High frequency of memory stem cells with a distinct gene signature in HIV patients with treatment interruption.

Reservoirs of HIV remain a major obstacle to the complete eradication of virus despite regular anti-retroviral therapy (ART). Memory stem cells (Tscm), one of the major reservoirs, are relatively less studied owing to their presence in lower numbers and inaccessible anatomical locations. We have evaluated the molecular characteristics of Tscms in patients with ART interruption (n = 15) versus patients on uninterrupted ART (n = 12) using flow cytometry. RNA sequencing was done in the sorted Tscms to study the differential gene expression. Patients with ART interruption had significantly lower baseline CD4+T-cell counts and high viral loads as compared to patients on ART. The former group had significantly higher frequency of CD4+ and CD8+Tscms with a higher expression of PD-1 on CD8+Tscms. The transcriptome profile of Tscm was significantly different among the patient groups. The main pathways were cellular and metabolic pathways, cellular development pathways, cell differentiation and negative regulation of cellular migratory pathways. An increased yet dysfunctional CD8+ memory stem cells describe HIV-1-infected patients with break-in ART and a distinct transcriptional signature of CD4+ Tscm as compared to those of patients on ART. A more detailed understanding of the biology and dynamics of Tscm in future studies is warranted. Strategies to improve the functionality of the CD8+ Tscm will help these patients to tackle the outburst of viral replication that occurs after the cessation of therapy.

[68Ga]Ga-Pentixafor PET/CT imaging for in vivo CXCR4 receptor mapping in different lung cancer histologic sub-types: correlation with quantitative receptors' density by immunochemistry techniques.

PURPOSE: In vivo CXCR4 receptor quantification in different lung cancer (LC) sub-types using [68Ga]Ga-Pentixafor PET/CT and to study correlation with quantitative CXCR4 receptors' tissue density by immunochemistry analyses. METHODS: [68Ga]Ga-Pentixafor PET/CT imaging was performed prospectively in 94 (77 M: 17F, mean age 60.1 ± 10.1 years) LC patients. CXCR4 receptors' expression on lung mass in all the patients was estimated by immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) analyses. SUVmax on PET, intensity score on IHC, and mean fluorescence index (MFI) on FACS analyses were measured. RESULTS: A total of 75/94 (79.8%) cases had non-small cell lung cancer (NSCLC), 14 (14.9%) had small cell lung cancer (SCLC), and 5 (5.3%) had lung neuroendocrine neoplasm (NEN). All LC types showed increased CXCR4 expression on PET (SUVmax) and FACS (MFI). However, both these parameters (mean SUVmax = 10.3 ± 5.0; mean MFI = 349.0 ± 99.0) were significantly (p = 0.005) higher in SCLC as compared to those in NSCLC and lung NEN. The mean SUVmax in adenocarcinoma (n = 16) was 8.0 ± 1.9 which was significantly (p = 0.003) higher than in squamous cell carcinoma (n = 54; 6.2 ± 2.1) and in not-otherwise specified (NOS) sub-types (n = 5; 5.8 ± 1.5) of NSCLC. A significant correlation (r = 0.697; p = 001) was seen between SUVmax and MFI values in squamous cell NSCLC as well as in NSCLC adenocarcinoma (r = 0.538, p = 0.031) which supports the specific in vivo uptake of [68Ga]Ga-Pentixafor by CXCR4 receptors. However, this correlation was not significant in SCLC (r = 0.435, p = 0.121) and NEN (r = 0.747, p = 0.147) which may be due to the small sample size. [68Ga]Ga-Pentixafor PET/CT provided good sensitivity (85.7%) and specificity (78.1%) for differentiating SCLC from NSCLC (ROC cutoff SUVmax = 7.2). This technique presented similar sensitivity (87.5%) and specificity (71.4%) (ROC cutoff SUVmax = 6.7) for differentiating adenocarcinoma and squamous cell variants of NSCLC. CONCLUSION: The high sensitivity and specificity of [68Ga]Ga-Pentixafor PET/CT for in vivo targeting of CXCR4 receptors in lung cancer can thus be used effectively for the response assessment and development of CXCR4-based radioligand therapies in LC.

