Histological Phenotype Research Articles

Inhibition of autophagy rescues muscle atrophy in a LGMDD2 Drosophila model.

Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. Here we report the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, we provide a proof of concept of autophagy as a target to treat disease phenotypes and propose a neurogenic component to explain mutant TNPO3 pathogenicity in diseased muscles.

Lack of 5-lipoxygenase in intramembranous and endochondral 129Sv mice skeleton and intramembranous healing

ObjectiveTo analyze the lack of 5-lipoxygenase (5LO) on dental socket healing and post-natal phenotype of intramembranous and endochondral bones. DesignWild type (WT) 129/SvEv (n = 20) and 5LO knockout (5LOKO) (n = 20) male mice underwent tooth extraction of the upper right incisor and were euthanized after 7, 14, and 30 day time points for the evaluation of dental socket healing and histological phenotyping of intramembranous (IM) and endochondral (EC) bones. Microscopic analysis of alveolar sockets included histopathological description, histomorphometry, and immunohistochemistry for 5LO, cyclooxygenase 2 (COX2), and tartrate resistant acid phosphatase (TRAP). ResultsHistological phenotyping revealed thicker cortical bone in EC bones (femur and vertebra) of 5LOKO mice compared to WTs, with no differences in collagenous content. Although dental socket healing was similarly observed in both groups, WT mice revealed increased numbers of COX-2+ and 5LO+ cells during bone maturing stage, with a decrease of TRAP+ cells at day 30. On the other hand, an increased quantity of fibroblasts was observed at day 7 in 5LOKO group, as well as increased inflammatory infiltrate and significantly decreased TRAP+ cells at final stages of alveolar socket healing in comparison to WTs. ConclusionsThe lack of 5LO in 5LOKO mice resulted in thicker cortical of EC, but not of IM post natal bones. Furthermore, genetic deletion of 5LO in the 5LOKO mice directly affected the inflammatory response during socket healing, influencing initial and late phases of bone repair in a model of post-tooth extraction in 129Sv WT and 5LOKO mice.

Single-cell RNA sequencing reveals the epithelial cell heterogeneity and invasive subpopulation in human bladder cancer.

Bladder cancer represents a highly heterogeneous disease characterized by distinct histological, molecular and clinical phenotypes, and a detailed analysis of tumor cell invasion and crosstalks within bladder tumor cells has not been determined. Here, we applied droplet-based single-cell RNA sequencing (scRNA-seq) to acquire transcriptional profiles of 36 619 single cells isolated from seven patients. Single cell transcriptional profiles matched well with the pathological basal/luminal subtypes. Notably, in T1 tumors diagnosed as luminal subtype, basal cells displayed characteristics of epithelial-mesenchymal transition (EMT) and mainly located at the tumor-stromal interface as well as micrometastases in the lamina propria. In one T3 tumor, muscle-invasive tumor showed significantly higher expression of cancer stem cell markers SOX9 and SOX2 than the primary tumor. We additionally analyzed communications between tumor cells and demonstrated its relevance to basal/luminal phenotypes. Overall, our single-cell study provides a deeper insight into the tumor cell heterogeneity associated with bladder cancer progression.

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT.

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

Infrequent RAS mutation is not associated with specific histological phenotype in gliomas

BackgroundMutations in driver genes such as IDH and BRAF have been identified in gliomas. Meanwhile, dysregulations in the p53, RB1, and MAPK and/or PI3K pathways are involved in the molecular pathogenesis of glioblastoma. RAS family genes activate MAPK through activation of RAF and PI3K to promote cell proliferation. RAS mutations are a well-known driver of mutation in many types of cancers, but knowledge of their significance for glioma is insufficient. The purpose of this study was to reveal the frequency and the clinical phenotype of RAS mutant in gliomas.MethodsThis study analysed RAS mutations and their clinical significance in 242 gliomas that were stored as unfixed or cryopreserved specimens removed at Kyoto University and Osaka National Hospital between May 2006 and October 2017. The hot spots mutation of IDH1/2, H3F3A, HIST1H3B, and TERT promoter and exon 2 and exon 3 of KRAS, HRAS, and NRAS were analysed with Sanger sequencing method, and 1p/19q codeletion was analysed with multiplex ligation-dependent probe amplification. DNA methylation array was performed in some RAS mutant tumours to improve accuracy of diagnosis.ResultsRAS mutations were identified in four gliomas with three KRAS mutations and one NRAS mutation in one anaplastic oligodendroglioma, two anaplastic astrocytomas (IDH wild-type in each), and one ganglioglioma. RAS-mutant gliomas were identified with various types of glioma histology.ConclusionRAS mutation appears infrequent, and it is not associated with any specific histological phenotype of glioma.

Case Report and Literature Review: Pulmonary Sclerosing Pneumocytoma With Multiple Metastases Harboring AKT1 E17K Somatic Mutation and TP53 C176Y Germline Mutation

Pulmonary Sclerosing Pneumocytoma (PSP) is considered as a benign tumor, although a few cases have been reported to have multiple lesions, recurrence, and even regional lymph nodes (LNs) metastasis. Here, we report a case of PSP with atypical histologic features and malignant biological behavior, and explore its molecular genetic changes. The 23-year-old male showed a 6.5-cm pulmonary nodule in the right middle lobe (RML) and enlarged media stinal LNs. He underwent thoracoscopic RML lobectomy, systematic LNs dissection, and mediastinal lymphadenectomy. The metastases to the cervical LNs and liver were detected in a short period and then resected. Postoperative pathological examination confirmed the diagnosis of PSP in all the lesions, based on the histological characteristics and immune phenotypes. Furthermore, whole-exome sequencing identified both AKT1 E17K somatic mutation and TP53 C176Y germline mutation in this case. Thus, we presented an extremely rare case of atypical PSP with rapid recurrence and multiply metastases, which can easily be misdiagnosed as primary lung cancer. In addition, PSP-specific AKT1 E17K somatic E17K somatic mutation accompanied with TP53 C176Y germline mutation may contribute to the malignant clinical course of this tumor.

Targeting S1PR1 May Result in Enhanced Migration of Cancer Cells in Bladder Carcinoma.

Simple SummaryMetastasis is critical to the prognosis of patients with bladder cancer, and it is important to understand the mechanism of its occurrence. S1PR1 expression is thought to be associated with poor prognosis, but it is unknown whether it is associated with tumor metastasis. Analysis of clinical gene expression data suggests that endothelial or immune cells in tumor tissue may be the source of S1PR1 expression. Comparative analysis of clinical tumor tissues with bladder cancer cells suggests that S1PR1 expression is associated with cellular adhesion. In vitro experiments demonstrated that S1PR1 expression was negatively correlated with cancer cell motility, and that S1PR1 inhibition by FTY-720 may cause an increase in cancer cell motility, suggesting that the use of S1PR1 inhibition as a synergistic therapy requires additional observations and considerations.Clinical bladder tumor histological analysis shows that high expression of S1PR1 is associated with poor patient prognosis. However, there are no studies that describe the underlying mechanism. To investigate the relative distribution and actual function of S1PR1 in bladder tumors, we analyzed multiple clinical databases in combination with tumor purity and immune cell infiltration simulations, as well as databases of well-defined histological phenotypes of bladder cancer, and single-cell sequencing of adjacent normal tissues and bladder tumors, and further compared them with bladder cancer cell lines. The results showed that S1PR1 expression was generally higher in normal tissues than in bladder cancer tissues, and its distribution was mainly in endothelial cells or immune cells. The association between high S1PR1 expression and poor prognosis may be due to tumor invasion of adjacent normal tissues, where highly expressed S1PR1 may affect prognostic interpretation. The effect of S1PR1 itself on cancer cells was associated with cell adhesion, and in bladder cancer cells, S1PR1 expression was negatively correlated with cell motility. Moreover, the use of FTY-720 will cause an increased metastatic ability of bladder cancer cells. In conclusion, we suggest that the use of S1PR1-specific inhibition as a synergistic treatment requires more observation and consideration.

