Engineering high affinity antigen-binders: Beyond conventional antibodies.

For decades, antibodies have remained the archetypal binding proteins that can be rapidly produced with high affinity and specificity against virtually any target. A conventional antibody is still considered the prototype of a binding molecule. It is therefore not surprising that antibodies are routinely used in basic scientific and biomedical research, analytical workflows, molecular diagnostics etc. and represent the fastest growing sector in the field of biotechnology. However, several limitations associated with conventional antibodies, including stringent requirement of animal immunizations, mammalian cells for expression, issues on stability and aggregation, bulkier size and the overall time and cost of production has propelled evolution of concepts along alternative antigen binders. Rapidly evolving protein engineering approaches and high throughput screening platforms have further complemented the development of myriads of classes of non-conventional protein binders including antibody derived as well as non-antibody based molecular scaffolds. These non-canonical binders are finding use across disciplines of which diagnostics and therapeutics are the most noteworthy.

Immunotherapy guided precision medicine in solid tumors.

Cancer is no longer recognized as a single disease but a collection of diseases each with its defining characteristics and behavior. Even within the same cancer type, there can be substantial heterogeneity at the molecular level. Cancer cells often accumulate various genetic mutations and epigenetic alterations over time, leading to a coexistence of distinct subpopulations of cells within the tumor. This tumor heterogeneity arises not only due to clonal outgrowth of cells with genetic mutations, but also due to interactions of tumor cells with the tumor microenvironment (TME). The latter is a dynamic ecosystem that includes cancer cells, immune cells, fibroblasts, endothelial cells, stromal cells, blood vessels, and extracellular matrix components, tumor-associated macrophages and secreted molecules. The complex interplay between tumor heterogeneity and the TME makes it difficult to develop one-size-fits-all treatments and is often the cause of therapeutic failure and resistance in solid cancers. Technological advances in the post-genomic era have given us cues regarding spatial and temporal tumor heterogeneity. Armed with this knowledge, oncologists are trying to target the unique genomic, epigenetic, and molecular landscape in the tumor cell that causes its oncogenic transformation in a particular patient. This has ushered in the era of personalized precision medicine (PPM). Immunotherapy, on the other hand, involves leveraging the body's immune system to recognize and attack cancer cells and spare healthy cells from the damage induced by radiation and chemotherapy. Combining PPM and immunotherapy represents a paradigm shift in cancer treatment and has emerged as a promising treatment modality for several solid cancers. In this chapter, we summarise major types of cancer immunotherapy and discuss how they are being used for precision medicine in different solid tumors.

A journey from omics to clinicomics in solid cancers: Success stories and challenges.

The word 'cancer' encompasses a heterogenous group of distinct disease types characterized by a spectrum of pathological features, genetic alterations and response to therapies. According to the World Health Organization, cancer is the second leading cause of death worldwide, responsible for one in six deaths and hence imposes a significant burden on global healthcare systems. High-throughput omics technologies combined with advanced imaging tools, have revolutionized our ability to interrogate the molecular landscape of tumors and has provided unprecedented understanding of the disease. Yet, there is a gap between basic research discoveries and their translation into clinically meaningful therapies for improving patient care. To bridge this gap, there is a need to analyse the vast amounts of high dimensional datasets from multi-omics platforms. The integration of multi-omics data with clinical information like patient history, histological examination and imaging has led to the novel concept of clinicomics and may expedite the bench-to-bedside transition in cancer. The journey from omics to clinicomics has gained momentum with development of radiomics which involves extracting quantitative features from medical imaging data with the help of deep learning and artificial intelligence (AI) tools. These features capture detailed information about the tumor's shape, texture, intensity, and spatial distribution. Together, the related fields of multiomics, translational bioinformatics, radiomics and clinicomics may provide evidence-based recommendations tailored to the individual cancer patient's molecular profile and clinical characteristics. In this chapter, we summarize multiomics studies in solid cancers with a specific focus on breast cancer. We also review machine learning and AI based algorithms and their use in cancer diagnosis, subtyping, prognosis and predicting treatment resistance and relapse.

Association of ATG10 rs1864183, ATG16L1 rs2241880 and miR-126 with esophageal cancer.