Natural history and profile of selective cytokines in patients of acute pancreatitis with acute kidney injury.

OBJECTIVES: To study the natural course of patients with acute pancreatitis (AP) with acute kidney injury (AKI) and their cytokine profile. METHODS: Natural course of patients with AP and AKI was studied in 97 individuals. Levels of TNFα, IL-6, IL-10, IL-8 and IL-1ß were measured at presentation and at 72 h in patients who developed AKI. RESULTS: Amongst the entire cohort, 16.4% patients developed AKI (persistent AKI - 11 patients, transient AKI - 5 patients). Mortality rate was 25% amongst patients with AKI. Levels of IL-6 (p = 0.035) and IL-8 (p = 0.002) were found to be significantly higher in the AKI group. On multivariate analysis, IL-8 levels at baseline were found to be an independent predictor of AKI. AKI group had significant rise of TNF-α (P < 0.001), IL-6 (P < 0.001) and IL- 1ß (P < 0.001) on day 3 whereas persistent-AKI group had significant rise of TNF-α (p = 0.031), IL-6 (p = 0.001) and IL-1ß on day 3 and significant decline of IL-10 (p = 0.015). Using a cut-off of 105 pg/ml, IL-8 levels at baseline could predict AKI with a sensitivity of 87.5% and specificity of 59.2%, with area under the curve being 0.744 (p = 0.002). CONCLUSION: AP patients developing AKI have poor prognosis. IL-8 levels can predict AKI in patients with AP.

A juggernaut of innate & adaptive immune cells in chronic hepatitis C.

Hepatitis C virus (HCV) is a small positive-sense, single-stranded RNA virus, the causal organism for chronic hepatitis. Chronic hepatitis leads to inflammation of liver, causing cirrhosis, fibrosis and steatosis, which may ultimately lead to liver cancer in a few cases. Innate and adaptive immune responses play an important role in the pathogenesis of HCV infection, thus acting as an important component in deciding the fate of the disease. Numerous studies have indicated that the derangement of these immune responses results in the persistence of infection leading to chronic state of the disease. Interactions between virus and host immune system generally result in the elimination of virus, but as the virus evolves with different evading mechanisms, it makes environment favourable for its survival and replication. It has been reported that HCV impairs the immune system by functional modulation of the cells of innate as well as adaptive immune responses, resulting in chronic state of the disease, influencing the response to antiviral therapy in these patients. These defects in the immune system lead to suboptimal immune responses and therefore, impaired effector functions. This review highlights the involvement or association of different immune cells such as natural killer cells, B cells, dendritic cells and T cells in HCV infection and how the virus plays a role in manipulating certain regulatory mechanisms to make these cells dysfunctional for its own persistence and survival.

Immunological markers for identifying recent HIV infection in North-West India.

Background & objectives: Being more efficient and widely used, limiting antigen (LAg)-avidity enzyme immunoassay (EIA) based on the recent infection testing algorithm (RITA) has been developed for differentiating recent and established HIV-1 infection. So far, LAg-avidity EIA has not been validated among the Indian population. Hence, the present study was planned to identify recent HIV infections in high risk patients in the North-West region of India using modified LAg-avidity RITA. Methods: Four hundred HIV-positive high risk patients registered on pre-antiretroviral therapy (ART) programme in the last one year, from five ART centres in North-Western States of India, were included for identifying the recent HIV infections. One hundred HIV-positive cases registered for pre-ART for greater than two years in ART centres were included for estimating false recent rate (FRR). Single-well LAg-avidity EIA-based modified RITA was used to identify recent HIV infection cases. Results: Of the 400 HIV-1-positive samples, 64 (16%) were found to have been infected within the past 130 days. The proportion of recent HIV infections was 16.8 per cent (18/107) among female sex workers, 10.7 per cent (9/84) among men who have sex with men and 17.7 per cent (37/209) among injecting drug users. The FRR was one per cent (1/100). Interpretation & conclusions: LAg-avidity EIA-based modified RITA provided good discrimination between recent and non-recent HIV infection, hence, it could be considered suitable for estimating HIV incidence in sentinel surveillance system in India.