MP22-12 MAGNETIC RESONANCE IMAGING FEATURES ASSOCIATED WITH HISTOLOGY OF BENIGN PROSTATIC HYPERPLASIA NODULES: A PREDICTIVE MODEL

You have accessJournal of UrologyImaging/Radiology: Uroradiology II (MP22)1 Sep 2021MP22-12 MAGNETIC RESONANCE IMAGING FEATURES ASSOCIATED WITH HISTOLOGY OF BENIGN PROSTATIC HYPERPLASIA NODULES: A PREDICTIVE MODEL Jessica Dai, Tara Morgan, Ramy Goueli, Daniel Parrott, Douglas Strand, Daniel Costa, Claus Roehrborn, and Jeffrey Gahan Jessica DaiJessica Dai More articles by this author , Tara MorganTara Morgan More articles by this author , Ramy GoueliRamy Goueli More articles by this author , Daniel ParrottDaniel Parrott More articles by this author , Douglas StrandDouglas Strand More articles by this author , Daniel CostaDaniel Costa More articles by this author , Claus RoehrbornClaus Roehrborn More articles by this author , and Jeffrey GahanJeffrey Gahan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002013.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Histologic phenotypic variation of benign prostatic hyperplasia (BPH) has been previously described and is hypothesized to underlie response to medical therapy. In this study, we evaluate pre-operative magnetic resonance imaging (MRI) of robotic simple prostatectomy (RASP) specimens and determine imaging features associated with histologic phenotype. METHODS: All patients undergoing RASP from 11/2015 to 11/2019 with multiparametric MRI ≤1 year before surgery were included. Those with no obvious BPH nodules on histologic specimens were excluded. Histology slides and corresponding MRI were reviewed independently by a blinded cell biology Ph.D. and radiologist, respectively. Each nodule was assigned a phenotypic score on a 5-point Likert scale (1=predominantly glandular, 5=predominantly stromal). Scores were compared using the sign test and univariate analysis. Signal intensity relative to transition zone and nodule texture were noted on T2, diffusion-weighted imaging (DWI), and dynamic contrast-enhanced imaging (DCE) sequences (Figure 1). Univariate and multiple stepwise linear regression analysis were conducted to identify MRI features associated with histology score. All analyses were performed using Statistical Analysis System (SAS v 9.4). RESULTS: 99 prostate nodules in 29 patients were included. Median phenotypic scores by histology and MRI were comparable (2, IQR 2-3 vs. 2, IQR 2-4, respectively; p=0.63). Histology scores were positively associated with MRI scores on univariate analysis (p <0.0001). Multiple stepwise linear regression analysis showed apparent diffusion coefficient (ADC) low signal intensity and DCE wash-in to be positively associated with more stromal histology score, and ADC SD, DCE wash-out, and heterogeneous T2 texture to be negatively associated with more stromal histology score (Table 1). CONCLUSIONS: There is strong correlation between MRI features and the histologic phenotype of BPH nodules. MRI may provide a non-invasive method to determine underlying BPH nodule histology. Source of Funding: DK115477 (DWS) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e395-e396 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jessica Dai More articles by this author Tara Morgan More articles by this author Ramy Goueli More articles by this author Daniel Parrott More articles by this author Douglas Strand More articles by this author Daniel Costa More articles by this author Claus Roehrborn More articles by this author Jeffrey Gahan More articles by this author Expand All Advertisement Loading ...

Eosinophilic features in clear cell renal cell carcinoma correlate with outcomes of immune checkpoint and angiogenesis blockade

BackgroundClear cell renal cell carcinoma (ccRCC) displays heterogeneity in appearance—a distinctive pale clear to eosinophilic cytoplasm; however, little is known about the underlying mechanisms and clinical implications. We investigated the...

Genetic ablation of Gpnmb does not alter synuclein-related pathology

The gene GPNMB is known to play roles in phagocytosis and tissue repair, and is upregulated in microglia in many mouse models of neurodegenerative disease as well as in human patients. Nearby genomic variants are associated with both elevated Parkinson's disease (PD) risk and higher expression of this gene, suggesting that inhibiting GPNMB activity might be protective in Parkinson's disease. We tested this hypothesis in three different mouse models of neurological diseases: a remyelination model and two models of alpha-synuclein pathology. We found that Gpnmb deletion had no effect on histological, cellular, behavioral, neurochemical or gene expression phenotypes in any of these models. These data suggest that Gpnmb does not play a major role in the development of pathology or functional defects in these models and that further work is necessary to study its role in the development or progression of Parkinson's disease.

O-118 New insight into the genetic contribution of common variants to the development of extreme phenotypes of unexplained male infertility: a multicenter genome-wide association study

Abstract Study question What is the contribution of the common genetic variation to the development of unexplained male infertility due to severe spermatogenic failure (SPGF)? Summary answer Genetic polymorphisms of key immune and spermatogenesis loci are involved in the etiology of the most severe SPGF cases, defined by Sertoli cell-only (SCO) phenotype. What is known already Male infertility is a rising worldwide concern that affects millions of couples. Non-obstructive azoospermia (NOA) and severe oligospermia (SO) are two extreme manifestations characterized by SPGF. A genetic cause can be established in only around 20% of affected men, with the remaining cases being classified as otherwise unexplained. To date, the genome-wide association study (GWAS) strategy, although already successfully applied in several other complex traits and diseases, was less fruitful in studies that attempted to decipher the genetic component of unexplained SPGF, mainly due to both a lack of well-powered samples in different ancestries and limitations in study design. Study design, size, duration We designed a GWAS for unexplained male infertility due to SPGF including a total of 1,274 affected cases and 1,951 fertile controls from the Iberian Peninsula (Spain and Portugal) and Germany. Different biostatistics and bioinformatics approaches were used to evaluate the possible effect of single-nucleotide polymorphisms (SNPs) across the whole genome in the susceptibility to specific subtypes of unexplained SPGF. Participants/materials, setting, methods The case cohort comprised 502 SO and 772 NOA patients, who were subdivided according to histological phenotypes (SCO, maturation arrest, and hypospermatogenesis) and the outcome of testicular sperm extraction techniques (TESE) from testis biopsies. Genotyping was performed with the GSA platform (Illumina). After quality-control and genotype imputation, 6,539,982 SNPs remained for the analysis, which was performed by logistic regression models. The datasets went through a meta-analysis by the inverse variance weighted method under fixed effects. Main results and the role of chance Genetic associations with SCO at the genome-wide-level of significance were identified in the major histocompatibility (MHC) class II region (rs1136759, OR = 1.80, P = 1.32E-08) and in a regulatory region of chromosome 14 nearby the vaccinia-related kinase 1 (VRK1) gene (rs115054029, OR = 3.14, P = 4.37-08). VRK1 is a relevant proliferative factor for spermatogenesis that causes progressive loss of spermatogonia when disrupted in mouse models. The role of the MHC system in SCO susceptibility was comprehensively evaluated through a validated imputation method that infers classical MHC alleles and polymorphic amino acid positions. A serine at position 13 of the HLA-DRβ1 protein (defined by the risk allele of the lead variant rs1136759) explained most of the SCO association signals within the MHC class II region. This residue is located in the binding pocket of the HLA-DR molecule and interacts directly with the presented antigen. Interestingly, position 13 of HLA-DRβ1 is the most relevant risk amino acid position for a wide spectrum of immune-mediated disorders. The HLA-DRB1*13 haplotype (which includes the serine at position 13 and represents the strongest NOA-associated marker in Asians to date) was the strongest signal amongst the classical MHC alleles in our study cohort (OR = 1.93, P = 9.90E-07). Limitations, reasons for caution Although the statistical power for the overall analysis was appropriate, the subphenotype analyses performed had considerably lower counts, which may influence the identification of genetic variants conferring low to moderate risk effects. Independent studies in larger SCO study cohorts should be performed to confirm our findings. Wider implications of the findings The molecular mechanisms underlying unexplained SPGF are largely unknown. Our data suggest a relevant role of common genetic variation in the development of SCO, the most extreme histological phenotype of NOA. SCO is characterized by the loss of germ cells and, therefore, implies a considerably higher probability of unsuccessful TESE. Trial registration number N/A

