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1.  A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens 
Mediators of Inflammation  2015;2015:364758.
Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%—with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases.
PMCID: PMC4546975  PMID: 26347586
2.  Effect of Vaginal Immunization with HIVgp140 and HSP70 on HIV-1 Replication and Innate and T Cell Adaptive Immunity in Women 
Journal of Virology  2014;88(20):11648-11657.
The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4+ T cells compared with preimmunization CD4+ T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4+ T cells. Indeed, a significant inverse correlation between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to have an inverse correlation with the viral load (r = −0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at under registration no. NCT01285141.)
IMPORTANCE Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4+ T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4+ T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4+ T cells and the stimulation of CD4+ or CD8+ T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4+ and CD8+ T cell proliferative responses.
PMCID: PMC4178755  PMID: 25008917
3.  Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis 
PLoS ONE  2015;10(3):e0120682.
Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.
PMCID: PMC4370629  PMID: 25798914
4.  Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B 
BMC Biotechnology  2014;14:68.
Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic.
In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection.
Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.
PMCID: PMC4119940  PMID: 25033887
5.  Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis 
BioMed Research International  2014;2014:140829.
The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.
PMCID: PMC4119628  PMID: 25110654
6.  PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress 
The Biochemical journal  2013;451(3):375-388.
Vascular injury and chronic arterial diseases result in exposure of vascular smooth muscle cells (VSMCs) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with platelet-derived growth factor (PDGF)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and upregulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1, or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 led to a remarkable stabilization α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix and abrogated VSMC hyperproliferation and migration. Interestingly, treatment of cells with PDGF prevented protein damage and cell death due to exposure to the lipid peroxidation product, 4-hydroxynonenal. These results demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under conditions of high oxidative stress found to occur in vascular lesions.
PMCID: PMC4040966  PMID: 23421427
autophagy; atherosclerosis; restenosis; 4-hydroxynonenal; growth factor; spautin
7.  Breastfeeding Reduces Breast Cancer Risk: A Case-Control Study in North India 
Worldwide, breast cancer is the most common cancer among women. In India and other developing countries, breast carcinoma ranks second only to cervical carcinoma among women. Although studies have been done globally, to find the association between breastfeeding and breast cancer, very few studies in India document such a benefit.
A case–control study was done from August 2009 to July 2010 in the wards of General Surgery and Oncosurgery at Pt. B. D. Sharma PGIMS, Rohtak, Haryana, India. A total of 128 histopathologically confirmed new cases of breast cancer during the study period were taken as cases. Equal numbers of controls were selected by simple random sampling. Controls were matched for age with a range of ± 2 years. Subjects were interviewed using a pretested questionnaire after obtaining written informed consent. The categorical data were analyzed statistically using the Chi-square test and odds ratio with a 95% confidence interval. Continuous variables were analyzed using an independent t-test. All the analysis was done using SPSS, version 17.
The age group of the cases was 25-78 years, while that of the controls was 24-79 years. The proportions of cases (56.3%) and controls (63.3%) living in rural areas were more than those living in urban areas. A significant association of breast cancer cases was found with caste, age at marriage, age at the first pregnancy, number of live births, and lifetime duration of breastfeeding.
Breastfeeding has a significant role in reducing breast cancer, and so information, education, and communication activities for the promotion of breastfeeding and creating awareness about this fatal disease are the need of the hour.
PMCID: PMC4085934  PMID: 25013701
Breast cancer; breastfeeding; case–control
8.  xRRM 
RNA Biology  2013;10(3):353-359.