Increased expression of negative regulators of cytokine signaling during chronic HIV disease cause functionally exhausted state of dendritic cells.

Mechanisms of functional impairment of dendritic cells (DCs) during chronic HIV-1 infection are not well understood. In order to understand this phenomenon, we aimed to study the expression of negative regulators of cytokine signaling and correlate with DC exhaustion during chronic HIV-1 disease. Monocyte-derived DCs (mo-DCs) from 27 HIV-1 infected patients (CD4+ T-cell counts: 429±44 cells/µL, plasma viral load: Log103.9±1.0copies/ml) and 19 healthy controls (HCs) were stimulated ex vivo with TLR4 agonist, lipopolysaccharide (LPS) for 2days to evaluate their functional fitness. The expression of a set of genes associated with cytokine signaling was evaluated in a custom designed PCR array by Real-Time PCR. The mo-DCs from HIV-1 infected patients depicted functional exhaustion as evident by decreased allo-stimulation index (mean±SD: 10±6 vs. 24±16) (p<0.05), decreased cytokine production (pg/ml) (IL-12: 4.6±16 vs. 25±85; TNF-α: 128±279 vs. 286±544; IL-10: 6±12 vs. 13±20; IL-8: 10,688±11,748 vs. 17,470±125,049) and retained endocytosis (1.1±0.3 vs. 1.0±0.29) (p<0.05) even after LPS-stimulation, as compared to HCs. Significantly upregulated expression of SOCS-1 (mean±SD fold change: 2.2±2vs.0.8±0.6), SOCS-3 (6.3±7.4vs.1.4±0.4), PIAS-1 (1.6±0.1vs.1.0±0.3) and SHP-1 (0.8±0.4vs.0.4±0.2) correlated positively with PD-L1 expression in these DCs (Spearman's coefficient, SOCS-1: 0.63, SOCS-3: 1.0 and PIAS-1: 0.7) (p<0.05). The expression of these molecules trended positively with plasma viral load and negatively with CD4+ T-cell counts. These findings suggest that the upregulation of negative regulatory factors during chronic HIV disease have profound down-modulatory effects on DC functions and establishment of an overall exhausted state. Understanding mechanisms causing upregulation of these factors may lead to the design of new generation therapeutics based on silencing of their gene expression.

Combination of low producer AA-genotypes in IFN-γ and IL-10 genes makes a high risk genetic variant for HIV disease progression.

Rate of HIV disease progression varies considerably among individuals, host genetic makeup be one of the possible reasons. We aimed to determine association of functional single nucleotide polymorphism (SNPs), (-179G/T and +874T/A) in IFN-γ and (-1082A/G, -819C/T and -592C/A) in IL-10 genes, with the rate of disease progression or susceptibility to HIV infection. Therapy naïve HIV infected individuals from North India, categorized as slow progressors or fast progressors and HIV exposed seronegative individuals were recruited for this study. Genotyping results revealed significantly higher frequencies of low producer AA genotype at +874T/A in IFN-γ gene and -592C/A position in IL-10 gene in FPs (p<0.002). Multifactor dimensional reduction (MDR) analysis revealed this to be a high risk combination for faster disease progression in HIV-1 infected individuals. Low producer AA genotype carriers at +874T/A in IFN-γ gene produced significantly low amounts of cellular IFN-γ. Low producing haplotype 'ATA' at -1082, -819 and -592 loci in IL-10 gene was significantly over-represented in FPs as compared to SPs (p<0.01) and these individuals showed poor response to therapy in terms of CD4 count gains after one year of ART, compared to high producing haplotype (GCC) carriers. Thus, a combination of genetic variations in IFN-γ and IL-10 cytokine genes in a single host associate with HIV disease progression and may help clinicians to better manage the HIV disease if known earlier.