Deep cross-view co-regularized representation learning for glioma subtype identification

The new subtypes of diffuse gliomas are recognized by the World Health Organization (WHO) on the basis of genotypes, e.g., isocitrate dehydrogenase and chromosome arms 1p/19q, in addition to the histologic phenotype. Glioma subtype identification can provide valid guidances for both risk-benefit assessment and clinical decision. The feature representations of gliomas in magnetic resonance imaging (MRI) have been prevalent for revealing underlying subtype status. However, since gliomas are highly heterogeneous tumors with quite variable imaging phenotypes, learning discriminative feature representations in MRI for gliomas remains challenging. In this paper, we propose a deep cross-view co-regularized representation learning framework for glioma subtype identification, in which view representation learning and multiple constraints are integrated into a unified paradigm. Specifically, we first learn latent view-specific representations based on cross-view images generated from MRI via a bi-directional mapping connecting original imaging space and latent space, and view-correlated regularizer and output-consistent regularizer in the latent space are employed to explore view correlation and derive view consistency, respectively. We further learn view-sharable representations which can explore complementary information of multiple views by projecting the view-specific representations into a holistically shared space and enhancing via adversary learning strategy. Finally, the view-specific and view-sharable representations are incorporated for identifying glioma subtype. Experimental results on multi-site datasets demonstrate the proposed method outperforms several state-of-the-art methods in detection of glioma subtype status.

Abstract 3014: A preclinical platform of PDX-derived cell lines as a tool for pharmacological screening and functional studies

Abstract Despite considerable progress in understanding the biology and genetics of cancer progression, the development of effective therapies against cancer need physiological and predictive preclinical models. In this context, patient-derived xenograft (PDX) models has become a standard tool as they reproduce accurately the behavior of tumor of origin, in term of histological and molecular phenotype and response to chemotherapy. Although PDXs in vivo models are indispensable for preclinical studies, they suffer from some limitations due to study costs related to tumor maintenance on mice, variable engraftment rate, growth delay and limited throughput for large-scale drug screening. To address this problem and propose a time a cost effective preclinical screening tool, we developed a panel of PDX-derived low-passage 2D cell lines as a convenient in vitro pre-screening platform to profile compound activity. Different PDX models including breast, lung, colon, melanoma, glioblastoma and hepatoblastoma were tested for their capacity to generate cell lines maintaining the characteristics of the parental PDX tumor and usable for in vitro assays. Today, we succeeded with a series of 50 PDX models with 83% success rate. Tumor cells isolated from PDX tumor tissue were cultured under different media and matrix conditions, allowing at least 5 passages in culture. A Short Tandem Repeat (STR) comparison profile was done with the parental PDX before performing a master bank. We performed short term 2D cytotoxicity assays and long term colony assays to compare cell lines in vitro drug sensitivity with their parental PDX in vivo drug response and overall, the results show that this panel reproduced the drug response profile of the original PDXs with chemotherapies, PARP inhibitors, an ADC (T-DM1) and FGFR-targeting therapies. Moreover, cellular models engrafted back onto mice showed in vivo response to chemotherapies similar to that of the parental PDX confirming the identical behavior of cell line/PDX couples. As the use of cellular models is still considered as a standard for early preclinical test to evaluate drug response before moving to in vivo assays, our PDX-derived cell line platform appeared to be a robust and relevant tool. Furthermore, since the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models, the similarities between cell lines and parental PDX should maximize success of further in vivo preclinical drug development. Citation Format: Olivier Deas, Sophie Banis, Kathleen Flosseau, Lea Sinayen, Enora Le Ven, Jean-Gabriel Judde, Stefano Cairo. A preclinical platform of PDX-derived cell lines as a tool for pharmacological screening and functional studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3014.

