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Background: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis., Methods and Results: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR., Conclusion: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (Copyright © 2024. Published by Elsevier Inc.)

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., (Copyright © 2024. Published by Elsevier Inc.)

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., (Copyright © 2024. Published by Elsevier Inc.)

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 I 2 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; p corr   =  0.011; OR = 0.55) and over dominant models (p = 0.010; OR = 0.59; p corr   =  0.001; OR = 0.46). Significant association of +3953C > T was also observed with POAG under dominant (p = 0.011; OR = 1.46; p corr   =  0.018; OR = 1.48) and PACG under recessive models (p < 0.0001; OR = 4.47; p corr <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., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Rajeev Kumar Pandey reports a relationship with Thermo Fisher Scientific that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: None

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., (© 2022. The Author(s).)

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., Competing Interests: The authors have no conflicts of interest to disclose., (© 2022 Harborside™.)

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., (© 2021 Wiley Periodicals LLC.)

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; p corr  = 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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 CoCl 2 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 CoCl 2 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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (©2021 American Association for Cancer Research.)

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.

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 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., (© 2020 Wiley Periodicals LLC.)

Nitric oxide (NO) is an anti-microbial effector of the innate immune system which plays major role in non-specific killing of various pathogens including protozoan parasites. However, due to subversion of the host's immune processes by pathogens, suboptimal production of NO is frequently found in many infection models. Previous studies have shown suppressed NO production during Leishmania donovani infection, the causative agent of visceral leishmaniasis (VL). Availability of L-Arginine, a semi-essential amino acid is required for inducible nitric oxide synthase (iNOS) mediated NO production. However, arginase is another enzyme, which if expressed concomitantly, may strongly compete for L-Arginine, and suppress NO production by iNOS. In the present study, plasma nitrite and arginase levels were measured in VL patients before and after successful drug treatment, endemic and non-endemic healthy donors. We observed significantly lower NO levels in the plasma of VL patients as compared to endemic controls, which improved significantly post-treatment. Significantly elevated arginase activity was also observed in the plasma of VL patients, which may be associated with NO deficiency. VL patients also showed significantly higher levels of IL-10 and TGF-β, which are known to regulate expression of arginase in various immune cells. In vitro studies with human peripheral blood mononuclear cells (PBMCs) further corroborated the role of IL-10 and TGF-β in arginase mediated suppression of NO production., Competing Interests: RK was employed by Thermo Fisher Scientific. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kupani, Sharma, Pandey, Kumar, Sundar and Mehrotra.)

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 p16 INK4A /p15 INK4B 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 (p corr  = 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.

Malakoplakia is a rare, granulomatous disease that affects a wide variety of organs and can have a clinical and radiographic presentation resembling that of malignancy. The genitourinary tract is the most commonly involved site. There are scant reported cases presenting as a locally advanced renal mass and even rarer, diagnosed by fine-needle aspiration (FNA) cytology. We report clinical, imaging, cytologic, and histological findings of an interesting case of malakoplakia initially diagnosed by FNA cytology. We also briefly review the literature and emphasize the importance of recognizing this entity when encountered in an aspirate material, which can help mitigate the diagnostic confusion of malakoplakia for clinicians., (© 2020 Wiley Periodicals, Inc.)

Background & Aims: Despite the widespread use of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to sample pancreatic lesions and the standardization of pancreaticobiliary cytopathologic nomenclature, there are few data on inter-observer agreement among cytopathologists evaluating pancreatic cytologic specimens obtained by EUS-FNA. We developed a scoring system to assess agreement among cytopathologists in overall diagnosis and quantitative and qualitative parameters, and evaluated factors associated with agreement., Methods: We performed a prospective study to validate results from our pilot study that demonstrated moderate to substantial inter-observer agreement among cytopathologists for the final cytologic diagnosis. In the first phase, 3 cytopathologists refined criteria for assessment of quantity and quality measures. During phase 2, EUS-FNA specimens of solid pancreatic lesions from 46 patients were evaluated by 11 cytopathologists at 5 tertiary care centers using a standardized scoring tool. Individual quantitative and qualitative measures were scored and an overall cytologic diagnosis was determined. Clinical and EUS parameters were assessed as predictors of unanimous agreement. Inter-observer agreement (IOA) was calculated using multi-rater kappa (κ) statistics and a logistic regression model was created to identify factors associated with unanimous agreement., Results: The IOA for final diagnoses, based on cytologic analysis, was moderate (κ = 0.56; 95% CI, 0.43-0.70). Kappa values did not increase when categories of suspicious for malignancy, malignant, and neoplasm were combined. IOA was slight to moderate for individual quantitative (κ = 0.007; 95% CI, -0.03 to -0.04) and qualitative parameters (κ = 0.5; 95% CI, 0.47-0.53). Jaundice was the only factor associated with agreement among all cytopathologists on multivariate analysis (odds ratio for unanimous agreement, 5.3; 95% CI, 1.1-26.89)., Conclusions: There is a suboptimal level of agreement among cytopathologists in the diagnosis of malignancy based on analysis of EUS-FNA specimens obtained from solid pancreatic masses. Strategies are needed to refine the cytologic criteria for diagnosis of malignancy and enhance tissue acquisition techniques to improve diagnostic reproducibility among cytopathologists., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Solid pseudopapillary neoplasm of pancreas is a rare tumor with a low potential for metastasis and recurrence. Long-term outcomes after surgical resection are excellent and recurrences after an R0 resection are extremely rare. Case Presentation: We present an unusual case of a 42-year-old man who had a recurrence of his solid pseudopapillary tumor 4 years after undergoing a distal pancreatectomy and splenectomy and then again a year after his reresection. Conclusions: The lack of histological features deemed to be suggestive of a malignant variant and the aggressive clinical course seen in this case is remarkable. It underscores the fact that despite the low incidence, recurrences of solid pseudopapillary neoplasms of the pancreas do occur and it can be very difficult to predict malignant potential based on radiological or histopathological features., Competing Interests: No competing financial interests exist.

MMP (Matrix metalloproteinase) 9 is reported to affect glaucoma pathogenesis by altering intraocular pressure (IOP) through its role in remodeling the extracellular matrix (ECM) in the trabecular meshwork. A genetic variant at the promoter region in the MMP9 gene (-1562C>T) has a putative role in regulating its transcription rate and hence can affect genetic predisposition to primary glaucoma. The present study examined the association of -1562C>T promoter polymorphism in the MMP9 gene with Primary Open Angle Glaucoma (POAG) and Primary Angle Closure Glaucoma (PACG) in a north Indian population. A total of 729 subjects (POAG = 224, PACG = 138 and 367 controls) were recruited for the study. Genotyping for the promoter sequence variant was done with PCR-RFLP method. Genotypic and allelic frequency distribution of the POAG and PACG data sets were compared to that of controls by chi-square test and genetic association was tested under different genetic models as implemented under PLINK. Statistically significant difference was observed in the genotype frequencies between PACG cases and controls (p = 0.030). However, in the POAG cases, this difference was only borderline (p = 0.052). Genetic model analysis, under the dominant model revealed 1.6 and 1.4 fold increased susceptibility to PACG and POAG (p = 0.012, p = 0.032) respectively. A higher frequency of CT genotype was observed in PACG as well as POAG males as compared to female subjects. According to the dominant model, CT+TT genotype conferred 1.8 fold higher risk of developing PACG among male patients as compared to the control group (p = 0.048, OR = 1.87;1.00-3.50). Current findings suggest significant association of MMP9 -1562C>T polymorphism with primary glaucoma in the targeted north Indian population and warrant further replication of the findings in other populations.

Oncocytic variant of medullary thyroid carcinoma is rare form of thyroid carcinoma that is easily misdiagnosed on fine needle aspiration specimens due to it is low incidence and cytomorphologic overlap with other more common Hurtle cell lesions. A correct initial diagnosis by fine needle aspiration is imperative as the clinical treatment for medullary carcinoma differs significantly from the mimickers. We present a case of this rare variant tumor that on initial fine needle aspiration was described as a Hurthle cell lesion and was subsequently correctly classified on the resection specimen. In this brief review, we describe the cytomorphologic features of medullary carcinoma, oncocytic variant of medullary carcinoma and it is most common mimickers, and we discuss the ancillary studies required to confirm the diagnosis. This case highlights the importance of a complete clinical history and radiologic correlation, which in conjunction with a careful attention to the cytologic features of the fine needle aspiration sample, should in most cases ensure a correct initial diagnosis., (© 2017 Wiley Periodicals, Inc.)

