2DL1(+)-HLA-C2(+) genotype as heritable risk factor in OSCC predisposing to OSCC at younger age. Interestingly, KIR2DL3(+)-HLA-C(+) genotype was seen to be protective in OSCC. This study may be useful towards cancer surveillance and early detection of oral cancer in patients with FHC.

The association of IL-8-251T/A polymorphism with complicated malaria in Karbi Anglong district of Assam.

Amongst host genetic factors, cytokine gene polymorphism can be anticipated to be an important factor as qualitative, quantitative and time of secretion play an important role in disease outcome. We have investigated association of cytokine promoter SNPs with risk of Plasmodium falciparum malaria and disease severity in a case control study in malaria endemic Karbi Anglong district of Assam, India. Frequency of IL-8-251T/A (p=0.03 and p=0.01) and TGF-ß1-509C/T (p=0.02 and p=0.03) was higher in malaria in comparison to control participants and non-malarial fever controls. Interestingly, a higher frequency of mutant allele of IL-10-819T/C was observed in non-malarial fever controls compared to malaria thus suggesting its role as a distinguishing marker of the two disease groups. Higher IL-8 expression and increased frequency of IL-8-251T/A in complicated malaria (p=0.002) was reported indicating its role in susceptibility to complicated malaria. In conclusion, our study suggests the role of mutant genotype of IL-8-251T/A as a marker of complicated malaria in our population. Surprisingly, decreased expression of TGF-ß1 in uncomplicated malaria even in presence of high expressing mutant genotype was observed and needs to be investigated in context of the pool of activated cells producing the cytokine.

Dysregulation of cytokines expression in complicated falciparum malaria with increased TGF-ß and IFN-γ and decreased IL-2 and IL-12.

BACKGROUND: In Plasmodium falciparum infections, proinflammatory cytokine response is implicated in control of parasite multiplication as well as in disease pathogenesis. However, the regulation of proinflammatory and anti-inflammatory cytokine balance and its relation to disease severity remains poorly understood. METHODS: We examined cytokines gene expression by quantitative real time-PCR technique in a case control study comprising of P. falciparum infected (n=58) and non infected (n=30) groups. P. falciparum infected were further stratified as complicated and uncomplicated as per WHO criterion and parasitaemia levels. RESULTS: Higher expression of IL-2, IL-12α and TGF-ß with decreased levels of IL-10 was seen in P. falciparum positivity. Complicated malaria was associated with enhanced expression of IFN-γ and TGF-ß but lower IL-2 and IL-12α in comparison to uncomplicated malaria. Modeling of data suggested higher expression of IL-12α to be predictive of uncomplicated malaria [Odds ratio=3.074, 95% CI (1.254-7.536)] and was negatively associated with complicated malaria outcome (p=0.014). Interestingly, the probability of complicated malaria in males with elevated TNF-α expression was three times higher [p=0.05; Odds ratio=3.412, 95% CI (0.98-11.848)]. Age was also seen to be a factor with higher IL-8 in diseased young (p=0.012). CONCLUSION: Our data suggested induction of balanced cytokine response in uncomplicated malaria while cytokine dysregulation with a role for TGF-ß was indicated in complicated malaria. TH cells did not appear to be the source of increased IFN-γ expression associated with malaria severity.

Polymorphisms and expression of TLR4 and 9 in malaria in two ethnic groups of Assam, northeast India.

Infectious diseases have been postulated to play an important role in exerting pressure and in selection of TLR polymorphisms. Single nucelotide polymorphisms (SNPs) of TLR4 have been reported to show unique distributions in populations from Africa, Asia and Europe, and malaria is suggested to influence these patterns. In this context, we examined association of TLR polymorphisms with the risk of malaria in two ethnic groups-the Austro-Asiatics and Tibeto-Burmans-from malaria endemic districts of Assam to understand the influence of malaria in selection of TLRs in these genetically-distinct populations. TLR9 (T-1237C) mutation was positively associated with complicated (P = 0.001) and frequent (P = 0.035) malaria in Austro-Asiatics (relative risk = 0.595 95% CI: 0.479-0.836), but not in Tibeto-Burmans. Nonetheless, these alleles were not in Hardy-Weinberg Equilibrium in Tibeto-Burmans (P < 0.001). In contrast, the TLR9 1486T/C genotype was favourable where it was negatively associated with complicated malaria (Fishers exact P = 0.014). Sequencing data revealed that the two populations differed in nucleotide diversity of the TLR9 promoter region. Enhanced expression of TLR4 (P = 0.05), but not of TLR9, was associated with complicated malaria. Austro-Asiatics appeared to have accumulated favourable genotypes of TLR9, perhaps because of their longer exposure to malaria.

Antibody response to allelic variants of 19kDa fragment of MSP-1: recognition of a variant and protection associated with ethnicity in Assam, India.

