E-cadherin Expression in Premalignant Lesions, Premalignant Conditions, Oral Squamous Cell Carcinoma, and Normal Mucosa: An Immunohistochemical Study.

Background Oral squamous cell carcinoma (OSCC) is a multi-step process. Epithelial-mesenchymal transition (EMT) is an important step in the progression of OSCC. One of the components that influence EMT is E-cadherin. The aim of this study was to determine the expression of E-cadherin in oral submucous fibrosis (OSMF), various grades of epithelial dysplasia, OSCC, and to compare it with the expression in the normal mucosa. Material and methods E-cadherin immunohistochemical detection was done using a monoclonal antibody of clone EP-6TM and the PolyExcel HRP/DAB chromogen detection system. A total of 100 samples, were divided into four groups, which included epithelial dysplasia (group 2) (30 cases), oral submucous fibrosis (group 3) (OSMF-30 cases), and oral squamous cell carcinoma (group 4) (OSCC-30 cases), which was compared with normal mucosa (group 1) (10 cases). The positive control used for E-cadherin was ductal breast carcinoma. Results All the cases of normal mucosa, epithelial dysplasia, and OSMF showed positivity for E-cadherin expression. In OSCC, 97% of cases expressed E-cadherin except one case. Out of 30 cases of epithelial dysplasia, 53% of mild epithelial dysplasia had a moderate intensity of expression and 75% had a mild intensity of E-cadherin expression. In moderately differentiated OSCC, 82% of cases showed mild intensity. Tissue localization of the E-cadherin stain in the basal layer decreased from normal mucosa to grades of epithelial dysplasia and OSCC. The pattern of E-cadherin staining in all the cases of group I, group II, and group III was membranous. In 97% of OSCC cases, both membranous and cytoplasmic staining were seen. Conclusion E-cadherin expression was reduced in increasing grades of epithelial dysplasia, OSCC, and OSMF compared to that of normal mucosa. E-cadherin expression is reduced as the lesions progress to malignancy. Hence, E-cadherin can be considered a surrogate marker of malignancy.

Unique cellular immune signatures of multisystem inflammatory syndrome in children.

The clinical presentation of MIS-C overlaps with other infectious/non-infectious diseases such as acute COVID-19, Kawasaki disease, acute dengue, enteric fever, and systemic lupus erythematosus. We examined the ex-vivo cellular parameters with the aim of distinguishing MIS-C from other syndromes with overlapping clinical presentations. MIS-C children differed from children with non-MIS-C conditions by having increased numbers of naïve CD8+ T cells, naïve, immature and atypical memory B cells and diminished numbers of transitional memory, stem cell memory, central and effector memory CD4+ and CD8+ T cells, classical, activated memory B and plasma cells and monocyte (intermediate and non-classical) and dendritic cell (plasmacytoid and myeloid) subsets. All of the above alterations were significantly reversed at 6-9 months post-recovery in MIS-C. Thus, MIS-C is characterized by a distinct cellular signature that distinguishes it from other syndromes with overlapping clinical presentations. Trial Registration: ClinicalTrials.gov clinicaltrial.gov. No: NCT04844242.

CRISPR-Cas system and its use in the diagnosis of infectious diseases.

Rapid and accurate diagnostic methods for detecting pathogens are needed for effective management and treatment of infectious diseases. The conventional pathogen detection approach based on culture is considered the gold standard method, but needs several days to corroborate its results. Using nucleic acids from pathogens as detection targets has a considerable advantage in overcoming these time-consuming issues. The development of several molecular techniques has started to change the landscape of infectious disease diagnosis. However, these require expensive reagents, equipment, and sophisticated infrastructure, as well as highly trained workers. In this context, it is necessary to identify new diagnostic strategies to overcome these issues. Recently, CRISPR/Cas based diagnosis has revolutionized the area of molecular diagnostics of pathogenic diseases. In this review, we have discussed the different classes of CRISPR-Cas systems and their functions, and then focused on recent advances in CRISPR-based diagnosis technologies and the perspective of using this as a potential biosensing platform to detect infectious disease.