Endoscopic ultrasound-guided fine-needle aspiration is increasingly utilized for the diagnosis of pancreatic lesions. Although operator dependent, the procedure has good overall performance characteristics and is minimally invasive; however, accuracy and sensitivity are reportedly lower for pancreatic neuroendocrine tumor (PanNET) compared with the more common pancreatic ductal adenocarcinoma (pACA). The underperformance is further exacerbated by the unusual cases of PanNET presenting with variant cytomorphology. We report two separate diagnostically challenging cases: a pigmented PanNET and a clear cell PanNET. We briefly review the literature and emphasize the importance of recognizing these uncommon variants when encountered in aspirate material. Diagn. Cytopathol. 2017;45:371-378. © 2016 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Members of the Bcl-2 family are major regulators of apoptosis in mammalian cells, and hence infection-induced perturbations in their expression could result into elimination of the parasites or creation of a niche favoring survival. In this investigation, we uncover a novel role of host Bcl-2 in sustaining Leishmania donovani infection. A rapid twofold increase in Bcl-2 expression occurred in response to parasite challenge. Downregulation of post infection Bcl-2 increase using siRNA or functional inhibition using Bcl-2 small molecule inhibitors interfered with intracellular parasite survival confirming the necessity of elevated Bcl-2 during infection. An increased nitric oxide (NO) response and reduced parasitic burden was observed upon Bcl-2 inhibition, where restitution of the NO response accounted for parasite mortality. Mechanistic insights revealed a major role of elevated Th 2 cytokine IL-13 in parasite-induced Bcl-2 expression via the transcription factor STAT-3, where blocking at the level of IL-13 receptor or downstream kinase JAK-2 dampened Bcl-2 induction. Increase in Bcl-2 was orchestrated through Toll like receptor (TLR)-2-MEK-ERK signaling, and changes in TLR-2 levels affected parasite uptake. In a mouse model of visceral leishmaniasis (VL), Bcl-2 inhibitors partially restored the antimicrobial NO response by at least a twofold increase that resulted in significantly reduced parasite burden. Interestingly, monocytes derived from the peripheral blood of six out of nine human VL subjects demonstrated Bcl-2 expression at significantly higher levels, and sera from these patients showed only marginally quantifiable nitrites. Collectively, our study for the first time reveals a pro-parasitic role of host Bcl-2 and the capacity of host-derived IL-13 to modulate NO levels during infection via Bcl-2. Here, we propose Bcl-2 inhibition as a possible therapeutic intervention for VL.

Background and Aims: Endoscopic ultrasound with fine needle aspiration (EUS-FNA) has become the standard of care in the evaluation of solid pancreatic lesions. Limited data exist on interobserver agreement (IOA) among cytopathologists in assessing solid pancreatic EUS-FNA specimens. This study aimed to evaluate IOA among cytopathologists in assessing EUS-FNA cytology specimens of solid pancreatic lesions using a novel standardized scoring system and to assess individual clinical and cytologic predictors of IOA., Methods: Consecutive patients who underwent EUS-FNA of solid pancreatic lesions at a tertiary care referral center were included. EUS-FNA slides were evaluated by four blinded cytopathologists using a standardized scoring system that assessed final cytologic diagnosis and quantitative (number of nucleated/diagnostic cells) and qualitative (bloodiness, inflammation/necrosis, contamination, artifact) cytologic parameters. Final clinical diagnosis was based on final cytology, surgical pathology, or 1-year clinical follow-up. IOA was calculated using multi-rater kappa (κ) statistics. Bivariate analyses were performed comparing cases with and without uniform agreement among the cytopathologists followed by logistic regression with backward elimination to model likelihood of uniform agreement., Results: Ninety-nine patients were included (49 % males, mean age 64 years, mean lesion size 26 mm). IOA for final diagnosis was moderate (κ = 0.45, 95 % confidence interval (CI) 0.4 - 0.49) with minimal improvement when combining suspicious and malignant diagnoses (κ = 0.54, 95 %CI 0.49 - 0.6). The weighted kappa value for overall diagnosis was 0.65 (95 %CI 0.54 - 0.76). IOA was slight to fair (κ = 0.04 - 0.32) for individual cytologic parameters. A final clinical diagnosis of malignancy was the most significant predictor of agreement [OR 3.99 (CI 1.52 - 10.49)]., Conclusions: Interobserver agreement among cytopathologists for pancreatic EUS-FNA specimens is moderate-substantial for the final cytologic diagnosis. The final clinical diagnosis of malignancy was the strongest predictor of agreement. These results have significant implications for patient management and need to be validated in future trials.

Histiocytic sarcoma (HS) is a rare malignant tumor, and in extraordinary circumstances it can be transdifferentiated from a low-grade B-cell lymphoma. In our report, a 62-year-old female was initially diagnosed with a low-grade follicular lymphoma, and 2 years later she presented with bilateral lung masses and lymphadenopathy. Fine needle aspiration (FNA) of the lung masses revealed solid nests of large pleomorphic epithelioid cells. By immunohistochemical studies, the tumor cells were positive for histiocytic markers (CD68 and CD163) and S100, supporting a diagnosis of HS with dendritic cell differentiation. Further fluorescence in situ hybridization (FISH) analyses detected IGH@/BCL2 rearrangement in both the previous follicular lymphoma and the current HS, which was highly suggestive of the clonal relationship between these two tumors. To the best of our knowledge, this is the first case of transformation of follicular lymphoma into HS diagnosed by FNA of lung masses., (© 2015 Wiley Periodicals, Inc.)