Rhabdoid Signet- Ring Mixed Cell Subtype Gastrointestinal Stromal Tumor from the Stomach - Pathohistological and Immunohistochemical Analysis, Differential Diagnosis, Prognosis and Complex Treatment

We present rarely diagnosed extremely malignant rhabdoid signet- ring mixed cell subtype gastrointestinal stromal tumor (GIST) from the stomach in a man of 67 years. This is a third rhabdoid histological phenotype GIST posted in the English medical literature. The diagnosis is placed after a pathochistological and immunohistochemical analysis of the lung biopsy material. The purpose of this presentation is to discuss the difficulty early diagnostics, the prognosis of this extremely malignant GIST, the differential pathohistological and immunohistochemical diagnosis with other benign and malignant tumors. The diagnosis of a GISTs is directly related to treatment with a tyrosine kinase inhibitor (Imatinib), which is indicated for inoperable and metastatic GIST.

Gastric neoplasms in patients with familial adenomatous polyposis: endoscopic and clinicopathologic features

Gastric neoplasms in patients with familial adenomatous polyposis (FAP) occur at a high rate and can cause death. The endoscopic findings of gastric neoplasms in these patients are characteristic but not well recognized. To identify the relevant characteristics to enable early detection, we retrospectively investigated endoscopic findings of gastric neoplasms in patients with FAP and then compared the clinical, histopathologic, and genetic features among subgroups. Of 234 patients with 171 pedigrees at 2 institutes, 56 cases (24%, 133 gastric neoplasms) with 44 pedigrees were examined. Immunostaining was performed for histopathologic evaluation by 1 blinded pathologist. According to the endoscopic findings, gastric neoplasms were divided into 4 types based on location (L: antrum and pylorus, UM: the rest of the stomach) and color (W: white, T: translucent, R: reddish) and their clinicopathologic features examined. Of the cases, 93% could be classified into a single type. Among histologic phenotypes, high-grade dysplasia was present in 26% (type L), 41% (type UM-W), 0% (type UM-T), and 22% (type UM-R). The immunologic phenotype comprised the gastric type in 69% (93% in Type UM) and the intestinal phenotype, including the mixed type, in 31% (61% in type L). Moreover, 96% of patients had concurrent duodenal neoplasms. Adenomatous polyposis coli gene status was identified in 93% of patients; the pathogenic variant was detected in 98% but did not influence any endoscopic features. Gastric neoplasms in patients with FAP were stratified into 4 types according to their endoscopic findings. The endoscopic phenotype was related to the histopathologic phenotype but not to germline variants.

Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher’s disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson’s disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.

Liver tissue microbiota in nonalcoholic liver disease: a change in the paradigm of host-bacterial interactions.

Nonalcoholic fatty liver disease (NAFLD) pathogenesis is explained by the complex relationship among diet and lifestyle-predisposing factors, the genetic variance of the nuclear and mitochondrial genome, associated phenotypic traits, and the yet not fully explored interactions with epigenetic and other environmental factors, including the microbiome. Despite the wealth of knowledge gained from molecular and genome-wide investigations in patients with NAFLD, the precise mechanisms that explain the variability of the histological phenotypes are not fully understood. Earlier studies of the gut microbiota in patients with NAFLD and nonalcoholic steatohepatitis (NASH) provided clues on the role of the fecal microbiome in the disease pathogenesis. Nevertheless, the composition of the gut microbiota does not fully explain tissue-specific mechanisms associated with the degree of disease severity, including liver inflammation, ballooning of hepatocytes, and fibrosis. The liver acts as a key filtration system of the whole body by receiving blood from the hepatic artery and the portal vein. Therefore, not only microbes would become entrapped in the complex liver anatomy but, more importantly, bacterial derived products that are likely to be potentially powerful stimuli for initiating the inflammatory response. Hence, the study of liver tissue microbiota offers the opportunity of changing the paradigm of host-NAFLD-microbial interactions from a "gut-centric" to a "liver-centric" approach. Here, we highlight the evidence on the role of liver tissue bacterial DNA in the biology of NAFLD and NASH. Besides, we provide evidence of metagenomic findings that can serve as the seed of further hypothesis-raising studies as well as can be leveraged to discover novel therapeutic targets.