Genuine La and La-related proteins group 7 (LARP7) bind to the non-coding RNAs transcribed by RNA polymerase III (RNAPIII), which end in UUU-3′OH. The La motif and RRM1 of these proteins (the La module) cooperate to bind the UUU-3′OH, protecting the RNA from degradation, while other domains may be important for RNA folding or other functions. Among the RNAPIII transcripts is ciliate telomerase RNA (TER). p65, a member of the LARP7 family, is an integral Tetrahymena thermophila telomerase holoenzyme protein required for TER biogenesis and telomerase RNP assembly. p65, together with TER and telomerase reverse transcriptase (TERT), form the Tetrahymena telomerase RNP catalytic core. p65 has an N-terminal domain followed by a La module and a C-terminal domain, which binds to the TER stem 4. We recently showed that the p65 C-terminal domain harbors a cryptic, atypical RRM, which uses a unique mode of single- and double-strand RNA binding and is required for telomerase RNP catalytic core assembly. This domain, which we named xRRM, appears to be present in and unique to genuine La and LARP7 proteins. Here we review the structure of the xRRM, discuss how this domain could recognize diverse substrates of La and LARP7 proteins and discuss the functional implications of the xRRM as an RNP chaperone.
PMCID: PMC3672277  PMID: 23328630
telomerase; Tetrahymena; telomerase RNA; p65; La protein; LARP7; xRRM2; RNA conformation
9.  Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa 
ACS Chemical Biology  2013;8(11):2518-2527.
3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH.
PMCID: PMC3833349  PMID: 24015914
10.  Microbial Heat Shock Protein 65 Attenuates Airway Hyperresponsiveness and Inflammation by Modulating the Function of Dendritic Cells 
Heat shock proteins (HSPs), produced in response to stress are suppressive in disease models. We previously showed that Mycobacterium leprae HSP65 prevented development of airway hyperresponsiveness and inflammation in mice. Our goal here was to define the mechanism responsible for the suppressive effects of HSP. In one in vivo approach, BALB/c mice were sensitized to ovalbumin (OVA) followed by primary OVA challenges. Several weeks later, HSP65 was administered prior to a single, provocative secondary challenge. In a second in vivo approach, the secondary challenge was replaced by intratracheal instillation of allergen-pulsed bone marrow-derived dendritic cells (BMDCs). The in vitro effects of HSP65 on BMDCs were examined in co-culture experiments with CD4+ T cells. In vivo, HSP65 prevented development of airway hyperresponsiveness and inflammation. As well, Th1 cytokine levels in bronchoalveolar lavage (BAL) fluid were increased. In vitro, HSP65 induced notch receptor ligand Delta1 expression on BMDCs and HSP65-treated BMDCs skewed CD4+ T cells to Th1 cytokine production. Thus, HSP65-induced effects on allergen-induced airway hyperresponsiveness and inflammation were associated with increased Delta 1 expression on DCs, modulation of DC function, and CD4+ Th1 cytokine production.
PMCID: PMC3448847  PMID: 22933632
HSP65; asthma; dendritic cells; T cells
11.  Structural basis for telomerase RNA recognition and RNP assembly by the holoenzyme La family protein p65 
Molecular Cell  2012;47(1):16-26.
Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a novel combination of single- and double-strand RNA binding, which induces a 105° bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an α-helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.
PMCID: PMC3398246  PMID: 22705372
12.  Association between body mass index and risk of breast cancer among females of north India 
South Asian Journal of Cancer  2013;2(3):121-125.
Worldwide, breast cancer is most common cancer among women. In India and other developing countries, breast carcinoma ranks second only to cervical carcinoma among women. Although studies have been done globally, to find association between BMI and breast cancer, very few studies in India document any such association.
To find out the association between BMI and breast cancer.
Materials and Methods:
A Case-control study was done from August 2009 - July 2010 in the wards of General Surgery and Oncosurgery at Pt.B.D.Sharma, PGIMS Rohtak, Haryana. A total of 128 histopathologically confirmed new cases of breast cancer during the study period were taken as cases. Equal number of controls was selected by simple random sampling. Controls were matched for age with range of ±2 years. Subjects were interviewed using a pretested questionnaire after obtaining written informed consent. Data were analyzed by applying appropriate statistical tests using SPSS version 17.