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients.

Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7%) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1% HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.

Intercellular adhesion molecule-1 as a drug target in asthma and rhinitis.

Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein receptor of the immunoglobulin superfamily. Endothelial cells, epithelial cells, leukocytes and neutrophils are the major cells expressing ICAM-1. Ligands of ICAM-1 are macrophage adhesion ligand-1, leukocyte function-associated antigen-1 and fibrinogen (extracellular matrix protein). In normal physiological conditions, engagement of ICAM-1 receptor with immunological cells surface ligands assists in homing and trafficking of inflammatory cells to distant tissues. ICAM-1 has also long been known to mediate cell-to-cell interaction during antigen presentation and outside-in cell signalling pathways. ICAM-1-mediated elevated inflammation is implicated in asthma. On respiratory epithelial cells surface, ICAM-1 acts as natural binding site for human rhinovirus (HRV), a common cold virus that ultimately causes exacerbation of asthma. This review presents the findings on the role of ICAM-1 in the complication of asthma and in particular asthma exacerbation by HRV.

Hepatitis B virus reverse transcriptase mutations in treatment Naïve chronic hepatitis B patients.

Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome lead to decreased susceptibility to nucleos(t)ide analogs approved for treatment of HBV infection. The aim of this study was to detect and analyze pre-existing HBV RT mutations in treatment naïve patients with chronic hepatitis B. Seventy one chronic HBV treatment naïve patients were enrolled from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi-nested PCR products. HBV genotypes were determined by multiplex PCR. Genotype D was found in 64 patients (90.1%) followed by genotype C and A which were present in 5 (7.0%) and 2 (2.8%) patients respectively. The results of the RT sequence analysis showed mutations in 34 (47.9%) patients. The rtH248N mutation was the most common mutation, accounting for 47.1% patients. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D, which were present in 26.5% (9/34), 29.4% (10/34), 20.6% (7/34), 20.6% (7/34), 20.6% (7/34), 17.6% (6/34), 14.7% (5/34), 14.7% (5/34), 11.8% (4/34), 11.8% (4/34) and 11.8% (4/34) patients respectively. The known primary drug resistance mutations were found in 3 (8.8%) patients. The present study shows the presence of RT amino acid substitutions in treatment-naïve patients with chronic hepatitis B, which may decrease susceptibility to available oral antiviral drugs. On the basis of the finding of this study, genotypic testing is recommended before the start of therapy in naïve patients, so that suitable antiviral drugs can be prescribed.

Success of antiviral therapy in chronic hepatitis C infection relates to functional status of myeloid dendritic cells.

Chronic hepatitis C infection poses a major global health predicament and appears to be potent threat to mankind. The treatment in wide use is interferon/ribavirin combination therapy which is generally effective in about 60-70 per cent of patients carrying genotype 3 and causes significant morbidity. The response to therapy is largely guided by limited number of factors such as genotype of virus, rapid virological response, ethnicity, pre-therapy viral load, etc. While involvement of host genetic factors has been a major focus of research in playing an important role in the outcome of disease, the role of immune system cannot be marginalized. Poor cellular trafficking and suboptimal T cell responses in liver, the hall marks of chronic hepatitis C virus infection, might be attributed to defective antigen presentation. Various immunological factors, both innate and adaptive, play role in the pathogenesis of the disease and become dysfunctional in active disease. Recent reports suggest the major impact of functional and numerical status of dendritic cells in deciding the fate of antiviral therapy. In this review we take a look at the involvement of dendritic cells in playing an important role in the response to therapy.

Functional reconstitution of defective myeloid dendritic cells in chronic hepatitis C infection on successful antiviral treatment.

