Genetic profiling of cell types in the pathogenesis of Nonalcoholic Steatohepatitis (NASH)

NASH is the progressive form of non-alcoholic fatty liver disease (NAFLD) which affects 25% of adults in the US. We are interested in identifying genetic markers for specific cell populations in the liver and hypothesize that cells undergo genetic reprogramming to handle the molecular stresses of the NASH disease state. Using previously published single-cell RNA sequencing datasets from mouse liver whole tissue, we identified the top 25 most highly variable genes per cell type in NASH liver tissue. Using R-programming language, datasets from both healthy and diseases livers were deconvoluted, normalized, linearized and dimensionally reduced to create a 3D map of cell clusters according to changes in gene expression. Subsequently, cell clusters were labeled according to the most differentially expressed gene per cell type. The final graph demonstrated that the most statistically significant differences in the emergence of the disease phenotype came from genetic markers associated with T-cells and dendritic cells. The meta-analysis led to a depiction of 13 cell clusters with varying degrees of involvement in NASH pathogenesis. The top two statistically significant cell populations were T-cells and dendritic cells with the most differentially expressed genes Trbc2 and Cst3, respectively. Our results support our hypothesis as both T-cells and dendritic cells are cell populations that can be involved in inflammation during disease states. In future studies, merging datasets from different papers can help to strengthen our previous associations and be used to create algorithms for predicting disease course as well as possible therapies for patients with NASH

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C

The Gram-positive bacterium Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8+ T-cell responses towards the dominant H-2Kd-restricted epitope, GUCY2C254-262. However, Lm-GUCY2C produced robust CD8+ T-cell responses towards Lm-derived peptides suggesting that GUCY2C254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C254-262 immunity completely suppressed GUCY2C254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2Kd compared to GUCY2C254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2Kd binding, producing GUCY2CF255Y, significantly improved stability with H-2Kd and rescued GUCY2C254-262 immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines

Immunohistochemistry for the Proliferation Markers Ki67 and PHH3 in Cell Block Material of Basaloid Salivary Gland Neoplasms is Predictive of Malignancy

Background: Basaloid salivary gland neoplasms (BSGNs) are notoriously difficult to classify in fine needle aspiration (FNA) specimens due to the morphologic overlap shown by the various benign and malignant entities in this category1,2,3. Adenoid cystic carcinoma (AdCC) represents a particular diagnostic challenge, as it typically shows low grade cytologic features despite its aggressive clinical behavior and need for more extensive surgery4. We examined whether the proliferation markers Ki67 and PHH3 could help predict malignancy in BSGNs, thus improving diagnostic accuracy and optimizing patient care.https://jdc.jefferson.edu/pacbposters/1020/thumbnail.jp

Novel compound heterozygous mutations in GPT2 linked to microcephaly, and intellectual developmental disability with or without spastic paraplegi

WOS: 000428318500022PubMed ID: 29226631We here describe novel compound heterozygous missense variants, NM_133443:c.[400C>T] and NM_133443:[1435G>A], in the glutamic-pyruvic transaminase 2 (GPT2) gene in a large consanguineous family with two affected siblings diagnosed with microcephaly intellectual disability and developmental delay (IDD). In addition to these clinical phenotypes, the male sibling has spastic paraplegia, and the female sibling has epilepsy. Their four extended family members have IDD and microcephaly. Both of these variants, c.400C>T (p.R134C) and c. 1435G>A (p.V479M), reside in the pyridoxal phosphate-dependent aminotransferase domain. The missense variants affect highly conserved amino acids and are classified to be disease-causing by meta-SVM. The candidate variants were not found in the Exome Aggregation Consortium (ExAC) dataset or in dbSNP. Both GPT2 variants have an allele frequency of 0% (0/ similar to 600) in the whole-exome sequenced Turkish cohort. Upon Sanger sequencing, we confirmed these mutations in all affected family members and showed that the index patient and his affected sister inherited one mutant allele from each unaffected parent. To the best of our knowledge, this is the first family in which a novel compound heterozygous variant in the GPT2 gene was identified.Yale Center for Mendelian DisordersYale Center for Mendelian Disorder

GRK2 Regulates ADP Signaling in Platelets Via P2Y1 and P2Y12.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for \u3e3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5\u27-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2-/- platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2-/- platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2-/- platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2-/- mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury

Malignant Cerebellar Edema Subsequent to Accidental Prescription Opioid Intoxication in Children

We present two recent cases of toddlers who developed malignant cerebellar edema subsequent to accidental ingestion of prescription opioids. Both children presented acute neurological decline, hydrocephalus, and tonsillar herniation requiring emergent ventricular drain placement, suboccipital craniectomy, and partial cerebellectomy. Together with several other reports, these cases suggest the existence of an uncommon yet severe syndrome of acute opioid-induced malignant cerebellar edema. We hypothesize that the condition results from a combination of primary opioid receptor-mediated changes in neuronal metabolism that are exacerbated by secondary hypoxic insult. If recognized promptly, this syndrome can be treated with emergent neurosurgical intervention with good clinical outcomes. These cases also illustrate the unintended consequences and innocent victims of the spiraling prescription opioid epidemic, which will likely increase in prevalence. Recognition of this syndrome by clinicians is thus critical

Pleiotropic role of TRAF7 in skull-base meningiomas and congenital heart disease

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects