Preclinical safety evaluation of calcineurin inhibitors delivered through an intraductal route to prevent post-ERCP pancreatitis demonstrates endocrine and systemic safety.

OBJECTIVE: There is an urgent need for safe and targeted interventions to mitigate post-ERCP pancreatitis (PEP). Calcineurin inhibitors (CnIs) offer therapeutic promise as calcineurin signaling within acinar cells is a key initiating event in PEP. In previous proof-of-concept studies using experimental models, we showed that concurrent intra-pancreatic ductal administration of the CnIs, tacrolimus (Tac) or cyclosporine A (CsA) with the ERCP radiocontrast agent (RC) prevented PEP. To translate this finding clinically, we investigated potential toxic effects of intraductal delivery of a single-dose RC-CnI formulation on endocrine pancreas function and systemic toxicities in a preclinical PEP model. METHODS: C57BL/6J mice underwent ductal cannulation and received a single, intra-pancreatic ductal infusion of RC or RC with Tac or CsA (treatment groups) or underwent ductal cannulation without infusion ('sham' group). To assess endocrine function, intraperitoneal glucose tolerance test (IPGTT) was performed at two days before infusion and on day 2 and 14 post-surgery. To evaluate off-target tissue toxicities, renal and hepatic function-related parameters including blood urea nitrogen, plasma creatinine, potassium, aspartate aminotransferase, alanine aminotransferase, and total bilirubin were measured at the same time-points as IPGTT. Histological and biochemical indicators of pancreas injury and inflammation were also evaluated. RESULTS: No abnormalities in glucose metabolism, hepatic or renal function were observed on day 2 or 14 in mice administered with intraductal RC or RC with Tac or CsA. CONCLUSION: Intraductal delivery of RC-CnI formulation was safe and well-tolerated with no significant acute or subacute endocrine or systemic toxicities, underscoring its clinical utility to prevent PEP.

Rectal administration of tacrolimus protects against post-ERCP pancreatitis in mice.

OBJECTIVE: There is an unmet clinical need for effective, targeted interventions to prevent post-ERCP pancreatitis (PEP). We previously demonstrated that the serine-threonine phosphatase, calcineurin (Cn) is a critical mediator of PEP and that the FDA-approved calcineurin inhibitors, tacrolimus (Tac) or cyclosporine A, prevented PEP. Our recent observations in preclinical PEP models demonstrating that Cn deletion in both pancreatic and hematopoietic compartments is required for maximal pancreas protection, highlighted the need to target both systemic and pancreas-specific Cn signaling. We hypothesized that rectal administration of Tac would effectively mitigate PEP by ensuring systemic and pancreatic bioavailability of Tac. We have tested the efficacy of rectal Tac in a preclinical PEP model and in cerulein-induced experimental pancreatitis. METHODS: C57BL/6 mice underwent ductal cannulation with saline infusion to simulate pressure-induced PEP or were given seven, hourly, cerulein injections to induce pancreatitis. To test the efficacy of rectal Tac in pancreatitis prevention, a rectal Tac suppository (1 mg/kg) was administered 10 min prior to cannulation or first cerulein injection. Histological and biochemical indicators of pancreatitis were evaluated post-treatment. Pharmacokinetic parameters of Tac in the blood after rectal delivery compared to intravenous and intragastric administration was evaluated. RESULTS: Rectal Tac was effective in reducing pancreatic injury and inflammation in both PEP and cerulein models. Pharmacokinetic studies revealed that the rectal administration of Tac helped achieve optimal blood levels of Tac over an extended time compared to intravenous or intragastric delivery. CONCLUSION: Our results underscore the effectiveness and clinical utility of rectal Tac for PEP prophylaxis.

Novel Circulating and Tissue Monocytes as Well as Macrophages in Pancreatitis and Recovery.