Age group of the cases was 25 - 78 years, while that of the controls was 24 - 79 years. Proportion of cases and controls living in rural areas were more than those living in urban areas. A significant association of breast cancer cases was found with high BMI and high fat intake
Obesity and high fat intake are the significant risk factors, which are modifiable. So women should be encouraged to take care of all these factors. Maximum cases presented in late stages so public awareness of this fatal disease must be developed.
PMCID: PMC3892536  PMID: 24455581
Association; body mass index; breast cancer
13.  Infected Mature Teratoma of Mesentery in a Child 
Mesenteric teratomas are extremely rare in children. We report a case of 5-year-old girl with abdominal mass and fever. At operation, a multicystic mass with variable consistency found within the leaves of the mesentery of jejunum with pus in it. Histopathology examination showed mature infected teratoma of the mesentery.
PMCID: PMC3754403  PMID: 24040596
Mature teratoma; Mesentery
14.  Prevalence of Latent and Active Tuberculosis among Dairy Farm Workers Exposed to Cattle Infected by Mycobacterium bovis 
Human tuberculosis caused by M. bovis is a zoonosis presently considered sporadic in developed countries, but remains a poorly studied problem in low and middle resource countries. The disease in humans is mainly attributed to unpasteurized dairy products consumption. However, transmission due to exposure of humans to infected animals has been also recognized. The prevalence of tuberculosis infection and associated risk factors have been insufficiently characterized among dairy farm workers (DFW) exposed in settings with poor control of bovine tuberculosis.
Methodology/Principal Findings
Tuberculin skin test (TST) and Interferon-gamma release assay (IGRA) were administered to 311 dairy farm and abattoir workers and their household contacts linked to a dairy production and livestock facility in Mexico. Sputa of individuals with respiratory symptoms and samples from routine cattle necropsies were cultured for M. bovis and resulting spoligotypes were compared. The overall prevalence of latent tuberculosis infection (LTBI) was 76.2% (95% CI, 71.4–80.9%) by TST and 58.5% (95% CI, 53.0–64.0%) by IGRA. Occupational exposure was associated to TST (OR 2.72; 95% CI, 1.31–5.64) and IGRA (OR 2.38; 95% CI, 1.31–4.30) adjusting for relevant variables. Two subjects were diagnosed with pulmonary tuberculosis, both caused by M. bovis. In one case, the spoligotype was identical to a strain isolated from bovines.
We documented a high prevalence of latent and pulmonary TB among workers exposed to cattle infected with M. bovis, and increased risk among those occupationally exposed in non-ventilated spaces. Interspecies transmission is frequent and represents an occupational hazard in this setting.
Author Summary
Mycobacterium tuberculosis complex causes tuberculosis in humans and other mammals. The complex includes M. bovis, which causes bovine tuberculosis. The main route of transmission of this zoonosis is the consumption of unpasteurized dairy products. Nevertheless, exposure to infected cattle while performing husbandry and farm activities may cause disease as well. In this study we were able to demonstrate: 1) A high prevalence of tuberculosis asymptomatic infection (latent tuberculosis) among workers exposed to infected cattle; 2) A higher probability of infection among individuals who are occupationally exposed in closed spaces; and 3) Cattle to human transmission confirmed by molecular methods (spoligotyping). We conclude that occupational exposure is frequent, and therefore strict prevention and control measures are required in these settings.