Mycobacterium tuberculosis Lineages Associated with Mutations and Drug Resistance in Isolates from India.

Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. In this study, we performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand the genomic diversity. A retrospective collection of 498 M. tuberculosis isolates submitted to the National Institute for Research in Tuberculosis for phenotypic susceptibility testing between 2014 to 2016 were sequenced. Genotypic resistance prediction was performed using known resistance-conferring determinants. Genotypic and phenotypic results for 12 antituberculosis drugs were compared, and sequence data were explored to characterize lineages and their association with drug resistance. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction. IMPORTANCE Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. We performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand genomic diversity. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction.

Antibody responses to the BBV152 vaccine in individuals previously infected with SARS-CoV-2: A pilot study.

Background & objectives: Vaccination against SARS-CoV-2 is a recommendation from the World Health Organization as the foremost preference in the current situation to control the COVID-19 pandemic. BBV152 is one of the approved vaccines against SARS-CoV-2 in India. In this study, we determined SARS-CoV-2-specific antibody levels at day 0 (baseline, before vaccination), day 28 ± 2 post-first dose (month 1) and day 56 ± 2 post-first dose (month 2) of BBV152 whole-virion-inactivated SARS-CoV-2 recipients, and compared the antibody responses of individuals with confirmed pre-vaccination SARS-CoV-2 infection to those individuals without prior evidence of infection. Methods: Blood samples were collected from 114 healthcare professionals and frontline workers who received BBV152 vaccine from February to May & June 2021. Prior infection with SARS-CoV-2 was determined at baseline. Serum samples were used to estimate SARS-CoV-2 nucleoprotein-specific IgG [IgG (N)], spike protein-specific IgG [IgG (S)] and neutralizing antibodies (NAb). Results: Participants with previous SARS-CoV-2 infection after a single vaccine dose elicited IgG (N) and IgG (S) antibody levels along with NAb binding inhibition responses levels were similar to infection-naïve vaccinated participants who had taken two doses of vaccine. Interpretation & conclusions: Our preliminary data suggested that a single dose of BBV152-induced humoral immunity in previously infected individuals was equivalent to two doses of the vaccine in infection-naïve individuals. However, these findings need to be confirmed with large sized cohort studies.

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube(MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

Evaluating and comparing the morphological and histopathological changes induced by erbium:yttrium-aluminum-garnet laser and diamond bur on enamel, dentin and pulp tissue.

AIM: Lasers are used for different types of dental treatments. Using the erbium:yttrium-aluminum-garnet (Er:YAG) laser to remove dental hard tissue is simple, advantageous and influences the type of cavity preparation, whether conventional or conservative in nature. The aim of the present study was to evaluate and compare the morphological and histopathological changes in the enamel, dentin and pulp tissue of the teeth treated by Er:YAG laser and conventional burs. METHODS: A conventional class I cavity was prepared in orthodontic patients by laser and bur. The teeth were extracted and analyzed for morphological changes using a scanning electron microscope, ground sections and histopathological changes under a light microscope. RESULTS: The time with laser was longer than the conventional methods. The lased cavity showed irregular appearance with absence of smear layer which is suitable for the resin restoration. The ground section and the histopathological study showed no differences between the groups. CONCLUSION: The Er:YAG laser is effective in the removal of dental hard tissue without damaging the pulp when coupled with ideal energy output. It is widely used in different dental fields. It needs time to be accepted by dentist and patients and further studies are required to explore its advantages.

Enhancing antimycobacterial activity of isoniazid and rifampicin incorporated norbornene nanoparticles.