To identify susceptibility loci for visceral leishmaniasis, we undertook genome-wide association studies in two populations: 989 cases and 1,089 controls from India and 357 cases in 308 Brazilian families (1,970 individuals). The HLA-DRB1-HLA-DQA1 locus was the only region to show strong evidence of association in both populations. Replication at this region was undertaken in a second Indian population comprising 941 cases and 990 controls, and combined analysis across the three cohorts for rs9271858 at this locus showed P(combined) = 2.76 × 10(-17) and odds ratio (OR) = 1.41, 95% confidence interval (CI) = 1.30-1.52. A conditional analysis provided evidence for multiple associations within the HLA-DRB1-HLA-DQA1 region, and a model in which risk differed between three groups of haplotypes better explained the signal and was significant in the Indian discovery and replication cohorts. In conclusion, the HLA-DRB1-HLA-DQA1 HLA class II region contributes to visceral leishmaniasis susceptibility in India and Brazil, suggesting shared genetic risk factors for visceral leishmaniasis that cross the epidemiological divides of geography and parasite species.

Regional variations in susceptibility of Leishmania donovani clinical isolates have been reported to antimonials but not other antileishmanial drugs. Therefore, we evaluated the susceptibility of four antileishmanial drugs in clinical use in 28 clinical isolates from endemic and non-endemic regions in the J774A.1 macrophage cell line, and we found increased tolerance of miltefosine and paromomycin in isolates from a patient from a high endemic region. Effective dose for 90% killing (ED(90)) values were significantly higher for miltefosine (P = 0.005) and paromomycin (P = 0.02) in isolates from the high endemic region, although there were no significant differences between ED(50) values for paromomycin, miltefosine, and amphotericin B in the non- versus endemic region isolates. This report is the first of higher ED(90) values for miltefosine and paromomycin indicating susceptibility difference between regions for these newly introduced drugs by the parasite, and their use should be carefully monitored through directly observed therapy or multidrug treatment to preserve their efficacy for longer periods.

Chromosome 6q26-27 is linked to susceptibility to visceral leishmaniasis (VL) in Brazil and Sudan. DLL1 encoding the Delta-like 1 ligand for Notch 3 was implicated as the etiological gene. DLL1 belongs to the family of Notch ligands known to selectively drive antigen-specific CD4 T helper 1 cell responses, which are important in protective immune response in leishmaniasis. Here we provide further genetic and functional evidence that supports a role for DLL1 in a well-powered population-based study centred in the largest global focus of VL in India. Twenty-one single nucleotide polymorphisms (SNPs) at PHF10/C6orf70/DLL1/FAM120B/PSMB1/TBP were genotyped in 941 cases and 992 controls. Logistic regression analysis under an additive model showed association between VL and variants at DLL1 and FAM120B, with top associations (rs9460106, OR=1.17, 95%CI 1.01-1.35, P=0.033; rs2103816, OR=1.16, 95%CI 1.01-1.34, P=0.039) robust to analysis using caste as a covariate to take account of population substructure. Haplotype analysis taking population substructure into account identified a common 2-SNP risk haplotype (frequency 0.43; P=0.028) at FAM120B, while the most significant protective haplotype (frequency 0.18; P=0.007) was a 5-SNP haplotype across the interval 5' of both DLL1 (negative strand) and FAM120B (positive strand) and extending to intron 4 of DLL1. Quantitative RT/PCR was used to compare expression of 6q27 genes in paired pre- and post-treatment splenic aspirates from VL patients (N=19). DLL1 was the only gene to show differential expression that was higher (P<0.0001) in pre- compared to post-treatment samples, suggesting that regulation of gene expression was important in disease pathogenesis. This well-powered genetic and functional study in an Indian population provides evidence supporting DLL1 as the etiological gene contributing to susceptibility to VL at Chromosome 6q27, confirming the potential for polymorphism at DLL1 to act as a genetic risk factor across the epidemiological divides of geography and parasite species., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Background: IL8RA and IL8RB, encoded by CXCR1 and CXCR2, are receptors for interleukin (IL)-8 and other CXC chemokines involved in chemotaxis and activation of polymorphonuclear neutrophils (PMN). Variants at CXCR1 and CXCR2 have been associated with susceptibility to cutaneous and mucocutaneous leishmaniasis in Brazil. Here we investigate the role of CXCR1/CXCR2 in visceral leishmaniasis (VL) in India., Methods: Three single nucleotide polymorphisms (SNPs) (rs4674259, rs2234671, rs3138060) that tag linkage disequilibrium blocks across CXCR1/CXCR2 were genotyped in primary family-based (313 cases; 176 nuclear families; 836 individuals) and replication (941 cases; 992 controls) samples. Family- and population-based analyses were performed to look for association between CXCR1/CXCR2 variants and VL. Quantitative RT/PCR was used to compare CXCR1/CXCR2 expression in mRNA from paired splenic aspirates taken before and after treatment from 19 VL patients., Results: Family-based analysis using FBAT showed association between VL and SNPs CXCR1&#95;rs2234671 (Z-score = 2.935, P = 0.003) and CXCR1&#95;rs3138060 (Z-score = 2.22, P = 0.026), but not with CXCR2&#95;rs4674259. Logistic regression analysis of the case-control data under an additive model of inheritance showed association between VL and SNPs CXCR2&#95;rs4674259 (OR = 1.15, 95%CI = 1.01-1.31, P = 0.027) and CXCR1&#95;rs3138060 (OR = 1.25, 95%CI = 1.02-1.53, P = 0.028), but not with CXCR1&#95;rs2234671. The 3-locus haplotype T&#95;G&#95;C across these SNPs was shown to be the risk haplotype in both family- (TRANSMIT; P = 0.014) and population- (OR = 1.16, P = 0.028) samples (combined P = 0.002). CXCR2, but not CXCR1, expression was down regulated in pre-treatment compared to post-treatment splenic aspirates (P = 0.021)., Conclusions: This well-powered primary and replication genetic study, together with functional analysis of gene expression, implicate CXCR2 in determining outcome of VL in India.