PMCID: PMC3636137  PMID: 23638198
15.  Goats Primed with Mycobacterium bovis BCG and Boosted with a Recombinant Adenovirus Expressing Ag85A Show Enhanced Protection against Tuberculosis 
This is the first efficacy study using the experimental goat model, a natural host of tuberculosis (TB), to evaluate the efficacy of heterologous Mycobacterium bovis bacillus Calmette-Guérin (BCG) prime followed by boosting with a replication-deficient adenovirus expressing the antigen Ag85A (AdAg85A). Three experimental groups of 11 goat kids each were used: BCG vaccinated, BCG vaccinated and AdAg85A boosted, and nonvaccinated. Twenty-two goat kids were vaccinated with ∼5 × 105 CFU of BCG (week 0), and 11 of them were boosted at week 8 with 109 PFU of AdAg85A. At week 14, all goats were challenged by the endobronchial route with ∼1.5 × 103 CFU of Mycobacterium caprae. The animals were euthanized at week 28. Cellular and humoral immunity induced by vaccination and M. caprae infection was measured throughout the study. After challenge BCG-AdAg85A-vaccinated animals exhibited reduced pathology compared to BCG-vaccinated animals in lungs and in pulmonary lymph nodes. There were significant reductions in bacterial load in both groups of vaccinated goats, but the reduction was more pronounced in prime-boosted animals. Antigen-specific gamma interferon (IFN-γ) and humoral responses were identified as prognostic biomarkers of vaccination outcome depending on their correlation with pathological and bacteriological results. As far as we know, this is the first report using multidetector computed tomography (MDCT) to measure vaccine efficacy against pulmonary TB in an animal model. The use in vaccine trials of animals that are natural hosts of TB may improve research into human TB vaccines.
PMCID: PMC3428397  PMID: 22761299
16.  Global Gene Transcriptome Analysis in Vaccinated Cattle Revealed a Dominant Role of IL-22 for Protection against Bovine Tuberculosis 
PLoS Pathogens  2012;8(12):e1003077.
Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.
Author Summary
Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of a complex group of pathogens that also includes the bacterium causing human tuberculosis, M. tuberculosis. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. In addition to the relevance for the veterinary field, cattle can also serve as useful pre-clinical model for the development of human vaccines. Identification of biomarkers in tuberculosis research remains elusive and the goal of this study was to identify host markers that allow the prediction of vaccine success. In this experiment, the outcome of vaccination was measured against relevant endpoints based on disease severity. Our results, generated using the state-of-the-art methodology of transcriptome sequencing defined a dominant role of the cytokine IL-22 for predicting vaccine success. We therefore identified a predictive biomarker that can be used in future experiments and also highlighted the role of T cells producing this cytokine in protective immunity.
PMCID: PMC3531513  PMID: 23300440
17.  Lipid Droplets and Mycobacterium leprae Infection 
Journal of Pathogens  2012;2012:361374.
Leprosy is a chronic infectious disease and is a major source of morbidity in developing countries. Leprosy is caused by the obligate intracellular bacterium Mycobacterium leprae, which infects as primary target Schwann cells. Lepromatous leprosy exhibits multiple lesions of the skin, eyes, nerves, and lymph nodes. The sites of infection are characterized by the presence of foamy macrophages, fully packed with lipid droplets (LDs), which are induced by M. leprae. In the last years, it has become evident that M. tuberculosis imports lipids from foamy macrophages and is dependent on fatty acids for growth in infected macrophages. M. leprae seems to have similar mechanisms for scavenging lipids from the host. But due to the inability to culture M. leprae on laboratory media, research progresses only slowly. However, in the last years, substantial progress has been made in the field of lipid metabolism in M. leprae. Herein, we will present and summarize the lipid droplets formation and the metabolism of lipids during M. leprae infection.
PMCID: PMC3503283  PMID: 23209912
18.  Monocytes and the 38kDa-antigen of mycobacterium tuberculosis modulate natural killer cell activity and their cytolysis directed against ovarian cancer cell lines 
BMC Cancer  2012;12:451.
Despite strong efforts to improve clinical outcome of ovarian cancer patients by conventional and targeted immuno-based therapies, the prognosis of advanced ovarian cancer is still poor. Natural killer (NK) cells mediate antibody-dependent cellular cytotoxicity (ADCC), release immunostimulatory cytokines and thus function as potent anti-tumour effector cells. However, tumour cells developed mechanisms to escape from an effective immune response. So highly immunogenic substances, like the 38 kDa-preparation of M. tuberculosis, PstS-1, are explored for their potential to enhance cancer-targeted immune responses. In this study we examined the modulation of different NK cell functions by accessory monocytes and PstS-1. We focussed on NK cell activation as well as natural and antibody-dependent cellular cytotoxicity directed against epidermal-growth-factor-receptor (EGFR)-positive ovarian cancer cell lines.