BACKGROUND: In India, esophageal cancer (EC) is among the major cause of cancer-related deaths in both sexes. In recent past, autophagy has emerged as one of the crucial process associated with cancer. In the development of EC, the role of autophagy and the precise molecular mechanism involved has yet to be fully understood. Recently, a small number of studies have proposed how variations in autophagy genes affect the growth and development of EC. Micro-RNA's are also known to play a critical role in the development of EC. Here, we examined the relationship between the risk of EC and two single-nucleotide polymorphisms (SNPs) in the key autophagy genes, ATG10 rs1864183 and ATG16L1 rs2241880. We also analyzed the association of miR-107 and miR-126 with EC as these miRNA's are associated with autophagy. METHODS AND RESULTS: A total of 230 EC patients and 230 healthy controls from North-west Indian population were enrolled. ATG10 rs1864183 and ATG16L1 rs2241880 polymorphism were analyzed using TaqMan genotyping assay. Expression levels of miR-107 and miR-126 were analyzed through quantitative PCR using SYBR green chemistry. We found significant association of CT + CC genotype (OR 0.64, p = 0.022) in recessive model for ATG10 rs1864183 polymorphism with decreased EC risk. For ATG16L1 rs2241880 polymorphism significant association for AG genotype (OR 1.48, p = 0.05) and G allele (OR 1.43, p = 0.025) was observed for increased EC risk. Expression levels of miR-126 were also found to be significantly up regulated (p = 0.008). CONCLUSION: Our results suggest that ATG10 rs1864183, ATG16L1 rs2241880 and miR-126 may be associated with esophageal carcinogenesis and warrant further investigation.

Genetic association of IL1B gene variants with primary glaucoma in a North Indian Punjabi cohort: An original study and meta-analysis.

Interleukin (IL) 1B is an important candidate gene in glaucoma pathogenesis as it affects the survival of retinal ganglion cells (RGCs). In the present study, -511T/C and +3953C/T polymorphisms in the IL1B were assessed as genetic risk factors for primary open angle (POAG) and angle closure glaucoma (PACG) in a North Indian Punjabi cohort comprising 867 samples (POAG cases = 307; PACG cases = 133 and controls = 427). Genetic association, diplotype and linkage disequilibrium (LD) analyses were performed. Corrections for confounding variables and multiple testing were applied. An updated meta-analysis was also performed. Pooled OR with 95% CI was calculated for dominant, over dominant, and recessive models. Level of heterozygosity among studies was tested using I2 statistic with fixed or random effect model based on the extent of heterogeneity. For -511T > C polymorphism, a positive association was observed with PACG under dominant (p = 0.038; OR = 0.65; pcorr = 0.011; OR = 0.55) and over dominant models (p = 0.010; OR = 0.59; pcorr = 0.001; OR = 0.46). Significant association of +3953C > T was also observed with POAG under dominant (p = 0.011; OR = 1.46; pcorr = 0.018; OR = 1.48) and PACG under recessive models (p < 0.0001; OR = 4.47; pcorr<0.0001; OR = 4.06). While C-C diplotype provided protection against primary glaucoma (0.67-fold; p = 0.0004), T-T and T-C diplotypes predisposed individuals to higher risk (1.31-fold; p = 0.030 and 1.36-fold; p = 0.022 respectively). In meta-analysis, a significant association between +3453 C>T and POAG was observed under dominant (pooled OR = 1.33, p = 0.0046) and over dominant (pooled OR = 1.25; p = 0.0269) models with overall heterogeneity of 15% and 0% respectively. The study provides strong evidence of IL1B variants in modifying genetic susceptibility to primary glaucoma in the targeted North Indian Punjabi population. Replication of the present findings in other populations, and functional studies are warranted to further assess the relevance of IL1B variants in the pathogenesis of primary glaucoma.

Vasculitis, CA19-9, and Perineural Invasion Differentially Predict Response and Surgical Outcome in Pancreatic Ductal Adenocarcinoma.