Background: SLC11A1 has pleiotropic effects on macrophage function and remains a strong candidate for infectious disease susceptibility. 5' and/or 3' polymorphisms have been associated with tuberculosis, leprosy, and visceral leishmaniasis (VL). Most studies undertaken to date were under-powered, and none has been replicated within a population. Association with tuberculosis has replicated variably across populations. Here we investigate SLC11A1 and VL in India., Methods: Nine polymorphisms (rs34448891, rs7573065, rs2276631, rs3731865, rs17221959, rs2279015, rs17235409, rs17235416, rs17229009) that tag linkage disequilibrium blocks across SLC11A1 were genotyped in primary family-based (313 cases; 176 families) and replication (941 cases; 992 controls) samples. Family- and population-based analyses were performed to look for association between SLC11A1 variants and VL. Quantitative RT/PCR was used to compare SLC11A1 expression in mRNA from paired splenic aspirates taken before and after treatment from 24 VL patients carrying different genotypes at the functional promoter GTn polymorphism (rs34448891)., Results: No associations were observed between VL and polymorphisms at SLC11A1 that were either robust to correction for multiple testing or replicated across primary and replication samples. No differences in expression of SLC11A1 were observed when comparing pre- and post-treatment samples, or between individuals carrying different genotypes at the GTn repeat., Conclusions: This is the first well-powered study of SLC11A1 as a candidate for VL, which we conclude does not have a major role in regulating VL susceptibility in India.

Previously developed methods for noninvasive PCR diagnosis of visceral leishmaniasis (VL) have significant limitations. Diagnosis of VL using PCR and buccal swabs was evaluated in 307 subjects, including 148 patients confirmed to have VL. This method is simple and well tolerated and has good potential for development, showing 83% sensitivity with 90.56% specificity in control groups.

Background: PCR based diagnosis for Visceral Leishmaniasis (VL), despite numerous published primers, remains far from being applied in the field. The present study was planned to design a Leishmania specific diagnostic assay and to evaluate its sensitivity and specificity on a sample size, which to the best of our knowledge is the largest ever screened in one study., Methods: Leishmania specific primers were developed using 18S rRNA gene and their sensitivity was evaluated on 500 parasitologically confirmed patients with VL and 25 Post Kala-azar Dermal Leishmaniasis (PKDL) patients. Specificity was calculated on 250 healthy endemic controls, 250 healthy non endemic controls and 250 non leishmanial diseases like malaria., Results: Our PCR assay had a sensitivity of 87.8% (95%CI: 84.1-89.8) using 200 µL of patient's peripheral-blood. Specificity was absolute in non-endemic healthy controls and in subjects with different diseases while in endemic controls it was 84% (95%CI: 78.9-88.0). Its overall specificity was 94.6% (95%CI-92.8-96.1)., Conclusions: The PCR assay developed is sensitive enough to detect the 18S rRNA gene in an amount equivalent to a single parasite or less in a one million human cell environment. The high sensitivity of this PCR diagnostic test with relatively non-invasive peripheral blood sampling method opens up the possibility of its deployment in field for the routine diagnosis of VL.