Background: Tuberculosis (TB) has been identified in skeletons over 6000 years old and still remains as the most prevalent infectious disease in the world; thus, there is a need for development of new drugs or tuning of old drugs. Nanotechnology, an advanced technology, plays a vital role in research for the diagnosis and treatment of TB, thus preventing adverse effects and drug resistance. The objective of this study was to enhance the antimycobacterial activity of isoniazid- (INH) and rifampicin (RIF)-incorporated norbornene (NOR) nanoparticles in comparison with plain INH and RIF without nanoparticles. Methods: Norbornene-polyethylene glycol - Isoniazid copolymer (NOR-PEG-INH) and norbornene polyethylene rifampicin Co polymer (NOR-PEG-RIF) were used for this study. The percentage of INH and RIF in NOR nanoparticles was 35% and 74%, respectively. Mycobacterium growth indicator tube containing Middlebrook 7H9 broth, the liquid medium, was used to analyze in vitro activity of the NOR-based drug and the plain drug. Minimum inhibitory concentration (MIC) of the drugs was determined from H37Rv control strain of mycobacterium TB (MTB). Results: The dosage of INH and RIF is minimal in the combination form with the NOR nanoparticles compared to the plain INH and RIF. The results indicate that the minimum concentration of NOR-PEG-INH and NOR-PEG-RIF required inhibiting H37Rv strain of MTB was 0.05 µg/ml and 0.5 µg/ml, respectively. The results were similar to plain INH and RIF MIC. Conclusion: Low dosage of INH and RIF along with NOR nanocarrier has similar activity to that of INH and RIF; thus this is expected to reduce adverse effects and NOR did not alter the functional activity of INH and RIF, thus becoming eligible for the newer drug carrier in TB treatment.

Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer.

Oestrogen controls Foxp3 expression in regulatory T cells (Treg cells) via a mechanism thought to involve oestrogen receptor alpha (ERα), but the molecular basis and functional impact of ERα signalling in Treg cells remain unclear. We report that ERα ligand oestradiol (E2) is significantly increased in human cervical cancer (CxCa) tissues and tumour-infiltrating Treg cells (CD4+CD25hiCD127low), whereas blocking ERα with the antagonist ICI 182,780 abolishes FOXP3 expression and impairs the function of CxCa infiltrating Treg cells. Using a novel approach of co-immunoprecipitation with antibodies to E2 for capture, we identified binding of E2:ERα complexes to FOXP3 protein in CxCa-derived Treg cells. Chromatin immunoprecipitation analyses of male blood Treg cells revealed ERα occupancy at the FOXP3 promoter and conserved non-coding DNA elements 2 and 3. Accordingly, computational analyses of the enriched regions uncovered eight putative oestrogen response elements predicted to form a loop that can activate the FOXP3 promoter. Together, these data suggest that E2-mediated ERα signalling is critical for the sustenance of FOXP3 expression and Treg cell function in human CxCa via direct interaction of ERα with FOXP3 promoter. Overall, our work gives a molecular insight into ERα signalling and highlights a fundamental role of E2 in controlling human Treg cell physiology.

A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis.

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.

Spoligotyping of Mycobacterium tuberculosis isolates at a tertiary care hospital in India.

OBJECTIVE: Spoligotyping is a valuable genotyping tool to study the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M. tb). The aim of this study was to analyse different spoligotype patterns of M. tb strains isolated from patients with tuberculosis from different parts of India. MATERIALS AND METHODS: A total of 163 M. tb isolates were spoligotyped between January 2014 and January 2015. About 47% (n = 77) were from patients with extrapulmonary tuberculosis; of these, 10 were MDR, and seven were Pre-XDR. Of the 86 M. tb isolates from patients with pulmonary tuberculosis, 25 were MDR, and 25 were Pre-XDR. RESULTS: We found 61 spoligo patterns, 128 clusters in the spoligotype data base (spoldb4 data base) with spoligo international type (SIT) number and 35 true unique isolates. The most pre-dominant spoligotype was EAI lineage (56), followed by Beijing (28), CAS (20), T(9), U(7), X(3), H(3), BOVIS_1 BCG(1) and LAM(1). CONCLUSION: Although our study identified EAI, CAS and Beijing strain lineages as pre-dominant, we also found a large number of orphan strains (20%) in our study. Beijing strains were more significantly associated with MDR TB than CAS and EAI lineages. Further studies on large sample sizes would help to clearly describe the epidemiology of M. tb in India.

Gastrointestinal Stromal Tumor Mimicking as Ovarian Tumor in Gynaecologic Oncology.