PURPOSE: Curative intent treatment of pancreatic adenocarcinoma (PDAC) relies on surgical resection. Modern treatment protocols focus on optimizing neoadjuvant therapy to increase resectability and improve oncologic outcomes. To elucidate differences in outcomes, we investigated the relationship between neoadjuvant chemotherapy (NAC), either with or without stereotactic body radiation therapy (SBRT), and vascular inflammation, surgical outcomes, and the resultant transcriptomic changes. METHODS AND MATERIALS: Clinical data were collected from patients with borderline resectable PDAC (clinical T3-T4N0-1) who underwent NAC or NAC-SBRT followed by curative intent resection between 2014 and 2019. Vascular structures on surgical specimens were histologically evaluated for vasculitis. RNA sequencing was used to evaluate differential gene expression and to generate enrichment maps. Multivariate analysis was used to analyze the relationship between patient characteristics and oncological outcome. RESULTS: In total, 46 patients met inclusion criteria (n = 12 NAC, n = 34 NAC-SBRT) with a median follow-up of 20.1 months. All patients underwent curative resection, with 91.3% achieving R0. There was no significant difference in patterns of failure, overall survival, or progression-free survival between NAC and NAC-SBRT groups. Patients with vasculitis had a lower median overall survival compared with those without (14.5 vs 28.3 months; hazard ratio, 12.96; 95% confidence interval, 3.55-47.28; P < .001). There was no significant correlation between inflammation and surgical complications or pathologic response. Neoadjuvant therapy did not have a significant effect on development of vasculitis (odds radio, 1.64 for NAC-SBRT; 95% confidence interval, 0.40-8.43; P = .52). Predictors of poor survival included perineural invasion and high baseline carbohydrate antigen 19-9 (CA19-9) (>191 U/mL). Patients with robust CA19-9 (>20% decrease) responses to neoadjuvant therapy had enrichment in immune response, chemotaxis, and cytotoxic T-cell and natural killer-cell proliferation. CONCLUSIONS: Vasculitis predicts for poor survival outcomes in patients with PDAC; NAC-SBRT did not increase the rate of vasculitis compared with NAC. Perineural invasion and CA19-9 remain strong prognosticators. Understanding and optimizing immune interactions remain a crucial hurdle in achieving response in pancreatic cancer.

Prediction of an immunogenic peptide ensemble and multi-subunit vaccine for Visceral leishmaniasis using bioinformatics approaches.

Visceral Leishmaniasis (VL) is a neglected tropical disease of public health importance in the Indian subcontinent. Despite consistent elimination initiatives, the disease has not yet been eliminated and there is an increased risk of resurgence from active VL reservoirs including asymptomatic, post kala azar dermatitis leishmaniasis (PKDL) and HIV-VL co-infected individuals. To achieve complete elimination and sustain it in the long term, a prophylactic vaccine, which can elicit long lasting immunity, is desirable. In this study, we employed immunoinformatic tools to design a multi-subunit epitope vaccine for the Indian population by targeting antigenic secretory proteins screened from the Leishmania donovani proteome. Out of 8014 proteins, 277 secretory proteins were screened for their cellular location and proteomic evidence. Through NCBI BlastP, unique fragments of the proteins were cropped, and their antigenicity was evaluated. B-cell, HTL and CTL epitopes as well as IFN-ɣ, IL-17, and IL-10 inducers were predicted, manually mapped to the fragments and common regions were tabulated forming a peptide ensemble. The ensemble was evaluated for Class I MHC immunogenicity and toxicity. Further, immunogenic peptides were randomly selected and used to design vaccine constructs. Eight vaccine constructs were generated by linking random peptides with GS linkers. Synthetic TLR-4 agonist, RS09 was used as an adjuvant and linked with the constructs using EAAK linkers. The predicted population coverage of the constructs was ∼99.8 % in the Indian as well as South Asian populations. The most antigenic, nontoxic, non-allergic construct was chosen for the prediction of secondary and tertiary structures. The 3D structures were refined and analyzed using Ramachandran plot and Z-scores. The construct was docked with TLR-4 receptor. Molecular dynamic simulation was performed to check for the stability of the docked complex. Comparative in silico immune simulation studies showed that the predicted construct elicited humoral and cell-mediated immunity in human host comparable to that elicited by Leish-F3, which is a promising vaccine candidate for human VL.

RAD51 135G>C polymorphism in esophageal cancer and meta-analysis in gastrointestinal tract cancers.