BACKGROUND AND AIMS: Acute pancreatitis (AP) is an inflammatory disease with mild to severe course that is associated with local and systemic complications and significant mortality. Uncovering inflammatory pathways that lead to progression and recovery will inform ways to monitor and/or develop effective therapies. METHODS: We performed single-cell mass Cytometry by Time Of Flight (CyTOF) analysis to identify pancreatic and systemic inflammatory signals during mild AP (referred to as AP), severe AP (SAP), and recovery using 2 independent experimental models and blood from patients with AP and recurrent AP. Flow cytometric validation of monocytes subsets identified using CyTOF analysis was performed independently. RESULTS: Ly6C+ inflammatory monocytes were the most altered cells in the pancreas during experimental AP, recovery, and SAP. Deep profiling uncovered heterogeneity among pancreatic and blood monocytes and identified 7 novel subsets during AP and recovery, and 6 monocyte subsets during SAP. Notably, a dynamic shift in pancreatic CD206+ macrophage population was observed during AP and recovery. Deeper profiling of the CD206+ macrophage identified 7 novel subsets during AP, recovery, and SAP. Differential expression analysis of these novel monocyte and CD206+ macrophage subsets revealed significantly altered surface (CD44, CD54, CD115, CD140a, CD196, podoplanin) and functional markers (interferon-γ, interleukin 4, interleukin 22, latency associated peptide-transforming growth factor-ß, tumor necrosis factor-α, T-bet, RoRγt) that were associated with recovery and SAP. Moreover, a targeted functional analysis further revealed distinct expression of pro- and anti-inflammatory cytokines by pancreatic CD206+ macrophage subsets as the disease either progressed or resolved. Similarly, we identified heterogeneity among circulating classical inflammatory monocytes (CD14+CD16-) and novel subsets in patients with AP and recurrent AP. CONCLUSIONS: We identified several novel monocyte/macrophage subsets with unique phenotype and functional characteristics that are associated with AP, recovery, and SAP. Our findings highlight differential innate immune responses during AP progression and recovery that can be leveraged for future disease monitoring and targeting.

Incidence, Risk Factors, and Outcomes of Retinal Detachment after Pediatric Cataract Surgery.

PURPOSE: To report the incidence of, and to estimate the long-term risk and predisposing factors and the surgical outcomes for, retinal detachment (RD) after pediatric cataract surgery. DESIGN: Retrospective consecutive interventional case series. PARTICIPANTS: During the study period 1996 to 2007 at a tertiary eye care institute, 481 eyes of 295 children aged below 16 years with no other ocular and systemic anomalies who underwent lensectomy, posterior capsulorrhexis, and anterior vitrectomy combined with primary intraocular lens implantation were included. The median follow-up was 66 months. METHODS: Kaplan-Meier estimates and Cox proportional hazard regression model were used for estimating cumulative risk and hazard ratio (HR), respectively. Difference between measured preoperative axial length and age-matched mean axial length (prior studies) was calculated, and was defined as age-adjusted axial length difference (ALD) (minus and plus denotes myopia and hypermetropia, respectively). MAIN OUTCOME MEASURES: Cumulative risk and potential risk factors for RD. RESULTS: Of the total, 12 eyes of 9 children developed RD after cataract surgery, with a median time of 70 months. The overall risk of RD was 5.5% at 10 years after cataract surgery. All 9 children were male. The multi-adjusted HR associated with increased risk of RD was 12.42 (95% confidence interval [CI], 2.91-53.01; P = 0.001) for eyes of children with intellectual disability and 21.93 (95% CI, 2.95-162.80; P = 0.003) for eyes of children with age-adjusted ALD < -1 mm (myopic). Retinal break associated with induction of posterior vitreous detachment was the most common (8 eyes) cause of RD. No surgical intervention was done in 2 eyes. Scleral buckle and vitrectomy combined with belt buckle were performed in 4 and 6 eyes, respectively. At final follow-up, 5 and 9 eyes had a visual acuity better than or equal to 6/18 and 6/60, respectively. CONCLUSIONS: A 5.5% risk for RD is estimated for the first 10 years after cataract surgery in children with no known ocular and systemic anomalies. The risk significantly increases in a male, myopic, and intellectual disabled child. We emphasize the need for regular and long-term follow-up after pediatric cataract surgery.

Influence of serum lipids on the incidence and progression of diabetic retinopathy and macular oedema: Sankara Nethralaya Diabetic Retinopathy Epidemiology And Molecular genetics Study-II.