Evidence suggests association of anti MSP-1(19) antibodies with protection from clinical malaria. However, the target epitope was reported to vary with respect to response to conserved or variant epitopes in different studies. We have investigated here humoral response of naturally exposed individuals of Tibeto-Burman and Austro-Asiatic ethnic groups to E-TSR and Q-KNG variants of MSP-1(19) in comparison to whole merozoite extract (WME) of local strain of Plasmodium falciparum in a longitudinal prospective cohort study. The association of antibodies in relation to risk of infection and disease severity was determined. A relatively lower seropositivity to MSP1(19) peptides derived from 3D7 and FVO strains in comparison to whole merozoite extract of local P. falciparum strain was observed. Recognition of Q-KNG variant was markedly lower in TB (p<0.0001) indicating a role of ethnicity. The Tea tribes of Austro-Asiatic affinity had higher antibody response (E-TSR; p=0.038 and Q-KNG; p=0.004) and equally recognized the two variants. A reduced risk of clinical infection in high transmission summer season was seen in presence of anti MSP-1(19) antibodies (p=0.013) and antibody level was predictive of risk of clinical malaria (ROC=0.729). Anti E-TSR antibodies were inversely associated to disease severity at KTE (λ(2)p=0.013; t-test p=0.032). The present study demonstrated antibody response to MSP-1(19) was associated with protection from frequent episodes of malaria and disease severity and that the host genetic background was important factor in response to MSP-1(19) allelic variant.

Differential association of KIR gene loci to risk of malaria in ethnic groups of Assam, Northeast India.

Receptors encoded within the Natural Killer Cell (NKC) complex and Killer Immunoglobulin like (KIRs) genomic regions have been suggested to influence malaria pathogenesis and infection susceptibility. We have examined KIR locus in relation to risk of infection and disease in Tea tribes (TT) of Austro Asiatic affinity and Tibeto-Burman (TB) populations from malaria endemic regions of Assam. Consistent with differences in their genetic background, KIR gene loci frequencies differed in studied groups. Surprisingly, KIR3DS1 frequency in TT was low (17%) and comparable to that reported from African populations. KIR3DL1 frequency was positively associated with malaria severity (Pearson phi, R(2) = 0.297 p = 0.006) and logistic regression modelling predicted KIR3DL1 as a risk factor in complicated malaria [Odds Ratio (95% C.I)] = [6.39 (1.34-30.60)]. An interaction between ethnicity and KIR3DL1 was also seen where higher proportion of KIR3DL1 positive and complicated malaria patients belonged to Tea tribes (p = 0.009). Notably, four activating genes protected from frequent malaria (p = 0.02) while six activating genes enhanced the risk of complicated malaria (p = 0.05). Combination of KIR2DS4, KIR2DS4del, KIR2DS5 negatively influenced disease outcome in Tea tribes (p = 0.048) but not in Tibeto-Burman. In conclusion our data indicates KIR gene loci differentially influenced malaria outcome in Tea tribes and Tibeto-Burman and that four activating genes appeared to provide optimal activation that protected from frequent episodes of malaria. Our data also indicated KIR3DS1 to be an ancestral genotype, maintained at low frequency possibly by malaria in the Austro Asiatic tribes.

Physical and biophysical characteristics of nanoscale tungsten oxide particles and their interaction with human genomic DNA.

Nanoscale tungsten oxide (WO3) particles were synthesized via a user-friendly solvothermal cum reduction route using sodium tungstanate (Na2WO4) and cetyl trimethyl ammonium bromide (C19H42NBr) as reactants. The X-ray diffraction and transmission electron microscopy studies have revealed monoclinic phase of WO3 with an average crystallite size of 40 nm and competitive crystallographic orientation along (002), (020), (200) planes. After extracting human genomic DNA from human blood by a standard protocol (Qiagen-Kit method), they were conjugated with nanoscale WO3 particles in varying molar concentrations. The biophysical interaction of DNA bound nanoparticles were characterized by Fourier transform infra-red spectroscopy, photoluminescence spectroscopy, agarose gel-electrophoresis and polymerase chain reaction. Understanding physical and biophysical aspects of unconjugated and DNA conjugated WO3 would provide scope for biosensing applications.

Temporal and spatial variation in MSP1 clonal composition of Plasmodium falciparum in districts of Assam, Northeast India.

Polymorphism in MSP1 gene generated by insertion/deletion of repeats causing repeat length polymorphisms is widely used as a marker for parasite genotyping. Elucidating Plasmodium falciparum clonal composition in relation to transmission intensity and other epidemiological factors in endemic areas is crucial to understanding the dynamics of host-parasite relationship and the development of immunity in malaria. We have examined here the allelic diversity of P. falciparum and attempted to understand the polymorphism and distribution of alleles of MSP1 with transition in transmission season and with differences in malaria epidemiology between sites. MSP1 diversity expressed as mean number of distinct alleles per isolate was 0.68 at Dimakusi and was much higher (p=0.007) than seen at Guabari (0.336) and Kondoli (0.45) as was multiplicity of infection at 4.12, indicating the highest diversity at this site. Size polymorphism of the allelic families at Guabari was distinctly different from Kondoli but shared similarity with Dimakusi. Infections in high transmission summer season tended to be more complex with higher number of alleles. The frequency of alleles of RO33 and MAD20 allelic families at Guabari was found to be different between the two transmission periods. A 380 base pair allele of RO33 was over represented in high transmission summer season and seen frequently in isolates with high parasitaemia. At Kondoli allele distribution of only MAD20 was found to be different in each study year. Study site and ethnicity but not age of the study population were identified as risk factors in infection complexity. The present study demonstrates that allelic composition of P. falciparum varied with study site and between periods of high and low transmission as well as in different years of study.