OBJECTIVE: Poor cellular trafficking and suboptimal T-cell responses in liver, the hall marks of chronic hepatitis C virus (CHC) infection, might be attributed to defective antigen presentation. Controversy exists regarding role of myeloid dendritic cells (DCs) in CHC and response to antiviral treatment. This study examines functional status of DCs before and after completion of treatment with the aim to find any modulatory effect. DESIGN: Frequency and functions of monocyte-derived DCs (mo-DCs) were evaluated in CHC (n = 25), before the start of therapy (CHC(0) ). These patients were then put on treatment with peg-interferon-α plus ribavirin for 24 or 48 weeks, and the mo-DC functions were evaluated after 6 months of completion of treatment (CHC(6) ) again, using multicolour flow cytometry, endocytosis assay, cytokine assay and mixed lymphocyte reaction. RESULTS: Pre-treatment frequency of mo-DCs in CHC(0) was lower than that in healthy controls, which became close to normal in patients who achieved virological response (SVR+, n = 20) but not in non-responders (SVR-, n = 5). Pre-treatment levels of CD83, CD80 and CD86 on mo-DC in SVR(0) +, but not SVR(0) -, got upregulated after lipopolysaccharide stimulation supporting the hypothesis that DCs play deciding role in response to therapy. Post-treatment allostimulatory and phagocytosing capacity of mo-DCs in SVR+ patients indicated regain in functional capacity in these patients but not in SVR- patients. CONCLUSIONS: Our results indicate that DCs in CHC patients exhibiting mature and functional phenotype prior to therapy achieve sustained virological response suggesting that functional modulation of defective DCs is directly associated with successful response to therapy.

Association of serotonin transporter promoter polymorphism (5-HTTLPR) with orocecal transit time in irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a multifactorial disorder with altered intestinal motility, secretion, and sensation. Serotonin (5-HT) stimulates gut motility and alters serotonin signaling that may lead to both intestinal and extraintestinal symptoms in IBS. AIM: The aim of this study was to examine the association of serotonin transporter gene promoter polymorphism (5-HTTLPR) in IBS with orocecal transit time (OCTT) measured by lactulose hydrogen breath test. METHOD: This prospective case-control study included 151 IBS patients (mean±SD 37.4±11.6 years, median 36, range 19-68). Ninety-two patients were diarrhea-predominant IBS (D-IBS), 44 constipation-predominant IBS (C-IBS), 15 alternating diarrhea and constipation IBS (M-IBS), and 100 healthy controls (mean±SD 37.2±11.4 years, median 36, range 20-64 years). 5-HTTLPR gene polymorphism was studied by polymerase chain reaction-based method. 5-HT levels were measured by enzyme-linked immunosorbent assay (ELISA). Orocecal transit time (OCTT) was measured by a non-invasive lactulose hydrogen breath test. OCTT was also compared with respect to 5-HTTLPR genotypes in different IBS phenotypes. RESULTS: Serum serotonin levels were significantly higher in overall IBS patients (152±77 ng/mL, p<0.001), D-IBS (184±76 ng/mL, p<0.001), compared to healthy controls (129±56 ng/mL). There was no difference in 5-HT levels between C-IBS (124±53 ng/mL) and controls. In the case of M-IBS, 5-HT levels were (88±49 ng/mL p<0.05) significantly lower than that of controls. OCTT was significantly shorter in D-IBS patients (95±36 min) as compared to controls (112±41 min). In contrast, C-IBS showed significantly prolonged OCTT (136±54 min). There was a significant difference in OCTT between D-IBS and C-IBS patients (p<0.001). There was no significant association found between OCTT and 5-HTTLPR. CONCLUSIONS: Serum serotonin concentrations were increased in D-IBS compared to controls and C-IBS. OCTT was shorter in D-IBS and delayed in C-IBS patients. There was no association of 5-HTLPR polymorphism with OCTT.

68Ga-Pentixafor PET/CT Demonstrating In Vivo CXCR4 Receptor Overexpression in Rare Lung Malignancies: Correlation with Histologic and Histochemical Findings.