IMPORTANCE: The importance of lipids on incidence and progression of diabetic retinopathy has not been studied in the Indian population. BACKGROUND: To elucidate the influence of serum lipid control on the incidence and progression of diabetic retinopathy and diabetic macular oedema in subjects with type 2 diabetes. DESIGN: Population-based longitudinal observational study in a hospital setting. PARTICIPANTS: Eight hundred ninety subjects were examined at baseline and follow-up. METHODS: Diabetic retinopathy was graded per Modified Early Treatment Diabetic Retinopathy Study scales; 45°, 4-field dilated stereoscopic digital photography was performed with an additional 30°, 7-field for those who had retinopathy. Macular oedema was evaluated per Proposed International Clinical Diabetic Retinopathy and Diabetic Macular Oedema Disease Severity Scales. MAIN OUTCOME MEASURES: Association of serum lipids and incidence and progression of diabetic retinopathy. RESULTS: Poor control of total cholesterol was associated with the incidence of sight-threatening retinopathy (odds ratio = 7.2 [95% confidence interval: 1.5-34.3], P = 0.012) and macular oedema (odds ratio = 5.5 [95% confidence interval: 1.4-27.4], P = 0.037) after adjusting for potential confounders. Poor control of triglycerides was associated with progression to proliferative diabetic retinopathy (odds ratio = 3.2 [95% confidence interval: 1.1-10.5], P = 0.048). Risk for incident macular oedema (P = 0.041) and progression to proliferative diabetic retinopathy (P = 0.028) was greater when all lipid types were abnormal. CONCLUSIONS AND RELEVANCE: Poor control of lipids is a risk factor for incidence of and progression to late stages of retinopathy. Abnormal levels of all lipid types are associated with risk of incident macular oedema and progression to proliferative diabetic retinopathy.

The association of smokeless tobacco use and pack-years of smokeless tobacco with age-related macular degeneration in Indian population.

AIM: To explore the association of use versus no use and the influence of pack-year use of smokeless tobacco with that of early and late age-related macular degeneration (AMD) in rural and urban south Indian population. We hypothesized that the use and pack-years of use would be significantly associated with both early and late AMD. We therefore sought to examine subjects who gave a history of using smokeless tobacco and we quantified the usage as pack-years, to examine the association with that of early and late AMD. MATERIALS AND METHODS: This was part of Sankara Nethralaya: Rural-Urban Age-related Macular degeneration study (SN-RAM study), which was conducted between 2007 and 2010. Subjects aged 60 years or older or those turning 60 in the present calendar year, with a history of using smokeless tobacco were noted along with duration and number of packs used per day. Smokeless tobacco was defined as chewed-tobacco (loose leaves) and/or snuff (finely chopped tobacco). Subjects underwent detailed ophthalmic evaluation including cataract grading using the Lens Opacities Classification System (LOCS III), 45° 4-field stereoscopic fundus photography and AMD evaluation. Pack-years of smokeless tobacco use was stratified as <15, 15-34 and ≥35 years; the association of tobacco use and pack-years of use with that of early and late AMD was examined. A p value of < 0.05 was considered statistically significant. RESULTS: The number of smokeless tobacco users was significantly higher in rural (n = 767) than in urban groups (n = 281), p < 0.001. Of the 1048 users, 238 subjects (23%) provided details regarding quantification of use. There were no significant differences in the pack-years between rural and urban areas, p = 0.756 or that between AMD and no AMD, p = 0.562. Use of smokeless tobacco compared with no use was significantly associated with late AMD, OR= 3.178, 95%CI: 1.095, 9.227, p = 0.033, when adjusted for age, gender, rural-urban differences, presence of diabetes, socioeconomic status, systolic and diastolic blood pressure, total cholesterol, low-density and high-density lipoprotein levels. The association was not significant for early AMD, p = 0.582. The pack-years of use did not show a statistically significant association with early or late AMD. Furthermore, out of the 1048 subjects, 547 reported as using areca nut. Of which, 415 (75.8%) subjects had no AMD, 119 (21.7%) showed evidence of early AMD and 13 (2.4%) had late AMD. There was no significant association between the use of areca nut and early AMD, (X2 (1, N = 930) = 2.345, p = 0.126) or with that of late AMD (X2 (1, N = 761) = 0.075, p = 0.785). CONCLUSIONS: Smokeless tobacco use compared with no use, is associated with late AMD, regardless of the pack-years of use. Tobacco use is a modifiable risk factor. Efforts to reduce or stop the use of smokeless tobacco is indicated in an effort to prevent vision loss with respect to late AMD.

Exploiting open source omics data to advance pancreas research.

