School of Medicine: Recent submissions
Now showing items 1-20 of 2550
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Digital Assessment of CMYC and Ki67 in Diffuse Large B-Cell LymphomaCMYC expression by immunohistochemistry has been shown to be an independent prognostic factor in DLBCL, although manual scoring methods suffer from low reproducibility and both intra- and interobserver variation. The critical cut-off value of 40% CMYC expression is often difficult to determine, due to variability in nuclear stain intensity in tumor cells. We sought to determine if digital image analysis demonstrates utility in CMYC quantification and prognostic impact, in new diagnostic samples of DLBCL. The pathology case files at our institution were reviewed for patients with a new diagnosis of DLBCL, obtained between 2010 and 2023. 30 such patients were selected, and CMYC immunohistochemical stains were scanned into the Aperio CS2 digital pathology scanning system (Leica Biosystems). Tumor areas were defined by manual region of interest selection, after review by 3 board certified hematopathologists. A customized image analysis algorithm was derived from Aperio ImageScope software’s preset nuclear macro algorithm, by setting a score of 1+/3 (weak nuclear staining) at a threshold of 220. Percentages of weak (1+/3), moderate (2+/3), and strong (3+/3) tumor nuclei were computed, and Hscores were calculated. Digitally quantified CMYC expression percentages varied widely in the 30 samples (mean 32.8%, standard deviation 12.9%, range 13.5 – 66.7%). The calculated H-scores were similarly variable (average 53.1, standard deviation 26.8, range 21.6 - 131.8). H-scores demonstrated a negative correlation with overall survival (r=0.27). When an H-score cut-off value of 75 was selected, the group with H-scores > 75 demonstrated lower overall survival (average 961 days, standard deviation 824 days) than the group with Hscores < 75 (average 2049 days, standard deviation 818 days); the relationship is statistically significant (p<0.05). Digital image analysis of CMYC expression demonstrated diagnostic utility in a group of 30 patients with newly diagnosed DLBCL. Calculated H-scores correlated inversely with patient survival, and an H-score cut-off value of 75 separated patients into longer and shorter survival groups.
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Decoding the mechanisms of mitochondrial electron transport chain components loss upon pro-inflammatory microglial activationAberrant or excessive pro-inflammatory microglial activation contributes to many neurodegenerative diseases. This activation is modeled in vitro by combined exposure to the Toll-like receptor 4 (TLR4)-activating molecule lipopolysaccharide (LPS) and the cytokine interferon-gamma (IFN-γ), which cause microglia to adopt a neurotoxic state. Inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production, mitochondrial electron transport chain (ETC) dysfunction, and NLRP3 inflammasome-dependent caspase-1 activation are implicated in the pro-inflammatory activation. The mitochondrial ETC dysfunction, which includes large decreases in multiple ETC complex subunits, is thought to depend on NO production. However, the precise mechanisms of subunit loss are unclear. Here, we tested the hypothesis that both TLR4-dependent caspase-1 protease activation and NO production are required for mitochondrial ETC subunits loss in proinflammatory microglia. Using an OXPHOS antibody cocktail, we examined the level of ETC complex proteins by western blot at 18 hours post LPS+IFN-γ stimulation in wild type (WT) and Nos2 knockout HAPI mouse microglial cells ± caspase-1 inhibitor and/or TLR4 inhibitor. Complex I subunit NDUFB8, Complex II subunit SDHB, and Complex IV subunit COX1 were all reduced relative to α-tubulin or total protein in LPS+IFN-γ-activated WT HAPI cells whereas Complex III UQCRC2 subunit and Complex V ATP5A subunit were unchanged. CRISPR knockout of Nos2 and inhibition of NO production in WT cells each rescued the decrease in Complex II and IV subunits but did not preserve the Complex I subunit NDUFB8. Addition of caspase-1 inhibitor VX765 or TLR4 antagonist TAK-242 failed to rescue any of the ETC complex proteins. However, each inhibitor moderately reduced iNOS protein expression and their combined addition led to a partial rescue of COX1. Unexpectedly, iNOS expression was partially suppressed by caspase-1 inhibitor and, also, a decreased level of the caspase-1 p20 active fragment was detected when NO production was prevented by the iNOS inhibitor 1400W or by Nos2 ablation. These results suggest that there is a positive feedback loop between caspase-1 activation and nitric oxide production. In addition, findings indicate that mitochondrial ETC dysfunction is mediated by multiple mechanisms, as SDHB and COX1 but not Complex I subunit NDUFB8 were rescued by nitric oxide elimination and only COX1 loss was sensitive to the caspase-1-TLR4 inhibitor combination. Additional work is needed to elucidate the TLR4-, caspase-1-, and nitric oxide-independent mechanisms recruited by LPS+ IFN-γ that impair the mitochondrial ETC in microglia.
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University of Maryland School of Medicine State of the School 2023University of Maryland, Baltimore. School of Medicine, 2023
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University of Maryland School of Medicine State of the School 2022University of Maryland, Baltimore. School of Medicine, 2022