To report the clinical presentation and outcomes of a series of patients who presented with abdominal/pelvic mass or pelvic pain and were diagnosed with a gastrointestinal stromal tumor (GIST). Retrospective data were collected of all patients who presented with an abdominal/pelvic mass or pelvic pain between January 2010 and July 2015 and who were ultimately diagnosed with a GIST. The patients' medical records were reviewed. A literature review was also conducted. The event free survival and overall survival was calculated for all patients using Kaplan Meier curve (SPSS19-SPSS Inc. USA). A total ten patients were identified with GIST during the study period. Eight of ten patients had a tumor in the small intestine, one in sigmoid colon and one in base of small bowel mesentry. The mean tumor size was 13.9 cm (range, 3.9 to 24 cm). A complete resection was achieved in all 10 patients. No patient had distance metastasis. There were no intraoperative complications. One patient developed postoperative intestinal fistula and was managed conservatively. All patients were treated with imatinib after surgery. The mean follow-up time was 18 months (range, 2 to 47 months). The seven of the 10 patients (70 %) with no evidence of disease, two (20 %) lost follow up and one patient developed recurrence during follow up period and was started on sunitinib and patient died during follow up period because of disease. Gastrointestinal stromal tumors should be considered in the differential diagnosis of patients presenting with an abdominal/pelvic mass or pelvic pain in Gynaecologic oncology department. In such unusual circumstances the complete resection and appropriate adjuvant treatment results in complete durable remission.

Management of Ovarian Cancer In Younger Women.

BACKGROUND: Ovarian Cancer is a broad spectrum of diseases comprising several subtypes. Three major categories in younger women are germ cell tumor, sex cord stromal tumor and epithelial ovarian neoplasia. OBJECTIVE: literature search was for an update on management of ovarian cancer in young women. CONTEXT: Germ cell tumor is suspected in young girls presenting with solid ovarian neoplasm as abdominal mass, discomfort, dyspnea or pain abdomen. Preoperative evaluation should include thorough clinical examination with biochemical profile tumor markers and imaging techniques. When prepubertal girls present with precocious puberty, clitoromegaly, development of secondary sexual character, one should suspect juvenile granulosa theca cell tumor. Often serum beta inhibin is elevated in these cases. Young women are not immune to other tumors. Surgery should be fertility sparing, salphingo -oopherectomy, omentectomy, peritoneal cytology, retro peritoneal lymphadenectomy whenever indicated. Except Stage I A puredysgerminoma, Stage IA grade 1 immature teratoma, Stage IA /B grade 1 epithelial ovarian carcinoma, all other histopathological types irrespective of the stage of the disease require adjuvant chemotherapy. CONCLUSION: Girls, young women can have ovarian cancer conservative therapy. However treatment needs to be individualized. Except stage IA disease all other patient require adjuvant chemotherapy apart from fertility sparing surgery.

Columnar-integrated aerosol optical properties and classification of different aerosol types over the semi-arid region, Anantapur, Andhra Pradesh.

This study presents a characterization of aerosol columnar properties measured at a semi-arid station Anantapur in the southern part of India during the period from October 2012 to September 2013. Aerosol optical depth (AOD) and Angstrom exponent (α) have been retrieved from Microtops II Sunphotometer over the observation site. The results show that a pronounced spectral and monthly variability in the optical properties of aerosols is mainly due to anthropogenic sources. The results show that the spectral curvature can effectively be used as a tool for aerosol type discrimination, since the fine-mode aerosols exhibit negative curvature, while the coarse-mode particles are positive. The classification of aerosols is also proposed by using the values of AOD at 500 nm and Angstrom exponent values (α(380-870)) by applying threshold values obtained from the frequency distribution of AOD. The results of the analysis were identified by four individual components (anthropogenic/biomass burning, coarse/dust, coarse/marine, clean continental) of different origin and compositions. The most frequent situations observed over the site are that due to the anthropogenic/biomass burning situations which account for about 45.37%, followed by coarse/dust (43.64%), clean continental (7.2%) and coarse/marine (3.82%) during summer. The identification of the aerosol source type and the modification processes are analyzed by using the Gobbi et al. (2007) classification scheme based on the measured scattering properties (α, dα) derived from the Microtops II Sunphotometer.

