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1.  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.
doi:10.4161/rna.23608
PMCID: PMC3672277  PMID: 23328630
telomerase; Tetrahymena; telomerase RNA; p65; La protein; LARP7; xRRM2; RNA conformation
2.  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.
doi:10.4049/jimmunol.1201138
PMCID: PMC3448847  PMID: 22933632
HSP65; asthma; dendritic cells; T cells
3.  Structural basis for telomerase RNA recognition and RNP assembly by the holoenzyme La family protein p65 
Molecular Cell  2012;47(1):16-26.
SUMMARY
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.
doi:10.1016/j.molcel.2012.05.018
PMCID: PMC3398246  PMID: 22705372
4.  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.
Background:
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.
Purpose:
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.
Results:
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
Conclusion:
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.
doi:10.4103/2278-330X.114108
PMCID: PMC3892536  PMID: 24455581
Association; body mass index; breast cancer
5.  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
6.  Prevalence of Latent and Active Tuberculosis among Dairy Farm Workers Exposed to Cattle Infected by Mycobacterium bovis 
Background
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.
Conclusions
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.
doi:10.1371/journal.pntd.0002177
PMCID: PMC3636137  PMID: 23638198
7.  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.
doi:10.1128/CVI.00275-12
PMCID: PMC3428397  PMID: 22761299
8.  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.
doi:10.1371/journal.ppat.1003077
PMCID: PMC3531513  PMID: 23300440
9.  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.
doi:10.1155/2012/361374
PMCID: PMC3503283  PMID: 23209912
10.  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.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2407-12-451
PMCID: PMC3517432  PMID: 23036052
NK cell; PstS-1; Ovarian cancer; BCG; Immunotherapy; Cetuximab
11.  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.
Background
Although currently available IGRA have been reported to be promising markers for TB infection, they cannot distinguish active tuberculosis (TB) from latent infection (LTBI).
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1371/journal.pone.0046041
PMCID: PMC3461005  PMID: 23029377
12.  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.
doi:10.1186/1556-276X-7-511
PMCID: PMC3475093  PMID: 22989217
Nanoparticles; Multiferroic; Dielectric constant; dc magnetization
14.  Correction: Characterisation of Bovine Leukocyte Ig-like Receptors 
PLoS ONE  2012;7(8):10.1371/annotation/cfb0e8b5-3815-46c1-997b-c6267ba4856b.
doi:10.1371/annotation/cfb0e8b5-3815-46c1-997b-c6267ba4856b
PMCID: PMC3414627
15.  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.
doi:10.1128/IAI.05724-11
PMCID: PMC3257930  PMID: 21844233
16.  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
17.  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.
doi:10.1371/journal.pone.0034433
PMCID: PMC3326050  PMID: 22514633
18.  Characterisation of Bovine Leukocyte Ig-like Receptors 
PLoS ONE  2012;7(4):e34291.
Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.
doi:10.1371/journal.pone.0034291
PMCID: PMC3317502  PMID: 22485161
19.  The Role of Alveolar Epithelial Cells in Initiating and Shaping Pulmonary Immune Responses: Communication between Innate and Adaptive Immune Systems 
PLoS ONE  2012;7(2):e32125.
Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.
doi:10.1371/journal.pone.0032125
PMCID: PMC3290547  PMID: 22393384
20.  Tetrahydrodipicolinate N-Succinyltransferase and Dihydrodipicolinate Synthase from Pseudomonas aeruginosa: Structure Analysis and Gene Deletion 
PLoS ONE  2012;7(2):e31133.
The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD) from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity.
doi:10.1371/journal.pone.0031133
PMCID: PMC3281039  PMID: 22359568
21.  Immunization with Recombinant HLA Classes I and II, HIV-1 gp140, and SIV p27 Elicits Protection against Heterologous SHIV Infection in Rhesus Macaques▿† 
Journal of Virology  2011;85(13):6442-6452.
Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID50) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.
doi:10.1128/JVI.00129-11
PMCID: PMC3126489  PMID: 21490092
22.  Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies Overcome Resistance of Ovarian Cancer Cells to Targeted Therapy and Natural Cytotoxicity 
The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.
doi:10.3390/ijms130912000
PMCID: PMC3472788  PMID: 23109896
ovarian cancer; NK cell; cetuximab; EGFR; PstS-1; antibody-dependent cellular cytotoxicity
24.  Congenital Anterior Urethral Diverticulum in Children: A Case Report and Review 
ISRN Surgery  2011;2011:120307.
Congenital anterior urethral diverticulum (CAUD) is an uncommon condition in children. We describe 2 patients of CAUD who presented with ventral penile swelling; in one, the site of swelling was just proximal to corona which is quite rare. The diagnosis was made on USG and MCU. Both patients had normal renal function. Open diverticulectomy and primary repair was done in both patients.
doi:10.5402/2011/120307
PMCID: PMC3195389  PMID: 22084741
25.  The M. tuberculosis Phosphate-Binding Lipoproteins PstS1 and PstS3 Induce Th1 and Th17 Responses That Are Not Associated with Protection against M. tuberculosis Infection 
The M. tuberculosis phosphate-binding transporter lipoproteins PstS1 and PstS3 were good immunogens inducing CD8+ T-cell activation and both Th1 and Th17 immunity in mice. However, this antigen-specific immunity, even when amplified by administration of the protein with the adjuvant LTK63 or by the DNA priming/protein boosting regimen, was not able to contain M. tuberculosis replication in the lungs of infected mice. The lack of protection might be ascribed with the scarce/absent capacity of PstS1/PstS3 antigens to modulate the IFN-γ response elicited by M. tuberculosis infection during which, however, PstS1-specific IL-17 secreting cells were generated in both unvaccinated and BCG-vaccinated mice. In spite of a lack of protection by PstS1/PstS3 immunizations, our results do show that PstS1 is able to induce IL-17 response upon M. tuberculosis infection which is of interest in the study of anti-M. tuberculosis immunity and as potential immunomodulator in combined vaccines.
doi:10.1155/2011/690328
PMCID: PMC3095447  PMID: 21603219

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