Inhibitors of Activin Receptor-like Kinase 5 Interfere with SARS-CoV-2 S-Protein Processing and Spike-Mediated Cell Fusion via Attenuation of Furin Expression

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor β (TGF-β) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-β1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-β1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell–cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell–cell spread via downregulation of furin which is most pronounced upon TGF-β stimulation. Since a TGF-β dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.     

Immunometabolic Reprogramming in Response to HIV Infection Is Not Fully Normalized by Suppressive Antiretroviral Therapy

Background: HIV infection results in immunometabolic reprogramming. While we are beginning to understand how this metabolic reprogramming regulates the immune response to HIV infection, we do not currently understand the impact of ART on immunometabolism in people with HIV (PWH). Methods: Serum obtained from HIV-infected (n = 278) and geographically matched HIV seronegative control subjects (n = 300) from Rakai Uganda were used in this study. Serum was obtained before and ~2 years following the initiation of ART from HIV-infected individuals. We conducted metabolomics profiling of the serum and focused our analysis on metabolic substrates and pathways assocaited with immunometabolism. Results: HIV infection was associated with metabolic adaptations that implicated hyperactive glycolysis, enhanced formation of lactate, increased activity of the pentose phosphate pathway (PPP), decreased β-oxidation of long-chain fatty acids, increased utilization of medium-chain fatty acids, and enhanced amino acid catabolism. Following ART, serum levels of ketone bodies, carnitine, and amino acid metabolism were normalized, however glycolysis, PPP, lactate production, and β-oxidation of long-chain fatty acids remained abnormal. Conclusion: Our findings suggest that HIV infection is associated with an increased immunometabolic demand that is satisfied through the utilization of alternative energetic substrates, including fatty acids and amino acids. ART alone was insufficient to completely restore this metabolic reprogramming to HIV infection, suggesting that a sustained impairment of immunometabolism may contribute to chronic immune activation and comorbid conditions in virally suppressed PWH.     

Diffusion-weighted MR imaging in acute ischemia: value of apparent diffusion coefficient and signal intensity thresholds in predicting tissue at risk and final infarct size

BACKGROUND AND PURPOSE: Identifying tissue at risk for infarction is an important goal of stroke imaging. This study was performed to determine whether pixel-based apparent diffusion coefficient (ADC) and signal intensity ratio are helpful diffusion-weighted (DW) imaging metrics to predict tissue at risk for infarction. METHODS: Twelve patients presenting with acute hemispheric strokes underwent DW imaging within 7 hours of symptom onset. Region of interest (ROI), pixel-based ADC, and signal intensity analyses were performed at initial DW imaging to assess area of infarct growth, final infarct area, and normal tissue. RESULTS: Pixel-based analysis was less accurate than ROI-based analysis for evaluating infarct growth or final infarct with ADC, ADC ratio, and signal intensity ratios. In pixel-based analysis, signal intensity ratios were better than ADCs or ADC ratios for identifying tissue at risk (accuracy, 67.4%) and for predicting final infarct (accuracy, 79.9%). Linear regression analysis demonstrated a strong correlation between lesion volume on quantitative DW images or ADC maps and final infarct volume (P < .001). When receiver operating characteristic (ROC) curves were used to determine optimal cutoffs for ADC and DW image values, the region of infarct growth was significantly correlated with only the mismatch between initial qualitative DW image and quantitative DW image signal intensity ratio (cutoff value, 1.19; R = 0.652; P = .022). CONCLUSION: Pixel-based thresholds applied to ADC or DW image signal intensity maps were not accurate prognostic measures of tissue at risk. Quantitative DW images or ADC maps may provide added information not obtained by visual inspection of the qualitative DW image map.     

Integrating gene expression profiling into NCCN high-risk cutaneous squamous cell carcinoma management recommendations: impact on patient management

Abstract

Objective: To integrate gene expression profiling into the management of high-risk cutaneous squamous cell carcinoma (cSCC) within the National Comprehensive Cancer Network (NCCN) guidelines to improve risk-aligned management recommendations.

Methods: A cohort of 300 NCCN-defined high-risk cSCC patients, along with the American Joint Committee on Cancer (AJCC) T stage, Brigham and Women’s Hospital (BWH) T stage, and known patient outcomes were analyzed. Risk classifications using a validated 40-gene expression profile (40-GEP) test and T stage were applied to NCCN patient management guidelines. Risk-directed patient management recommendations within the NCCN guidelines framework were aligned based on risk for metastasis.