Background: A functional single-nucleotide polymorphism (SNP), 135G>C in the 5'UTR of the RAD51 gene, affects gene transcription activity with implications for the repair of damaged DNA related to tumorigenesis. Previous limited reported genetic studies to link the 135G>C polymorphism of RAD51 gene to the risk of gastrointestinal tract (GIT) cancers, especially esophageal cancer (EC), have been inconclusive. Materials and Methods: The polymorphism was evaluated by RFLP-PCR in 252 EC patients and 252 healthy controls from Amritsar, Punjab, India, for case-control study. For a meta-analysis, a total of 78 studies on GIT cancers were assessed, out of which 14 eligible studies (including the present study) comprising 2842 cases and 3224 controls were included. Odds ratios (ORs) with 95% confidence intervals (CIs) and Chi-square test were used to assess the association in different inheritance models. Results: The GC genotype (OR: 0.45, 95% CI: 0.29-0.68) and C allele (OR: 0.52, 95% CI: 0.36-0.75) were significantly lower (P = 0.0005) in cases as compared to controls. There was no significant association with any genetic model in the meta-analysis. Conclusion: C allele provides protection for EC in the studied population contrary to previous reports in Polish, Chinese population probably due to ethic differences. Compared with previous meta-analysis on individual GIT cancers, present meta-analysis included all GIT cancers but found no association.

A phase I/Ib trial and biological correlate analysis of neoadjuvant SBRT with single-dose durvalumab in HPV-unrelated locally advanced HNSCC.

Five-year survival for human papilloma virus-unrelated head and neck squamous cell carcinomas remain below 50%. We assessed the safety of administering combination hypofractionated stereotactic body radiation therapy with single-dose durvalumab (anti-PD-L1) neoadjuvantly (n = 21) ( NCT03635164 ). The primary endpoint of the study was safety, which was met. Secondary endpoints included radiographic, pathologic and objective response; locoregional control; progression-free survival; and overall survival. Among evaluable patients at an early median follow-up of 16 months (448 d or 64 weeks), overall survival was 80.1% with 95% confidence interval (95% CI) (62.0%, 100.0%), locoregional control and progression-free survival were 75.8% with 95% CI (57.5%, 99.8%), and major pathological response or complete response was 75% with 95% exact CI (51.6%, 100.0%). For patients treated with 24 Gy, 89% with 95% CI (57.1%, 100.0%) had MPR or CR. Using high-dimensional multi-omics and spatial data as well as biological correlatives, we show that responders had: (1) an increase in effector T cells; (2) a decrease in immunosuppressive cells; and (3) an increase in antigen presentation post-treatment.

Solid Pseudopapillary Neoplasm: A Single Institutional Case Series of a Rare Pancreatic Tumor.

Purpose: Solid pseudopapillary neoplasms (SPN) are rare pancreatic cystic neoplasms with low malignant potential that tend to occur in young women. Due to the rarity of this disease, there are few large case series in the literature, and the exact pathophysiology remains unknown. In this article, we aim to share our institutional experience. Methods: Retrospective clinical data collection and analysis was performed on all patients with a diagnosis of SPN at the University of Colorado Hospital and Children's Hospital of Colorado (n = 28). Results: Twenty-eight patients were diagnosed with SPN during the study period. The median age was 21.5 years, and the majority of patients were female (89.3%) and Caucasian (60.7%). Six patients were diagnosed incidentally (21.4%). The majority of tumors were in the pancreatic tail (46.4%), and most underwent distal pancreatectomy (64.3%). The mean tumor size was 5.4 cm, and R0 resection was achieved in 25 patients (89.3%). Ten patients underwent laparoscopic resection (35.7%). The median hospital length of stay was 8.5 days, and postoperative complication rate was 39.3%. Median follow-up was 41 months, with 78.6% of patients alive without evidence of disease, while 2 patients were lost to follow-up. Two patients developed recurrence/metastases, which were resected; both are alive without evidence of disease. Conclusion: SPN are rare pancreatic tumors diagnosed most frequently in young women. Surgical resection is the mainstay of treatment, and outcomes are excellent if complete resection is achieved. Predictors of malignant disease are inconsistent in current literature. Considerations should be made for a minimally invasive approach in patients with SPN. Multidisciplinary clinics may be helpful in the diagnosis, management, and surveillance of pancreatic cystic lesions, with major potential for the advanced practitioner role.

Metastatic tumors to the pancreas, a 12-year single institution review.