68Ga-pentixafor PET/CT imaging allows noninvasive assessment of C-X-C chemokine receptor type 4 (CXCR4) expression in various malignancies, but its use in rare lung cancer variants has not been reported. Methods: 68Ga-pentixafor PET/CT imaging was performed on 6 patients (3 men, 3 women; mean age, 57.0 ± 16.8 y) with suspected lung masses. Whole-body PET/CT images were acquired 1 h after intravenous injection of 148.0-185.0 MBq of the tracer. PET/CT images were reconstructed and analyzed. The image findings were correlated with histopathologic and quantitative (CXCR4) fluorescence-activated cell sorting analysis. Results: Histopathologic diagnosis of hemangioendothelioma, sarcomatoid carcinoma, and hemangiopericytoma was confirmed in 1 patient each. Lung metastasis was diagnosed in the remaining 3 of 6 patients with primary sarcoma (n = 1), renal cell carcinoma (n = 1), and unknown primary (n = 1). Increased uptake in the primary lung mass, with an SUVmax of 3.0, 6.34, and 13.0, was noted in the hemangiopericytoma, sarcomatoid carcinoma and hemangioendothelioma cases, respectively. The mean SUVmax, mean fluorescence intensity, and percentage of stained cells were highest in hemangioendothelioma. Among 3 patients with lung metastases, the highest SUVmax, 9.5, was in the primary sarcoma patient. Conclusion: 68Ga-pentixafor selectively targets the in vivo whole-body disease burden of CXCR4 receptors. This approach thus holds promise for developing suitable radiotheranostics for lung cancers expressing these targets.

Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis.

The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmania donovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund's adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1×10(7) L. donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.

A T-Cell Epitope-Based Multi-Epitope Vaccine Designed Using Human HLA Specific T Cell Epitopes Induces a Near-Sterile Immunity against Experimental Visceral Leishmaniasis in Hamsters.

Visceral leishmaniasis is a neglected tropical disease affecting 12 million people annually. Even in the second decade of the 21st century, it has remained without an effective vaccine for human use. In the current study, we designed three multiepitope vaccine candidates by the selection of multiple IFN-γ inducing MHC-I and MHC-II binder T-cell specific epitopes from three previously identified antigen genes of Leishmania donovani from our lab by an immuno-informatic approach using IFNepitope, the Immune Epitope Database (IEDB) T cell epitope identification tools, NET-MHC-1, and NET MHC-2 webservers. We tested the protective potential of these three multiepitope proteins as a vaccine in a hamster model of visceral leishmaniasis. The immunization data revealed that the vaccine candidates induced a very high level of Th1 biased protective immune response in-vivo in a hamster model of experimental visceral leishmaniasis, with one of the candidates inducing a near-sterile immunity. The vaccinated animals displayed highly activated monocyte macrophages with the capability of clearing intracellular parasites due to increased respiratory burst. Additionally, these proteins induced activation of polyfunctional T cells secreting INF-γ, TNF-α, and IL-2 in an ex-vivo stimulation of human peripheral blood mononuclear cells, further supporting the protective nature of the designed candidates.

Human Immunodeficiency Virus-1 Subtype-C Genetically Diversify to Acquire Higher Replication Competence in Human Host with Comorbidities.

Unusual disease progression is observed in the HIV-1 infected patients who are either coinfected with Mycobacterium tuberculosis (Mtb) or concomitantly on intravenous drug use (IDU). The mechanism involved in the breakdown of host immune defense and the synergistic effect in both the conditions are still not well understood. In this study, we aimed to highlight the emergence of genetically diversified variants of virus in these two cohorts among HIV-1 subtype-C infected population from a Northern state of India. A cross-sectional study was performed on treatment-naïve HIV-1 subtype-C infected individuals constituting three different cohorts of HIV-1 monoinfected, HIV-1-M. tuberculosis (HIV-TB) coinfected, and HIV-1 infected individuals on substance abuse (HIV-IDU) for acquisition of genetic alterations in terms of frequency of drug resistance (DR) mutations in reverse transcriptase gene. The data reveal a significantly higher viral load, higher death rate, and higher frequency of major DR mutations in the genome of viral isolates from HIV-TB and HIV-IDU cohorts as compared with HIV monoinfected. Majority of the mutations found in the HIV-TB coinfected and HIV-IDU cohorts conferred high level of resistance to the first-line treatment regimen (Lamivudine with Tenofovir or zidovudine or Abacavir and Nevirapine or Efavirenz). Our findings support the hypothesis that the HIV-1 evolve while replicating in the host with Mtb coinfection or substance abuse, with the emergence and accumulation of genetically diversified quasi-species. Further studies are warranted to understand the association of such genetic variations with increased replication competence and faster rate of disease progression in such individuals.