Activation, cytokine release and cytotoxicity of NK cells stimulated by monocytes and PstS-1 were determined by FACS-analysis, ELISA, Bioplex assay and quantitative polymerase-chain reaction (qPCR). Transwell assays were used to discriminate cell-cell contact-dependent from contact-independent mechanisms. Five ovarian cancer cell lines (A2780, IGROV-1, OVCAR-3, OVCAR-4 and SKOV-3) with different EGFR-expression were used as target cells for natural and antibody-dependent cellular cytotoxicity assays. Cetuximab (anti-EGFR-antibody) was used for ADCC studies.
Our data show that monocytes effectively enhance activation as well natural and antibody-dependent cytolytic activity of NK cells. PstS-1 directly stimulated monocytes and further activated monocyte-NK-co-cultures. However, PstS-1 did not directly influence purified NK cells and did also not affect natural and antibody-dependent cellular cytotoxicity directed against EGFR-positive ovarian cancer cells, even in presence of monocytes. Direct cell-cell contact between NK cells and monocytes was required for NK activation, while released cytokines seemed to play a minor role.
Our data suggest that monocytes enhance natural and antibody-dependent cytotoxic activity of NK cells in a cell-cell contact dependent manner. The TLR-agonist PstS-1 provides additional monocyte activation and induces NK activation markers, while NK cytotoxicity remains unaffected. We conclude that monocytes provide accessory function for ADCC exerted by NK during antibody-based cancer immunotherapy directed against EGFR-positive ovarian cancer cells.
PMCID: PMC3517432  PMID: 23036052
NK cell; PstS-1; Ovarian cancer; BCG; Immunotherapy; Cetuximab
19.  Potential Role of M. tuberculosis Specific IFN-γ and IL-2 ELISPOT Assays in Discriminating Children with Active or Latent Tuberculosis 
PLoS ONE  2012;7(9):e46041.
Although currently available IGRA have been reported to be promising markers for TB infection, they cannot distinguish active tuberculosis (TB) from latent infection (LTBI).
Children with LTBI, active TB disease or uninfected were prospectively evaluated by an in-house ELISPOT assay in order to investigate possible immunological markers for a differential diagnosis between LTBI and active TB.
Children at risk for TB infection prospectively enrolled in our infectious disease unit were evaluated by in-house IFN-γ and IL-2 based ELISPOT assays using a panel of Mycobacterium tuberculosis antigens.
Twenty-nine children were classified as uninfected, 21 as LTBI and 25 as active TB cases (including 5 definite and 20 probable cases). Significantly higher IFN-γ ELISPOT responses were observed in infected vs. uninfected children for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p = 0.003), and AlaDH (p = 0.001), while differences were not significant considering Ag85B (p = 0.063), PstS1 (p = 0.512), and HspX (16 kDa) (p = 0.139). IL-2 ELISPOT assay responses were different for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p<0.0001), HspX (16 kDa) (p<0.0001), PstS1 (p<0.0001) and AlaDH (p = 0.001); but not for Ag85B (p = 0.063). Comparing results between children with LTBI and those with TB disease differences were significant for IFN-γ ELISPOT only for AlaDH antigen (p = 0.021) and for IL-2 ELISPOT assay for AlaDH (p<0.0001) and TB 10.3 antigen (p = 0.043). ROC analyses demonstrated sensitivity of 100% and specificity of 81% of AlaDH-IL-2 ELISPOT assay in discriminating between latent and active TB using a cut off of 12.5 SCF per million PBMCs.
Our data suggest that IL-2 based ELISPOT with AlaDH antigen may be of help in discriminating children with active from those with latent TB.
PMCID: PMC3461005  PMID: 23029377
20.  Ferroelectric and magnetic properties of Nd-doped Bi4 − xFeTi3O12 nanoparticles prepared through the egg-white method 
Nanoscale Research Letters  2012;7(1):511.