Metabolomics analysis underlay mechanisms in the renal impairment of mice caused by combination of aflatoxin M1 and ochratoxin A

Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are pernicious mycotoxins widely co-existing in the environment. However, nephrotoxicity and underlying mechanism induced by AFM1 coupled with OTA still remain to be explored. In this study, CD-1 mice were treated with 3.5 mg/kg b.w. AFM1, OTA, and AFM1 + OTA for 35 days, and UPLC-MS-based metabolomics method was effectuated to investigate metabolomic profiles of mice kidney. Subsequent experiments on human renal proximal tubular (HK-2) cells were performed to dig out the causal connections between distinguished differential metabolites and nephrotoxicity. Compared with DMSO vehicle group, all three toxin treatments (AFM1 and OTA alone, and in combination) significantly reduced final body weight, and remarkably elevated the concentration of serum creatinine (SCr) and caused abnormal histological phenotypes (shown by histopathological slices). OTA, AFM1 + OTA but not AFM1 reduced the relative weight index of kidney. These phenotypic results indicated that AFM1 and OTA were both toxic to the body, and it seemed that OTA exhibited a notable impairment to kidney while AFM1 had similar but limited effect compared with OTA. Further metabolomics analysis showed that when AFM1 and OTA were combined together, OTA exerted dominant effect on the alteration of metabolic processes. There were few differences in the number of changed metabolites between OTA and AFM1 + OTA group. Among the differentially expressed metabolites affected by OTA, and AFM1 + OTA, lysophosphatidylcholines (LysoPCs) were identified as the main type with significant upregulation, in which LysoPC (16:0) accounted for the most prime proportion. Western blotting results of HK-2 cells showed that single OTA and AFM1 + OTA increased the apoptotic protein expressions of Bax, caspase 3 and PARP, and decreased the expression of Bcl-2; while AFM1 only raised the expression of caspase 3. LysoPC (16:0) but not LysoPC (18:1) lifted the protein level of caspase 3 and PARP in HK-2 cells, and reduced the level of Bcl-2. Taken together, this study is the first effort trying to assess nephrotoxicity of AFM1 with OTA, and we guessed that OTA had a more pronounced toxicity to kidney in contrast to AFM1. No obvious synergism between AFM1 and OTA was found to contribute to the occurrence or development of nephropathy. LysoPC (16:0) might be the pivotal metabolite in response to single OTA and combined AFM1 + OTA engendering renal injury.

MO268GLOMERULAR C3 DEPOSITS IN ANCA ASSOCIATED PATIENTS – IS IT AN IMPORTANT FINDING?