BACKGROUND: Tumors of the pancreas are often primary in nature, but are occasionally proven to be secondary. We report a 12-year review of all pancreatic tumors shown to be metastatic at the University of Colorado. METHOD: The electronic pathology database was searched between 2008 and 2020, for all pancreatic fine-needle aspiration cases at the University of Colorado to identify and review metastatic tumors to the pancreas. Additional data points to include age, sex, radiographic features, and clinical management were analyzed. RESULTS: Our data retrieval identified 39 metastatic tumors to the pancreas comprised of 12 (31%) renal cell carcinomas, 6 (15%) lung carcinomas, and 5 (13%) melanomas with the remainder of tumors originating from a variety of other sites. The most common location of these metastases within the pancreas was the pancreatic head (49%) followed by the body (20%). Eighty-five percentage of cases presented with a single radiographically identified mass. CONCLUSION: While the majority of pancreatic tumors represent primary processes, metastatic tumors to the pancreas represent a possible pitfall. The most common tumors to metastasize to the pancreas in this series were renal cell carcinoma, lung carcinoma, and melanoma. General awareness of the most frequent tumors to metastasize to the pancreas is practical when faced with a pancreatic lesion that could represent a metastasis, or in the absence of clinical history.

Signal transducer and activator of transcription-3 mediated neuroprotective effect of interleukin-6 on cobalt chloride mimetic hypoxic cell death in R28 cells.

BACKGROUND: Hypoxic injury to retinal ganglionic cells and adjoining glia is implicated in glaucomatous optic neuropathy. The present study evaluates the effect of IL-6 on R28 retinal precursor cell line exposed to hypoxic injury. METHODS AND RESULTS: Apoptotic cell death induced by hypoxia mimetic CoCl2 in R28 cells with or without IL-6 treatment was measured using cell viability assays and apoptotic markers. Oxidative stress was also measured. Hypoxia induced by mimetic CoCl2 led to a time and concentration dependent apoptosis of cells mediated by disruption of mitochondrial membrane potential and activation of caspase 3. Cells pre-treated with IL-6 demonstrated significantly higher viability and mitochondrial integrity under hypoxic conditions. A critical role of STAT3 was observed in mediating the cytoprotective effects of IL-6. Treatment of cells with IL-6 led to STAT3-mediated expression of the Bcl-2 family proteins and MnSOD. CONCLUSIONS: The data from the present study indicate cytoprotective role of IL-6 and suggest a previously unreported mechanism of neuroprotection via STAT3 mediated signaling.

Suggestive evidence of genetic association of -572G > C polymorphism with primary open angle glaucoma in a North Indian Punjabi population.

BACKGROUND: IL6 is an important candidate gene implicated in the pathogenesis of glaucoma. The present study assessed the genetic association of -174G > C and -572G > C polymorphisms in the IL6 promoter region with primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) in a north Indian Punjabi cohort. METHODS: 910 subjects (313 POAG, 148 PACG cases and 449 controls) were recruited. Genotyping was done by TaqMan assays. Genetic association was tested under different genetic models using Plink. Diplotype and linkage disequilibrium (LD) analysis was done through Haploview. Association of clinical parameters with the genotypes was assessed by one-way ANOVA. Adjustment for potential confounding variables was done by binary logistic regression. IL6 levels were measured in POAG patients and controls. RESULTS: 572G > C variant showed marginal difference in genotype frequency between pooled cases and POAG subgroup with respect to controls (p = 0.042; OR = 1.33; and p = 0.041; OR = 1.37). The GC genotype conferred 1.37-fold protection under codominant model in POAG cases (p = 0.034, OR = 1.37, 95% CI = 1.02-1.85; pcorr = 0.025, OR = 1.45, 95% CI = 1.04-2.02). The mean value for IOP was elevated among cases having 'CC' genotype at the -572G > C locus (p = 0.037). Lower levels of IL6 were detected in POAG patients in plasma samples (p = 0.0001). CONCLUSION: The study reports suggestive evidence for -572G > C variant in IL6 in affecting genetic susceptibility to POAG in the targeted North Indian Punjabi cohort. A correlation of IL6 levels in aqueous humor (AH) and systemic circulation in POAG was observed, the functional and diagnostic relevance of which may be further investigated.

Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Coregulator in Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) is the most common special histologic subtype of breast cancer, and nearly all ILC tumors express estrogen receptor alpha (ER). However, clinical and laboratory data suggest ILC are strongly estrogen-driven but not equally antiestrogen-sensitive. We hypothesized ILC-specific ER coregulators mediate ER functions and antiestrogen resistance in ILC, and profiled ER-associated proteins by mass spectrometry. Three ER+ ILC cell lines (MDA MB 134VI, SUM44PE, and BCK4) were compared with ER+ invasive ductal carcinoma (IDC) line data, and we examined whether siRNA of identified proteins suppressed ER-driven proliferation in ILC cells. This identified mediator of DNA damage checkpoint 1 (MDC1), a tumor suppressor in DNA damage response (DDR), as a novel ER coregulator in ILC. We confirmed ER:MDC1 interaction was specific to ILC versus IDC cells, and found MDC1 knockdown suppressed ILC cell proliferation and tamoxifen resistance. Using RNA-sequencing, we found in ILC cells MDC1 knockdown broadly dysregulates the ER transcriptome, with ER:MDC1 target genes enriched for promoter hormone response elements. Importantly, our data are inconsistent with MDC1 tumor suppressor functions in DDR, but suggest a novel oncogenic role for MDC1 as an ER coregulator. Supporting this, in breast tumor tissue microarrays, MDC1 protein was frequently low or absent in IDC, but MDC1 loss was rare in ER+ ILC. ER:MDC1 interaction and MDC1 coregulator functions may underlie ER function in ILC and serve as targets to overcome antiestrogen resistance in ILC. IMPLICATIONS: MDC1 has novel ER coregulator activity in ILC, which may underlie ILC-specific ER functions, estrogen response, and antiestrogen resistance.

Association of TGFB -509C>T promoter polymorphism with primary angle closure glaucoma in a North Indian Punjabi cohort.

PURPOSE: Transforming growth factor beta (TGFB) is an important candidate gene implicated in glaucoma pathogenesis because it affects retinal ganglionic cell survival. The present study assessed the genetic association of -509C > T variant in the TGFB promoter region with primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) in a North Indian Punjabi population. METHOD: A total of 867 subjects (307 POAG, 133 PACG cases and 427 controls) were recruited from the targeted population. Genotyping was done by PCR-RFLP method and the data was analyzed using PLINK software (v1.07). Logistic regression under different genetic models was applied and genotype phenotype correlation was assessed by one-way ANOVA. RESULT: A statistically significant difference in the frequency of heterozygotes among PACG cases (53.16%) and controls (30.07%) (p = 0.0002) was observed. Genetic model analysis revealed that mutant "TT" genotype conferred 2-fold risk towards PACG development under recessive model (p = 0.0019) while dominant model and co-dominant model provided 0.62 and 0.37 fold protection against PACG (p = 0.025 and p = 0.0001, respectively). Data segregation based on sex revealed a strong protective effect of heterozygous 'CT' genotype against progression of PACG among females (p = 0.002, OR = 0.37, 95% CI = 0.19-0.70), but conferred 2.14-fold risk among female POAG subjects (p = 0.013). CONCLUSION: The study revealed a strong genetic association of -509C > T variant in TGFB with PACG in females. There is a need to replicate the results in a larger PACG cohort in other populations and further assess the contribution of sex specific factors in modifying genetic susceptibility to PACG.

Genetic association between CDKN2B/CDKN2B-AS1 gene polymorphisms with primary glaucoma in a North Indian cohort: an original study and an updated meta-analysis.

BACKGROUND: Variants in CDKN2B/CDKN2B-AS1 have been reported to modulate glaucoma risk in several GWAS across different populations. CDKN2B/CDKN2A encodes tumor suppressor proteins p16INK4A/p15INK4B which influences cell proliferation/senescence in RGCs, the degeneration of which is a risk factor for glaucoma. CDKN2B-AS1 codes a long non-coding RNA in antisense direction and is involved in influencing nearby CDKN2A/CDKN2B via regulatory mechanisms. METHODS: Current study investigated four SNPs (rs2157719, rs3217992, rs4977756, rs1063192) of aforementioned genes in a case-control study in a North Indian cohort. Genotyping was done with Taqman chemistry. In addition, an updated meta-analysis was performed. RESULTS: Two SNPs, rs3217992 and rs2157719 were found to be significantly associated with the disease. The frequency of 'T' allele of rs3217992 was significantly lower in cases (POAG/PACG) [p = 0.045; OR = 0.80(CI = 0.65-0.99) and p = 0.024; OR = 0.73(CI = 0.55-0.96)], respectively than in controls. Genetic model analysis revealed that TT + CT genotype confers 0.73-fold protection against POAG [p = 0.047; OR = 0.73(CI = 0.54-0.99)] and trend assumed additive model gives 0.53 times higher protection against PACG progression. However the association of rs3217992 with POAG and PACG did not remain significant after Bonferroni correction. For rs2157719, the 'C' allele was found to be less prevalent among cases (POAG/PACG) with respect to controls. Cochran Armitage trend test assuming additive model revealed 0.77 and 0.64-fold protection against POAG and PACG respectively. Bonferroni correction (pcorr = 0.003) was applied and the association of rs2157719 remained significant in PACG cases but not among POAG cases (p = 0.024). The 'CC' genotype also confers protection against primary glaucoma (POAG/PACG) among males and female subjects. The frequency rs1063192 and rs4977756 did not vary significantly among subjects, however the haplotype 'CATA' was found to be associated with increased glaucoma risk. An updated meta-analysis conducted on pooled studies on POAG cases and controls revealed significant association between rs1063192, rs2157719, rs4977756 and POAG except rs3217992. CONCLUSION: The study concludes significant association between INK4 variants and primary glaucoma in the targeted North Indian Punjabi cohort. We believe that deep-sequencing of INK4 locus may help in identifying novel variants modifying susceptibility to glaucoma. Functional studies can further delineate the role of CDKN2B and CDKN2B-AS1 in primary glaucoma for therapeutic intervention.