Leishmaniasis is a vector-borne disease with up to 350 million people at risk of infection worldwide. Among its different clinical manifestations, visceral is the most severe form. Since clinical features of visceral leishmaniasis (VL) mimic several other common diseases, accurate diagnosis is crucial as the treatment is associated with significant toxicity. Invasive and risky techniques involving demonstration of the parasites in stained preparations from splenic and bone marrow aspirate is still the gold standard for VL diagnosis. Serological tests using rK39 in ELISA or rapid immunochromatographic format, Direct Agglutination Test (DAT), immunoblotting have issues related to a significant proportion of asymptomatic individuals being positive with these tests and their inability to diagnose relapses as these remain positive for several months to years after cure. PCR is the most common molecular technique successfully used for diagnosis and differentiation of species. Through this review we focus extensively on the comparative utilities of the various diagnostic tools currently available for VL, describing in depth their advantages and disadvantages, addressing the recent advances attained in the field. A simple, rapid, non invasive, accurate and cost effective marker of active VL, which can be used in field conditions, is necessary to improve diagnosis of VL., (Copyright © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Serial dilution of blood and spleen biopsy specimens, plated on Novy-MacNeal-Nicolle (NNN) blood agar using microtiter culture plates, is a sensitive and reproducible method for detection and growth of Leishmania parasites. Plates could be easily monitored, and growth could be rapidly detected. Moreover, parasite number may be estimated using this technique.

Background: Cyclooxygenase-2 (COX-2), implicated in carcinogenesis and tumour progression in many cancers including breast cancer, is hypothesised to cause progression by promoting angiogenesis. The exact mechanism of such action is not known and the clinical evidence of such interaction is weak. We studied COX-2 expression and microvessel density (MVD) in malignant breast tissues., Methods: COX-2 expression was analysed by immunohistochemistry in 89 breast cancer cases. MVD was assessed by CD31 immunohistochemistry using the Chalkey count method. COX-2 expression and MVD data were correlated with each other and with other prognostic factors., Results: COX-2 expression, observed in 70 (79%) cases, correlated positively with tumour type (p = 0.037) and tumour grade (p = 0.045), but negatively with oestrogen receptor (p = 0.013). It did not correlate with tumour size, axillary lymph node status, progesterone receptor and HER-2 status. MVD varied from 2.09 to 40.38, correlated positively with tumour grade (p = 0.050) and tumour size (p = 0.044), but negatively with progesterone receptor (p = 0.040). MVD did not correlate with tumour type, axillary lymph node status, oestrogen receptor and HER-2. There was no correlation between COX-2 expression and MVD (p = 0.702)., Conclusions: COX-2 expression does not correlate with angiogenesis in breast cancer. Angiogenesis in breast cancer may be dependent on multiple genes, rather than on COX-2 alone., (Copyright 2009 S. Karger AG, Basel.)

Cyclooxygenase enzymes play an important role in carcinogenesis, and increased expression of cyclooxygenase enzymes has been reported in cancers arising at a number of different sites. Most, if not all of these actions are thought to be mediated by prostaglandin E2 (PGE2). The actions of PGE2 are mediated via four main prostanoid receptors, designated EP1, EP2, EP3 and EP4, based on their different pharmacological properties and secondary messenger pathways. Recently, expression of EP1 has been reported in rat mammary gland and the inhibition of this receptor has been documented to have chemopreventive effect in this animal model. EP1 has also been shown to decrease the incidence of colon cancer in mouse models. In this study, we analysed the expression of EP1 in normal and malignant breast tissues. Expression of EP1 was analysed in breast (benign and cancer) cell lines by reverse-transcriptase polymerase chain reaction and by western blot analyses. Expression was also analysed by immunohistochemistry in normal breast tissues and in 89 cases of breast cancer. Semiquantitative analysis of the staining was performed. The data were compared with and correlated with other prognostic factors like tumour size, tumour grade, lymph node status, oestrogen receptor, progesterone receptor (PR), HER2/neu and cyclooxygenase-2. EP1 expression was demonstrated in human breast cancer by immunohistochemistry. Expression of EP1 was seen both in the cytoplasm and/or in the nuclear membrane in majority of cases. Nuclear EP1 expression correlated with PR (P=0.032) and inversely with node positivity (P=0.025). However, EP1 expression did not correlate with expression of cyclooxygenase-2 (P=0.059). Expression of EP1 is frequently seen in human breast cancers. Nuclear expression of EP1 correlates with good prognosis markers like node negative status and PR expression.