The "omics" revolution has transformed the biomedical research landscape by equipping scientists with the ability to interrogate complex biological phenomenon and disease processes at an unprecedented level. The volume of "big" data generated by the different omics studies such as genomics, transcriptomics, proteomics, and metabolomics has led to the concurrent development of computational tools to enable in silico analysis and aid data deconvolution. Considering the intensive resources and high costs required to generate and analyze big data, there has been centralized, collaborative efforts to make the data and analysis tools freely available as "Open Source," to benefit the wider research community. Pancreatology research studies have contributed to this "big data rush" and have additionally benefitted from utilizing the open source data as evidenced by the increasing number of new research findings and publications that stem from such data. In this review, we briefly introduce the evolution of open source omics data, data types, the "FAIR" guiding principles for data management and reuse, and centralized platforms that enable free and fair data accessibility, availability, and provide tools for omics data analysis. We illustrate, through the case study of our own experience in mining pancreatitis omics data, the power of repurposing open source data to answer translationally relevant questions in pancreas research.

Impaired innate and adaptive immune responses to BNT162b2 SARS-CoV-2 vaccination in systemic lupus erythematosus.

Understanding the immune responses to SARS-CoV-2 vaccination is critical to optimizing vaccination strategies for individuals with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here, we comprehensively analyzed innate and adaptive immune responses in 19 patients with SLE receiving a complete 2-dose Pfizer-BioNTech mRNA vaccine (BNT162b2) regimen compared with a control cohort of 56 healthy control (HC) volunteers. Patients with SLE exhibited impaired neutralizing antibody production and antigen-specific CD4+ and CD8+ T cell responses relative to HC. Interestingly, antibody responses were only altered in patients with SLE treated with immunosuppressive therapies, whereas impairment of antigen-specific CD4+ and CD8+ T cell numbers was independent of medication. Patients with SLE also displayed reduced levels of circulating CXC motif chemokine ligands, CXCL9, CXCL10, CXCL11, and IFN-γ after secondary vaccination as well as downregulation of gene expression pathways indicative of compromised innate immune responses. Single-cell RNA-Seq analysis reveals that patients with SLE showed reduced levels of a vaccine-inducible monocyte population characterized by overexpression of IFN-response transcription factors. Thus, although 2 doses of BNT162b2 induced relatively robust immune responses in patients with SLE, our data demonstrate impairment of both innate and adaptive immune responses relative to HC, highlighting a need for population-specific vaccination studies.

GPR15 in colon cancer development and anti-tumor immune responses.

Introduction: The chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies. Methods: In human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry. Results: Our analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to 'tumor-free' surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15 +-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden. Discussion: Our findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Autoantibodies targeting cytokines and connective tissue disease autoantigens are common in acute non-SARS-CoV-2 infections.

The widespread presence of autoantibodies in acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is increasingly recognized, but the prevalence of autoantibodies in infections with organisms other than SARS-CoV-2 has not yet been reported. We used protein arrays to profile IgG autoantibodies from 317 samples from 268 patients across a spectrum of non-SARS-CoV-2 infections, many of whom were critically ill with pneumonia. Anti-cytokine antibodies (ACA) were identified in > 50% of patients infected with non-SARS-CoV-2 viruses and other pathogens, including patients with pneumonia attributed to bacterial causes. In cell-based functional assays, some ACA blocked binding to surface receptors for type I interferons (Type I IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). Autoantibodies against traditional autoantigens associated with connective tissue diseases (CTDs) were also commonly observed in these cohorts, including newly-detected antibodies that emerged in longitudinal samples from patients infected with influenza. We conclude that autoantibodies, some of which are functionally active, may be much more prevalent than previously appreciated in patients who are symptomatically infected with diverse pathogens.

The aryl hydrocarbon receptor regulates expression of mucosal trafficking receptor GPR15.