Short Communication: Acquisition of Additional Nuclear Factor Kappa B Binding Sites in Long Terminal Repeat of Genetically Evolving HIV-1 Subtype C Viral Species in Host with Comorbidities.

HIV-1 causes millions of deaths around the world. Higher disease progression and mortality are seen in HIV positive individuals with comorbidities. Two of the most pertinent conditions are coinfection with Mycobacterium tuberculosis and Intravenous Drug abuse. The mechanisms involved, however, still remain unresolved. To elucidate the mechanisms involved, we evaluated the genetic alterations in terms of additional nuclear factor kappa B (NF-κB) sites in the long terminal repeat (LTR) of HIV-1 subtype-C isolates from infected human individuals from North India, supposedly acquired by the emerging viral quasi-species in the infected host in presence of these two comorbid conditions. Interestingly the results indicate higher number of NF-κB sites in the viral isolates from HIV-tuberculosis coinfected (n = 26, 16 isolates with 3 sites and 10 isolates with 2 sites) and intravenous drug users (n = 20, 13 isolates with 3 sites and 7 isolates with 2 sites) compared to the mono-infected hosts (n = 30, 10 isolates with 3 sites, 18 isolates with 2 sites, 2 isolates with 1 site). The biological relevance of these alterations in the NF-κB sites within the HIV-1 LTR with respect to viral replicative capacity and HIV disease progression needs to be studied further.

Evaluation of breast cancer stem cells in human primary breast carcinoma and their role in aggressive behavior of the disease.

BACKGROUND AND AIM: To delineate the underlying molecular mechanisms responsible for the intratumoral enrichment of breast cancer stem cells (BCSCs) in aggressive breast tumors, we evaluated the frequency and characteristics of BCSCs within the tumor tissue in primary human breast carcinomas. We assessed the expression profiles of various genes in cancer cells (CC) and stromal cells (SC) from these tumors to delineate the role played by the cellular niche in de novo origin or expansion of intra-tumoral cancer stem cells (CSC). METHOD: The study included primary tumor and adjacent normal breast tissue specimens from chemotherapy-naïve breast carcinoma patients. The BCSCs, identified as Lin-CD44+CD24- and aldehyde dehydrogenase 1 A1 positive, were enumerated. The flow-cytometrically sorted stromal, and CC were processed for gene expression profiling using a custom-designed polymerase chain reaction array of genes known to facilitate disease progression. RESULTS: The frequency of BCSCs within the tumor mass correlated significantly with histopathological and molecular grades of tumors, indicating a direct relationship of BCSC with the aggressive behavior of breast cancer. Further, a significantly increased expression of the genes associated with growth factors, cytokines and matricellular proteins in tumors were found in high BCSCs compared to Lo-BCSC tumors, suggesting the possible contribution of stromal and CC in an intratumoral expansion of CSCs. Similarly, a significant upregulation of genes associated with hypoxia and angiogenesis in Hi-BCSCs tumors further supported the role of a hypoxic environment. CONCLUSION: Overall, the findings suggest the molecular crosstalk between SC and CC potentially (directly or indirectly) contributes to the expansion of CSC. RELEVANCE FOR PATIENTS: The current study highlights the importance of CSC as a potential future predictive/prognostic marker for aggressive breast cancer. The present study predicts the potential risk stratification based on the frequency of BCSCs in primary breast tumors and existing prognostic factors.