The anti-tumour actions of cyclooxygenases (COX) are thought to be mediated by inhibition of prostaglandin E(2) (PGE(2)) synthesis. However, COX-2 inhibition also alters cellular production of other prostaglandins such as prostacyclin (PGI(2)). The latter action is believed to be important for the development of adverse cardio-vascular events. Microsomal PGES (mPGES-1) is an enzyme downstream to COX-2 and affects PGE(2) production only. It is possible that targeting mPGES-1 could decrease PGE(2) production without affecting PGI(2) production. In order to assess the potential of mPGES-1 as a target for therapy, we analysed its expression in breast cell lines and normal and malignant breast tissues. The expression of mPGES-1 and COX-2 was correlated in tumour cells and vascular endothelium, and with prognostic parameters in breast cancer. Although not detectable in normal epithelial cells, expression was noted in areas of fibrocystic change and in situ carcinoma. mPGES-1 expression was noted in 79% of breast cancer tissues. Its expression did not correlate with COX-2 overexpression or with prognostic markers of breast cancer. Endothelial cells did not show mPGES-1 expression. Upregulation of mPGES-1 is therefore frequent in pre-malignant and malignant breast disease. In this study, coordinate over-expression of COX-2 and mPGES-1 was not observed, particularly in the endothelial cells of blood vessels. Targeting mPGES-1 might prove to be an alternative therapeutic strategy to inhibit PGE2 production.

Background: This pilot study combined physiologic imaging, microcomputed tomography, and histologic tumor evaluation with a xenograft model of breast cancer to identify surrogates likely to correlate with response to AZD2171, an inhibitor of the vascular endothelial growth factor (VEGF) receptor tyrosine kinases., Experimental Design: MCF-7 cells transfected with vector (MCF-7neo) or VEGF (MCF(VEGF)) were implanted in the right and left mammary fat pads of 75 athymic mice. Treatment with AZD2171 (5 mg/kg/d) or vehicle control was initiated once tumors were established. Positron emission tomography with [11C]carbon monoxide to measure blood volume, [18F]fluoromethane to measure perfusion, and [18F]fluorodeoxyglucose to measure glucose utilization was done at baseline, and after 24 hours, 72 hours, and 4 weeks of treatment. After imaging, tumors were analyzed for microvessel density, proliferation, and VEGF expression., Results: AZD2171 induced significant inhibition of tumor growth in established MCF-7(neo) xenografts and regression of established MCF-7(VEGF) xenografts. An acute decrease in blood flow was detected in MCF-7(VEGF) tumors at 24 hours (P = 0.05). Tumor blood volume was increased in the MCF-7(VEGF) tumors but correlated with tumor size; blood volume did not change with AZD2171 therapy. Glucose utilization correlated with tumor size and did not change with acute or chronic AZD2171 therapy. Unlike blood flow and blood volume, glucose utilization was similar in MCF-7neo and MCF-7(VEGF) tumors. Microvessel density and proliferation acutely decreased in MCF-7(VEGF) tumors but returned to baseline during chronic therapy., Conclusions: [18F]Fluoromethane imaging may be a useful surrogate for biological activity of AZD2171 with changes identified within 24 hours of starting therapy.

Introduction: A subpopulation (CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells has the unique ability to invade, home, and proliferate at sites of metastasis., Methods: CD44 and CD24 expression was determined by flow cytometry. Northern blotting was used to determine the expression of proinvasive and 'bone and lung metastasis signature' genes. A matrigel invasion assay and intracardiac inoculation into nude mice were used to evaluate invasion, and homing and proliferation at sites of metastasis, respectively., Results: Five among 13 breast cancer cell lines examined (MDA-MB-231, MDA-MB-436, Hs578T, SUM1315, and HBL-100) contained a higher percentage (>30%) of CD44+/CD24- cells. Cell lines with high CD44+/CD24- cell numbers express basal/mesenchymal or myoepithelial but not luminal markers. Expression levels of proinvasive genes (IL-1alpha, IL-6, IL-8, and urokinase plasminogen activator [UPA]) were higher in cell lines with a significant CD44+/CD24- population than in other cell lines. Among the CD44+/CD24(-)-positive cell lines, MDA-MB-231 has the unique property of expressing a broad range of genes that favor bone and lung metastasis. Consistent with previous studies in nude mice, cell lines with CD44+/CD24- subpopulation were more invasive than other cell lines. However, only a subset of CD44+/CD24(-)-positive cell lines was able to home and proliferate in lungs., Conclusion: Breast cancer cells with CD44+/CD24- subpopulation express higher levels of proinvasive genes and have highly invasive properties. However, this phenotype is not sufficient to predict capacity for pulmonary metastasis.