Results: Of the 300 NCCN high-risk cSCC patients, 159 (53.0%) were 40-GEP Class 1 and AJCC T1-T2, and 173 (57.7%) were Class 1 and BWH T1-2a, indicating low risk for metastasis and, thereby, suggesting low management intensity. The 40-GEP integration suggested high intensity management for only 24 (8.0%) patients (all Class 2B), and moderate intensity management for the remainder of the cohort.

Conclusions: The 40-GEP test can be integrated within existing NCCN guideline recommendations for managing cSCC patients to help refine risk-directed management decisions. Integration of the 40-GEP test would allow >50% of this NCCN-defined high-risk cohort to be managed with the lowest intensity recommendations within the broad NCCN guidelines. High intensity management was deemed risk-appropriate for a small subpopulation (8.0%). This study demonstrates that the 40-GEP test, in combination with T stage, has clinical utility to impact patient management decisions in NCCN high-risk cSCC for improving risk-aligned management within the NCCN guidelines framework.     

Impact of Kaposi sarcoma-associated herpesvirus (KSHV) burden and HIV coinfection on the detection of T cell responses to KSHV ORF73 and ORF65 proteins

Cellular immune responses to Kaposi sarcoma-associated herpesvirus (KSHV), the etiological agent of KS and several other malignancies, are incompletely characterized. We assessed KSHV-specific interferon- gamma enzyme-linked immunospot responses in a cohort of 154 individuals, using overlapping peptide sets spanning the KSHV-encoded latency-associated nuclear antigen (ORF73) and the minor capsid glycoprotein (ORF65). Among KSHV-seropositive subjects, ORF73-specific responses dominated over responses to ORF65 and were preferentially detected in human immunodeficiency virus-coinfected individuals who had elevated levels of cell-associated KSHV DNA, indicating that the viral antigen burden may have been driving these responses. Responses to both ORF73 and ORF65 were also detected in several KSHV-seronegative subjects who were at increased risk for KSHV infection, which demonstrates that cellular immunity can be found in the absence of detectable humoral responses. These data have implications for the reliable identification of KSHV infection and may help guide the design of immune-based therapeutic and prophylactic interventions.     

Thrombophilic polymorphisms in Israel

Three thrombophilic polymorphisms, FV G1691A, FII G20210A and MTHFR C677T were investigated in Israeli populations by FRET, (fluorescence resonance energy transfer) real-time PCR. We observe extensive variability in the frequencies of each of the polymorphisms, as has been observed in the study of other polymorphisms in these populations. Very high allele frequencies for FV G1691A (the highest 0.087 in Turkish and Greek Jews) and FII G20210A (the highest 0.061 in Georgian Jews) in some of the Israeli populations justify a clinical investigation to assess their risk for venous thrombosis. Principal Coordinates Analysis demonstrates that the Jewish populations are interspersed among the non-Jewish populations. The resemblance of some Jewish populations to certain non-Jewish populations coincides with findings based on classical markers.     

Wash‐out of the non‐heart‐beating donor liver: a matter of flush solution and temperature?

BACKGROUND AND AIMS: Ischemically damaged donor livers are prone to graft non-function. This can in part be explained by a suboptimal wash-out during procurement. An enriched machine perfusion (MP) preservation solution for livers, named Polysol, was developed. The aim of this study was to investigate the type of flush solution, temperature and anticoagulant content on the wash-out of the non-heart-beating donor (NHBD) rat liver. METHODS: Rat livers were flushed after 30 min warm ischemia. After excision, livers were reperfused at 37 degrees C, with analysis of damage and function, concerning (1) solutions (University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK) and Polysol); (2) temperature (4 degrees C, 18 degrees C and 37 degrees C); (3) addition of heparin and (4) wash-out followed by 24 h MP. RESULTS: (1) Reperfusion results were inferior in the UW group; (2) less damage and improved function were seen after wash-out using Polysol at 37 degrees C; (3) No effects were seen of the addition of heparin to Polysol; (4) MP after wash-out using HTK resulted in more liver damage and decreased liver function as compared with wash-out using Polysol. CONCLUSIONS: Polysol is applicable as a flush solution for the NHBD liver, resulting in equal to better wash-out as compared with UW and HTK. The best temperature for this NHBD wash-out is 37 degrees C.