Abstract Background and Aims In recent years there were many studies explaining the role of alternative complement pathway (aCP) in etiopathogenesis of the ANCA associated vasculitis (AAV) and weather it affects patient outcomes. We hypothesized the importance of the C3 positive staining in glomeruli and that there could be differences between clinical, serological and histological phenotypes in AAV patients. Method This study included 106 consecutive AAV patients with renal involvement in the period from 2007-2017. We performed renal biopsy on patients using automatic 16 Gauge needle. Light, immunofluorescent and electronic microscopy were performed. Category variables were analysed with Fisher Exact testom and continuous with Kruskal-Wallis testom. Statistical difference was then analysed posthoc with Chi-square test. Primary outcomes were combined outcome progression to end-stage renal disease, defined as persistent (more than three months) need for renal replacement therapy or permanent reduction of EGFR to &amp;lt;15ml/minute (according to CKD EPI formula) and/or death (ESRDD), death (D) and ESRD alone, and disease relapse. Kaplan Meyer survival analysis and multivariate Cox proportional hazard regression analysis were used to explore difference between phenotypes and finding significant predictors regarding outcomes. Results The study included 106 AAV patients with renal involvement: 66 (61,1%) MPA, 20 (18,5%) GPA, 20 (18,5%) RLV. There were 14 (13%) PR3-ANCA positive patients, 57 (52,8%) MPO ANCA positive, 5 (4,6%) PR3-ANCA+MPO-ANCA and 32 (29,6%) ANCA negative patients. Average SCr was 316,5 μmol/l (IQR 207,0-548,5), 24-hour proteinuria median was 1,7g/24h (IQR 0,8-2,8). Histologicaly (Berden classification) 43 (39,8%) patients had crescentic, 19 (17,6%) focal, 34 (31,5%) mixed and 12 (11,1%) sclerotic class. Out of all, 48,5% MPA patients had C3 deposits in glomeruli compared to 25% GPA and 35% RLV though statistical significance was not reached (p=0,1). Same applied for serological phenotypes: MPO-ANCA and ANCA negatives had more C3 deposits but statistical significance was not reached (p=0,6). When comparing histopathological classes there was strong tendency of crescentic class having more C3 deposits (p=0,076) compared to focal and mixed. In sclerotic class 50% had C3 deposits but C3 deposits can be ordinary found in sclerotic lesions so the interpretation is more challenging. Interestingly patients requiring haemodialysis had strong tendency of having more C3 deposits compared to those not needing haemodialysis treatment (p=0,09). Conclusion Glomerular C3 deposits in kidney tissue samples could prove to be a useful tool for perhaps predicting the severity and the course of the disease. Though differences between various groups in our cohort didn't have statistical significance, there was a tendency for MPA patients, those with MPO-ANCA and those with crescentic class of having higher proportion of C3 deposits in glomeruli compared to other groups. This could suggest more aCP activation in MPO positive AAV. Also there was strong tendency of patients requiring dialysis having higher proportion of C3 deposits compared to non-dialysis patients which could signify C3 deposition of being the hallmark of more severe renal involvement in AAV patients. This data needs further confirmation from future studies.

Diagnostic and therapeutic challenge of unclassifiable enteropathies with increased intraepithelial CD103+ CD8+ T lymphocytes: a single center case series

Objectives Chronic diarrhea, villous atrophy and/or increased intraepithelial T-lymphocytes (IEL) occur in many inflammatory disorders including celiac disease (CD). However, a definite diagnosis is difficult to make in some patients despite an extensive diagnostic work-up. Clinical outcomes and histological phenotypes of such patients we refer to as unclassifiable enteropathy (UEP) remain unclear. Material and methods We performed a retrospective single-center analysis of patients with chronic diarrhea, weight loss and increased IEL. Patients with defined etiologies including infections, CD, drugs, immunodeficiencies or neoplasms were excluded. Clinical and histologic/immunophenotypic parameters were analyzed. Results Nine patients with UEP were identified. Small intestinal damage ranged from minor villous abnormalities to complete atrophy while all patients displayed high numbers of CD103+ CD8+ IELs. In contrast to CD, these CD8+ T cells were not confined to the surface epithelium, but also infiltrated the crypts. Additional histological features included apoptotic crypt epithelial cells and mixed inflammatory infiltrates in the tunica propria. Involvement of other segments of the gastrointestinal tract was observed in 7/9 patients. A clonal intestinal T-cell lymphoproliferative disorder developed in 2 patients, one of which had a fatal disease course. The majority of patients responded to corticosteroids, while response to immunosuppressive medications yielded heterogeneous results. Conclusions We report a patient population with ‘difficult-to-classify’ enteropathies characterized by various degrees of villous atrophy and strongly increased intraepithelial CD103+ CD8+ T cells in the small intestine which harbor an increased risk for T-cell lymphoproliferative disorders. Clinical course, histology, and response to immunosuppressive therapy all suggest an autoimmune pathogenesis.