Breast cancer metastasis predominantly occurs via lymphatic vessels. However, the study of lymphatic vessels and lymphangiogenesis has been hampered by lack of specific markers. Recently, antibodies directed against M2A (D2-40), Podoplanin, and Prox-1 that specifically mark lymphatic vessels in paraffin-embedded sections have become available. These were used to study lymphangiogenesis in archival paraffin sections of normal breast (n = 23), fibrocystic disease (n = 7), ductal carcinoma in situ (n = 32), invasive ductal carcinoma (n = 50), and invasive lobular carcinoma (n = 5). In addition, endothelial proliferation in lymphatic vessels was analyzed by dual-color immunohistochemistry with D2-40 and proliferating cell nuclear antigen (PCNA). Expression of D2-40, Prox-1, and Podoplanin was seen in lymphatic vessels but not in blood vessels. Lymphatic vessels were seen in the peritumoral area and as "entrapped" intratumoral vessels adjacent to preexisting normal lobules and ducts. Unlike angiogenesis, there was no increase of lymphatic vessel density in association with neoplastic transformation. On the contrary, a marked reduction in intratumoral lymphatic vessel density was seen in comparison to normal breast tissue, fibrocystic disease, and ductal carcinoma in situ (P = 0.0001). There was an increase in peritumoral lymphatic vessel density as compared with normal breast (P = 0.0001). However, the endothelial cells in the "entrapped" or the peritumoral lymphatic vessels did not show any expression of PCNA indicating minimal or no proliferative activity. This was in contrast to the strong expression seen in adjacent tumor cells and blood vessel endothelial cells. Thus, lymphangiogenesis was not evident when studied by lymphatic vessel density or by lymph vessel endothelial proliferation.

Parthenolide, a sesquiterpene lactone, shows antitumor activity in vitro, which correlates with its ability to inhibit the DNA binding of the antiapoptotic transcription factor nuclear factor kappaB (NF-kappaB) and activation of the c-Jun NH(2)-terminal kinase. In this study, we investigated the chemosensitizing activity of parthenolide in vitro as well as in MDA-MB-231 cell-derived xenograft metastasis model of breast cancer. HBL-100 and MDA-MB-231 cells were used to measure the antitumor and chemosensitizing activity of parthenolide in vitro. Parthenolide was effective either alone or in combination with docetaxel in reducing colony formation, inducing apoptosis and reducing the expression of prometastatic genes IL-8 and the antiapoptotic gene GADD45beta1 in vitro. In an adjuvant setting, animals treated with parthenolide and docetaxel combination showed significantly enhanced survival compared with untreated animals or animals treated with either drug. The enhanced survival in the combination arm was associated with reduced lung metastases. In addition, nuclear NF-kappaB levels were lower in residual tumors and lung metastasis of animals treated with parthenolide, docetaxel, or both. In the established orthotopic model, there was a trend toward slower growth in the parthenolide-treated animals but no statistically significant findings were seen. These results for the first time reveal the significant in vivo chemosensitizing properties of parthenolide in the metastatic breast cancer setting and support the contention that metastases are very reliant on activation of NF-kappaB.

Macrophage inhibitory cytokine 1 (MIC-1), a divergent member of the transforming growth factor beta superfamily, plays a role in the progression of a number of cancers, including breast, gastric, prostate and colorectal carcinomas. Serum MIC-1 levels are elevated in patients with metastatic prostate, breast and colorectal carcinomas. In vitro studies have revealed a cell type-specific role for MIC-1 in senescence and apoptosis. MIC-1 activates the survival kinase AKT/PKB in neuronal cells. Depending on the cell type, it activates or represses the MAP kinases ERK1/2. Mechanisms responsible for an increased MIC-1 expression in cancers and the consequences of MIC-1 overexpression, however, are not known. In this study, we show that AKT/PKB directly regulates the expression of MIC-1 in breast cancer cells. Sequences within -88 to +30 of the MIC-1 promoter are required for the AKT-mediated induction of MIC-1. This region of the promoter contains two SP-1 binding sites (SP-1B and SP-1C), which bind to the SP-1 and SP-3 proteins. Mutation of SP-1C but not SP-1B reduced the AKT-mediated activation of MIC-1. MIC-1 increased the basal ERK1 phosphorylation and prolonged the estrogen-stimulated ERK1 phosphorylation in MCF-7 breast cancer cells without altering the phosphorylation status of AKT/PKB. Immunohistochemistry with MIC-1 antibody revealed an MIC-1 expression within the cancer cells of primary breast cancer and in the MCF-7 xenografts. Furthermore, a limited analysis of RNA from primary breast cancers revealed an overexpression of MIC-1 in tumors, compared with normal tissues. These results suggest that AKT/PKB through MIC-1 could regulate the ERK1 activity and the MIC-1 expression levels may serve as a surrogate marker for the AKT activation in tumors.