GPR15 is a chemoattractant receptor that facilitates colon homing of regulatory and effector CD4+ T cells in health and colitis. The molecular mechanisms that control GPR15 expression are not fully known. Here we report the presence of two highly conserved aryl hydrocarbon receptor (AHR) binding sequences in a 3' enhancer of GPR15, leading us to investigate AHR function in regulating GPR15 expression. Using luciferase reporter assays, we show that AHR activation increased GPR15 expression and requires both the AHR binding sites. Consistent with a transcriptional regulatory role, treatment with AHR agonists induce GPR15 expression on human CD4+ T cells. Using AHR-deficient mice, we demonstrate that the lack of AHR signaling drastically reduces GPR15 expression on effector/memory and Foxp3+ CD4+ T cells. In mixed bone marrow chimeras of AHR-deficient and wildtype cells, GPR15 expression was similarly diminished on AHR-deficient CD4+ effector/memory and regulatory T cells in the colon and small intestine. Furthermore, administration of AHR agonists upregulated GPR15 expression on CD4+ effector/memory T cells and increased their homing capability, especially to the colon. Collectively, our studies reveal a novel function of the AHR in regulation of GPR15 expression and increased colon trafficking of CD4+ T cells expressing GPR15.

The prevalence and risk factors for cataract in rural and urban India.

PURPOSE: To report the prevalence and risk factors of cataract and its subtypes in older age group. METHODS: A total of 6617 subjects were recruited from both rural and urban areas. A detailed history including data on demographic, socioeconomic and ocular history was obtained. Lens opacity was graded according to the Lens Opacity Classification System III (LOCS III). RESULTS: Cataract was present in 1094 of the rural and 649 subjects in the urban population. Monotype subtype cataracts were found in 32% and 25% in rural and urban population and 12.68% and 18.6% were mixed cataracts in the rural and urban groups. In baseline characteristics history of diabetes, alcohol intake and presence of age-related macular degeneration were the risk factors in urban group. On multivariate analysis, the only significant risk factors for any cataract in subjects ≥60 years were increasing age in both rural [odds ratio (OR), 1.07] and urban (OR, 1.08) population, and HbA1c (OR, 1.14) in rural population. Overweight (OR, 0.6) was found to be a protective factor, and lower social economic status (OR, 1.52) a risk factor for cataract in urban population. A significant urban-rural difference was found in the prevalence of cataract and its subtypes (P ≤ 0.05). CONCLUSION: We found the risk factors for any cataract in older age group to be increasing age and HbA1c in rural group. Age and lower social economic status were found to be the risk factors in urban arm. A statistically significant difference was found on comparison of the prevalence of cataract and its subtypes between the rural and urban population.

Pupillary Abnormalities with Varying Severity of Diabetic Retinopathy.

Our aim is to study the dynamics of pupillary abnormalities in varying severity of diabetic retinopathy. A non-interventional case-control study with 405 eyes of 244 subjects with diabetes, and 41 eyes of 26 subjects with no history of diabetes was done. Diabetes group was classified according to retinopathy severity: no retinopathy, mild non-proliferative diabetic retinopathy (NPDR), moderate NPDR, severe NPDR and proliferative diabetic retinopathy (PDR). After dark adaptation, pupil size and flashlight response were captured with an infrared camera. Baseline Pupil Diameter (BPD), Amplitude of Pupillary Constriction (APC), Velocity of Pupillary Constriction (VPC) and Velocity of Pupillary Dilatation (VPD). Compared to controls, mean BPD decreased with increasing severity of diabetic retinopathy. Mean APC in control group was 1.73 ± 0.37 mm and reduced in mild NPDR (1.57 ± 0.39, p = 1.000), moderate NPDR (1.51 ± 0.44, p = 0.152) and found to be significant reduced in severe NPDR (1.43 ± 0.48, p = 0.001) and PDR (1.29 ± 0.43, p = 0.008). Compared to controls, mean VPC decreased progressively with increasing severity of retinopathy, with a maximal difference in the PDR group. Mean VPD as compared to the control group was significantly reduced in the no DR (p = 0.03), mild NPDR (p = 0.038), moderate NPDR (p = 0.05), PDR group (p = 0.02). We found pupillary dynamics are abnormal in early stages of diabetic retinopathy and progress with increasing retinopathy severity.

Association of obesity and age-related macular degeneration in Indian population.