Multiferroic behavior of Bi4 − xNdxFeTi3O12 (0.0 ≤ × ≤ 0.25, × = 0.05) ceramic nanoparticles prepared through the egg-white method was investigated. The dielectric properties of the samples show normal behavior and are explained in the light of space charge polarization. Room temperature polarization-electric field (P-E) curves show that the samples are not saturated with maximum remanence polarization, Pr = 0.110 μC/cm2, and a relatively low coercive field, Ec = of 7.918 kV/cm, at an applied field of 1 kV/cm was observed for 5% Nd doping. The room temperature M-H hysteresis curve shows that the samples exhibit intrinsic antiferromagnetism with a weak ferromagnetism. These properties entitle the grown nanoparticles of BNFT as one of the few multiferroic materials that exhibit decent magnetization and electric polarization.
PMCID: PMC3475093  PMID: 22989217
Nanoparticles; Multiferroic; Dielectric constant; dc magnetization
22.  Correction: Characterisation of Bovine Leukocyte Ig-like Receptors 
PLoS ONE  2012;7(8):10.1371/annotation/cfb0e8b5-3815-46c1-997b-c6267ba4856b.
PMCID: PMC3414627
23.  Impact of Toll-Like Receptor 2 Deficiency on Immune Responses to Mycobacterial Antigens▿ † 
Infection and Immunity  2011;79(11):4649-4656.
In the present study, we addressed the question of whether Toll-like receptor 2 (TLR2)-mediated innate immunity can contribute to the development of acquired immune responses. We immunized TLR2−/− and wild-type (WT) mice three times subcutaneously with the mycobacterial antigen (Ag19kDa) (a TLR2 ligand) or Ag85A (not a TLR2 ligand). One week after the last immunization, sera and spleens were collected. To evaluate cellular responses, we measured gamma interferon (IFN-γ) after in vitro restimulation of spleen cells with antigen alone or antigen-pulsed bone marrow-derived macrophages (BMMAg) or pulmonary macrophages (PuMAg). Antibody responses were comparable in the two mouse strains, but we observed differences in the cellular responses. Recall responses to Ag85A were similar in the two strains, but responses to Ag19kDa given alone or presented by BMM or PuM were lower in TLR2−/− than in WT mice. The largest differences in cellular responses were observed when Ag19kDa was presented by PuM. To understand this, we analyzed phenotypic and functional differences between BMM and PuM upon stimulation with various ligands. Generally, PuM had a lower response to the TLR2 ligand Pam3Cys-Ser-(Lys)4 trihydrochloride and to anti-CD40 than BMM, as measured by cytokine secretion and upregulation of costimulatory molecules. This might provide a partial explanation for the lower capacity of PuM when pulsed with Ag19kDa, also a TLR2 ligand. Altogether, our results revealed weaknesses in the T cell and antigen-presenting cell (APC) compartments of the Ag19kDa-immunized TLR2−/− mice but indicated that specific immune responses could be generated in the absence of TLR2 regardless of the characteristics of the antigen used.
PMCID: PMC3257930  PMID: 21844233
24.  Heterotopic Pancreas Leading to Ileo-Ileal Intussusception 
A heterotopic pancreas as the lead point of ileo-ileal intussusception is extremely rare. A 12-year-old previously healthy boy, presented to the emergency room with the complaint of severe abdominal pain for the last 6-8 hours. A preoperative diagnosis of ileo-ileal intussusception was made on ultrasound and an emergency exploratory laparotomy was done. At laparotomy an ileo-ileal intussusception was found and a polyp noted as a lead point. On histopathology this polyp was found to be heterotopic pancreas.
PMCID: PMC3418041  PMID: 22953306
Intussusception;  Heterotopic pancreas;  Lead point
25.  The Role of Innate APOBEC3G and Adaptive AID Immune Responses in HLA-HIV/SIV Immunized SHIV Infected Macaques 
PLoS ONE  2012;7(4):e34433.
The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread.
PMCID: PMC3326050  PMID: 22514633

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