Combination of ERCP-Based Modalities Increases Diagnostic Yield for Biliary Strictures.

BACKGROUND: Biliary stricture evaluation with brush cytology and intraductal forceps biopsy carries a low sensitivity, but the combination of newer modalities may improve sensitivity. AIM: To determine whether the addition of advanced modalities increases diagnostic yield of ERCP-based sampling. METHODS: This single-center retrospective study evaluates patients with biliary strictures sampled using brush cytology. Operating characteristics were calculated for individual and combinations of modalities including cholangioscopy, fluoroscopy- and cholangioscopy-directed intraductal biopsy, fluorescence in situ hybridization (FISH), and confocal laser endomicroscopy. Analyses under Standard Criteria (SC) included malignant results as "positive" and Expanded Criteria (EC) included "suspicious" and "high-grade dysplasia" results as "positive." RESULTS: A total of 614 patients were included, and 354 (57.8%) received brush cytology alone, which had a sensitivity of 38.5% (SC) to 40.3% (EC) and a specificity of 97.8% (EC) to 99.3% (SC). Combining brush cytology with fluoroscopy-guided biopsy (n = 259, 42.2%) had a sensitivity of 62.5% (SC) to 67.9% (EC) and specificity of 90.2% (EC) to 96.7% (SC). Adding FISH to brush cytology had a sensitivity of 84.2% (SC) to 87.5% (EC) and specificity of 54.1% (SC and EC), while cholangioscopy visualization addition resulted in a sensitivity of 80.4% (SC) to 92.2% (EC) and specificity of 67.3% (EC) to 89.1% (SC). There were no significant differences in sensitivity and specificity using SC and EC. CONCLUSIONS: Brush cytology has a low sensitivity, but the addition of other modalities increases sensitivity. There was no difference in specificity between the SC and the EC, supporting the inclusion of "suspicious" impressions with malignant results at our center.

Neutrophils and Visceral Leishmaniasis: Impact on innate immune response and cross-talks with macrophages and dendritic cells.

Neutrophils with their array of microbicidal activities are the first innate immune cells to guard against infection. They are also most crucial for the host's initial defense against Leishmania parasites which cause clinically diverse diseases ranging from self-healing cutaneous leishmaniasis (CL) to a more severe visceral form, visceral leishmaniasis (VL). Neutrophils are recruited in large numbers at the infection site after bite of sandfly, which is the vector for the disease. The initial interaction of neutrophils with the parasites may modulate the subsequent innate and adaptive immune responses and hence affect the disease outcome. The purpose of this review is to comprehensively appraise the role of neutrophils during the early stages of Leishmania infection with a focus on the visceral form of the disease. In the past decade, new insights regarding the role of neutrophils in VL have surfaced which have been extensively elaborated in the present review. In addition, since much of the information regarding neutrophil-Leishmania early interaction has accumulated through studies on mouse models of CL, these studies are also revisited. We begin by reviewing the factors which drive the recruitment of neutrophils at the site of injection by the sandfly. We then discuss the studies delineating the molecular mechanisms involved in the uptake of the Leishmania parasite by neutrophils and how the parasite subverts their microbicidal functions. In the end, the interaction of infected neutrophils with macrophages and dendritic cells is summarized.