Immunohistochemical study of p53 and proliferating cell nuclear antigen expression in odontogenic keratocyst and periapical cyst.

INTRODUCTION: p53 protein is a product of p53 gene, which is now classified as a tumor suppressor gene. The gene is a frequent target for mutation, being seen as a common step in the pathogenesis of many human cancers. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and plays a critical role in initiation of cell proliferation. AIM: The aim of this study is to assess and compare the expression of p53 and PCNA in lining epithelium of odontogenic keratocyst (OKC) and periapical cyst (PA). MATERIALS AND METHODS: A total of 20 cases comprising 10 OKC and 10 PA were included in retrospective study. Three paraffin section of 4 µm were cut, one was used for routine hematoxylin and eosin stain, while the other two were used for immunohistochemistry. Statistical analysis was performed using Chi-square test. RESULTS: The level of staining and intensity were assessed in all these cases. OKC showed PCNA expression in all cases (100%), whereas in perapical cyst only 60% of cases exhibited PCNA staining. (1) OKC showed p53 expression in 6 cases (60%) whereas in PA only 10% of the cases exhibited p53 staining. Chi-square test showed PCNA staining intensity was more significant than p53 in OKC. (2) The staining intensity of PA using p53, PCNA revealed that PCNA stating intensity was more significant than p53. CONCLUSION: OKC shows significant proliferative activity than PA using PCNA and p53. PCNA staining was more intense when compared with p53 in both OKC and PA.

A study of sister chromatid exchange in patients with dental amalgam restorations.

STUDY BACKGROUND: Dental amalgam is still widely used as a restorative material in developing countries due to its low cost and ease of manipulation. The health risks associated with the components of this restorative material has always been a matter of concern. Our study was designed to address this question regarding dental amalgam. OBJECTIVE: To study sister chromatid exchange (SCE) as an indicator of systemic genotoxicity, due to the exposure from the components of amalgam restorations during its placement and chronic use. MATERIALS AND METHODS: Systemic genotoxicity in subjects exposed to amalgam during its placement (Group II; n=5) and subjects with chronic exposure to amalgam (Group III; n=5) were compared with controls (Group I; n=5) by SCE assay in cultured peripheral blood lymphocytes. RESULT: Subjects exposed to amalgam during its placement and subjects having chronic exposure to amalgam showed an increase in the frequency of SCE, but the change was not statistically significant (P=0.84, P=0.123 respectively). CONCLUSION: Systemic genotoxicity was not observed due to the components of amalgam restorations released during its placement and chronic use. The findings of this study can be considered as preliminary information on the systemic toxicity due to the components of amalgam restorations.

Risk strata-based therapy and outcome in stage Ib-IIa carcinoma cervix: single-centre ten-year experience.

AIM: To review the outcome of stage (Ib, IIa), cervical cancer patients were primarily treated with radical hysterectomy and risk-based postoperative therapy. MATERIAL AND METHODS: Between January 2001 and December 2011, 601 cases underwent surgery followed by tailored therapy. Patients were classified into low risk (pelvic lymph node negative, tumour less than 4 cm, no evidence of lympho-vascular invasion, less than one-third of thickness of surgical stoma involved), intermediate risk (positive lympho-vascular space invasion, tumour size more than 4 cm, and deep invasion of cervical stroma), and high risk (pelvic lymph node involved, positive parametrial, or vaginal margins) groups. Postoperative adju-vant therapy in the form of radiotherapy alone to those with intermediate risk and chemo-radiotherapy to those with high risk was given to patients. The median follow-up was 60 months. RESULTS: The majority of patients had intermediate risk. The overall event-free survival (EFS) at five years was 74.37%, with EFS of 86.5% in those from the low-risk group, 73% in those from the intermediate-risk group, and 64% in those from the high-risk group. In conclusion, risk strata-based adjuvant postoperative therapy is able to provide a favourable outcome in patients with stage Ib-IIa cervical cancer with a nearly 11% improvement in survival compared with historical control.