Purpose: The aim of this study was to establish the prevalence and association of age-related macular degeneration (AMD) and obesity which was not studied extensively in Indian population over 60 years of age. Methods: This was a cross-sectional, population-based study. A total of 4791 patients with gradable fundus photography were included. All patients underwent detailed ophthalmic examination and AMD was graded with retinal photographs. Grading of AMD was done according to the International ARM Epidemiological Study Group and staged based on grading in worse eye. The association of AMD severity and obesity (based on body mass index, waist-hip ratio, waist circumference, isolated abdominal obesity, isolated generalized obesity, and combined obesity) was assessed. The main outcome variable was an association between the presence and severity of AMD with different grades of obesity. Results: No direct significant association was noted between the presence and severity of AMD and any obesity indices. Subgroup analyses based on lifestyle patterns and common systemic pathologies in AMD population were done. Late AMD was significantly associated with tobacco consumption in population with combined obesity (P = 0.033 and odds ratio = 2.998). Conclusion: No direct association was noted between the presence or severity of AMD and obesity in South Indian population. However, indirect associations between the severity of AMD and combined obesity were found.

Incidence, Progression, and Risk Factors for Cataract in Type 2 Diabetes.

Purpose: We report the 4-year incidence, progression, and risk factors of cataract subtypes in type 2 diabetes. Methods: A total of 779 subjects completed baseline and 4-year follow-up. Results: The incidences of nuclear opalescence (NO), nuclear color (NC), cortical cataract (CC), and posterior subcapsular cataract (PSC) were 70%, 55.2%, 25.7%, and 7.3%, respectively. One-step progressions of NO, NC, CC, and PSC were 14.3%, 16.1%, 8.8%, and 8.1%, respectively, and two-step or more progressions were 5.0%, 6.0%, 0.8%, and 6.0%, respectively. Incident NO was seen in patients 50 to 59 (odds ratio [OR] = 3.3), NC in those 50 to 59 (OR = 2.7) and 60 to 69 (OR = 3.9), and CC in those 60 to 69 (OR = 3.3) years old. A lower hemoglobin A1c (HbA1c; OR = 0.7), longer diabetes duration (OR = 1.1), and hyperopia (OR = 4.0) were associated with incident PSC. Women (OR = 1.7) and patients with higher total cholesterol (OR = 1.3) at baseline showed one-step NO progression. Patients 60 to 69 (OR = 2.8) and ≥70 (OR = 3.8) years old showed one-step NC progression, while those 60 to 69 years old showed one-step CC progression (OR = 6.3). A lower HbA1c (OR = 0.3) was associated with one-step PSC progression. A higher low-density lipoprotein (OR = 1.6) was associated with two-step or more NO progression. Patients 60 to 69 years old (OR = 6.7) had a greater risk, while those with hyperopia at baseline (OR = 0.2) had lower risk of two-step or more NC progression. Patients 40 to 49 years old constituted the reference group for all. Conclusions: The 4-year cumulative incidence of cataract is higher than that of progression. Greater age is a risk factor for incidence and progression of most types of cataract.

BECN1/Beclin 1 sorts cell-surface APP/amyloid ß precursor protein for lysosomal degradation.

The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid ß precursor protein), whose metabolism to amyloid-ß, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267-337). Deletion of a BECN1 ECD subregion (amino acids 285-299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.

TULA: an SH3- and UBA-containing protein that binds to c-Cbl and ubiquitin.

Downregulation of protein tyrosine kinases is a major function of the multidomain protein c-Cbl. This effect of c-Cbl is critical for both negative regulation of normal physiological stimuli and suppression of cellular transformation. In spite of the apparent importance of these effects of c-Cbl, their own regulation is poorly understood. To search for possible novel regulators of c-Cbl, we purified a number of c-Cbl-associated proteins by affinity chromatography and identified them by mass spectrometry. Among them, we identified the UBA- and SH3-containing protein T-cell Ubiquitin LigAnd (TULA), which can also bind to ubiquitin. Functional studies in a model system based on co-expression of TULA, c-Cbl, and EGF receptor in 293T cells demonstrate that TULA is capable of inhibiting c-Cbl-mediated downregulation of EGF receptor. Furthermore, modulation of TULA concentration in Jurkat T-lymphoblastoid cells demonstrates that TULA upregulates the activity of both Zap kinase and NF-AT transcription factor. Therefore, our study indicates that TULA counters the inhibitory effect of c-Cbl on protein tyrosine kinases and, thus, may be involved in the regulation of biological effects of c-Cbl. Finally, our results suggest that TULA-mediated inhibition of the effects of c-Cbl on protein tyrosine kinases is caused by TULA-induced ubiquitylation and degradation of c-Cbl.

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.