Toll-like receptors 7, 8, and 9 expression and function in primary human cervical cancer Langerhans cells: evidence of anergy.

OBJECTIVE: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. METHODOLOGY: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a*CD207* cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1ß, IL-6, IL-10, IL-12p70, interferon (IFN) α, interferon γ, and tumor necrosis factor (TNF) α by Luminex multiplex bead array. Human papillomavirus was genotyped. RESULTS: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1ß, and tumor necrosis factor α to TLR8 ligand and interferon α in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. CONCLUSIONS: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

A neonatal oral Mycobacterium tuberculosis-SIV prime / intramuscular MVA-SIV boost combination vaccine induces both SIV and Mtb-specific immune responses in infant macaques.

Mother-to-child-transmission of HIV by breast-feeding remains a major obstacle in the eradication of HIV infection. Compared to adults, HIV-infected infants have more rapid disease and show higher susceptibility to co-infections like tuberculosis (TB). Although the Bacille Calmette-Guérin vaccine can be administered at birth to protect against TB, BCG can disseminate in HIV-infected infants and increase mortality. Thus, a pediatric combination vaccine to stop both HIV and TB infection in infants is urgently needed. Towards the goal of developing a pediatric combination HIV-TB vaccine to prevent both oral HIV acquisition by breast-feeding and TB infection, we tested and optimized an immunization regimen using a novel live attenuated Mycobacterium tuberculosis vaccine engineered to express simian immunodeficiency (SIV) antigens followed by heterologous MVA-SIV boosting in the infant macaque model. A single oral dose of the attenuated Mtb-SIV vaccine strain mc26435 during the first week of life was sufficient to induce persistent TB-specific immune responses. SIV-specific immunity was induced at low but comparable magnitudes after oral or intradermal priming, and was enhanced following MVA-SIV boosts. T cell responses were most pronounced in intestinal tissues and oral lymph nodes. Importantly, in addition to plasma SIV-specific IgG and IgA antibodies, infant macaques developed mucosal SIV-specific IgA in saliva and intestinal IgA and IgG. While future SIV and Mtb challenge studies will be needed to determine the protective efficacy of the Mtb-SIV / MVA-SIV vaccine, infants at high risk for oral HIV acquisition by breast-feeding and TB infection could profoundly benefit from an effective combination vaccine.

A recombinant attenuated Mycobacterium tuberculosis vaccine strain is safe in immunosuppressed simian immunodeficiency virus-infected infant macaques.

Many resource-poor countries are faced with concurrent epidemics of AIDS and tuberculosis (TB) caused by human immunodeficiency virus (HIV) and Mycobacterium tuberculosis, respectively. Dual infections with HIV and M. tuberculosis are especially severe in infants. There is, however, no effective HIV vaccine, and the only licensed TB vaccine, the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine, can cause disseminated mycobacterial disease in HIV-infected children. Thus, a pediatric vaccine to prevent HIV and M. tuberculosis infections is urgently needed. We hypothesized that a highly attenuated M. tuberculosis strain containing HIV antigens could be safely administered at birth and induce mucosal and systemic immune responses to protect against HIV and TB infections, and we rationalized that vaccine safety could be most rigorously assessed in immunocompromised hosts. Of three vaccine candidates tested, the recombinant attenuated M. tuberculosis strain mc(2)6435 carrying a simian immunodeficiency virus (SIV) Gag expression plasmid and harboring attenuations of genes critical for replication (panCD and leuCD) and immune evasion (secA2), was found to be safe for oral or intradermal administration to non-SIV-infected and SIV-infected infant macaques. Safety was defined as the absence of clinical symptoms, a lack of histopathological changes indicative of M. tuberculosis infection, and a lack of mycobacterial dissemination. These data represent an important step in the development of novel TB vaccines and suggest that a combination recombinant attenuated M. tuberculosis-HIV vaccine could be a safe alternative to BCG for the pediatric population as a whole and, more importantly, for the extreme at-risk group of HIV-infected infants.