Plasma Proteins Associated with COVID-19 Severity in Puerto Rico.

Viral strains, age, and host factors are associated with variable immune responses against SARS-CoV-2 and disease severity. Puerto Ricans have a genetic mixture of races: European, African, and Native American. We hypothesized that unique host proteins/pathways are associated with COVID-19 disease severity in Puerto Rico. Following IRB approval, a total of 95 unvaccinated men and women aged 21-71 years old were recruited in Puerto Rico from 2020-2021. Plasma samples were collected from COVID-19-positive subjects (n = 39) and COVID-19-negative individuals (n = 56) during acute disease. COVID-19-positive individuals were stratified based on symptomatology as follows: mild (n = 18), moderate (n = 13), and severe (n = 8). Quantitative proteomics was performed in plasma samples using tandem mass tag (TMT) labeling. Labeled peptides were subjected to LC/MS/MS and analyzed by Proteome Discoverer (version 2.5), Limma software (version 3.41.15), and Ingenuity Pathways Analysis (IPA, version 22.0.2). Cytokines were quantified using a human cytokine array. Proteomics analyses of severely affected COVID-19-positive individuals revealed 58 differentially expressed proteins. Cadherin-13, which participates in synaptogenesis, was downregulated in severe patients and validated by ELISA. Cytokine immunoassay showed that TNF-α levels decreased with disease severity. This study uncovers potential host predictors of COVID-19 severity and new avenues for treatment in Puerto Ricans.

Quantitative Proteomics Reveal That CB2R Agonist JWH-133 Downregulates NF-κB Activation, Oxidative Stress, and Lysosomal Exocytosis from HIV-Infected Macrophages.

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.

The Characterization and Evaluation of the Soluble Triggering Receptor Expressed on Myeloid Cells-like Transcript-1 in Stable Coronary Artery Disease.

Platelets play crucial roles in the development and progression of coronary artery disease (CAD). The triggering receptor expressed in myeloid cells-like transcript-1 (TLT-1) is stored in platelet α granules, and activated platelets release a soluble fragment (sTLT-1). We set out to better characterize the constituent amino acids of sTLT-1 and to evaluate sTLT-1 for use as a biomarker in patients with stable CAD. We evaluated sTLT-1 release using immunoprecipitation and mass spectrometry and employed statistical methods to retrospectively correlate sTLT-1 concentrations, utilizing ELISA in plasma samples from 1510 patients with documented stable CAD. We identified TLT-1 residues to 133 in platelet releasates. ADAM17 cuts TLT-1, suggesting that S136 is the C-terminal amino acid in sTLT-1. Our results revealed that for CAD patients, sTLT-1 levels did not differ significantly according to primary outcomes of death or major cardiac event; however, patients with left ventricular (LV) dysfunction had significantly lower plasma sTLT-1 levels as compared to those with normal LV function (981.62 ± 1141 pg/mL vs. 1247.48 ± 1589 pg/mL; p = 0.003). When patients were stratified based on sTLT-1 peak frequency distribution (544 pg/mL), a significant association with congestive heart failure was identified (OR = 2.94; 1.040-8.282; p = 0.042), which could be explained by LV dysfunction.

Decreased CSTB, RAGE, and Axl Receptor Are Associated with Zika Infection in the Human Placenta.

Zika virus (ZIKV) compromises placental integrity, infecting the fetus. However, the mechanisms associated with ZIKV penetration into the placenta leading to fetal infection are unknown. Cystatin B (CSTB), the receptor for advanced glycation end products (RAGE), and tyrosine-protein kinase receptor UFO (AXL) have been implicated in ZIKV infection and inflammation. This work investigates CSTB, RAGE, and AXL receptor expression and activation pathways in ZIKV-infected placental tissues at term. The hypothesis is that there is overexpression of CSTB and increased inflammation affecting RAGE and AXL receptor expression in ZIKV-infected placentas. Pathological analyses of 22 placentas were performed to determine changes caused by ZIKV infection. Quantitative proteomics, immunofluorescence, and western blot were performed to analyze proteins and pathways affected by ZIKV infection in frozen placentas. The pathological analysis confirmed decreased size of capillaries, hyperplasia of Hofbauer cells, disruption in the trophoblast layer, cell agglutination, and ZIKV localization to the trophoblast layer. In addition, there was a significant decrease in CSTB, RAGE, and AXL expression and upregulation of caspase 1, tubulin beta, and heat shock protein 27. Modulation of these proteins and activation of inflammasome and pyroptosis pathways suggest targets for modulation of ZIKV infection in the placenta.

Inhibition of Cathepsin B and SAPC Secreted by HIV-Infected Macrophages Reverses Common and Unique Apoptosis Pathways.

Human immunodeficiency virus 1 (HIV-1) infects blood monocytes that cross the blood-brain barrier to the central nervous system, inducing neuronal damage. This is prompted by the secretion of viral and neurotoxic factors by HIV-infected macrophages, resulting in HIV-associated neurocognitive disorders. One of these neurotoxic factors is cathepsin B (CATB), a lysosomal cysteine protease that plays an important role in neurodegeneration. CATB interacts with the serum amyloid P component (SAPC), contributing to HIV-induced neurotoxicity. However, the neuronal apoptosis pathways triggered by CATB and the SAPC remain unknown. We aimed to elucidate these pathways in neurons exposed to HIV-infected macrophage-conditioned media before and after the inhibition of CATB or the SAPC with antibodies using tandem mass tag proteomics labeling. Based on the significant fold change (FC) ≥ |2| and p-value < 0.05 criteria, a total of 10, 48, and 13 proteins were deregulated after inhibiting CATB, SAPC antibodies, and the CATB inhibitor CA-074, respectively. We found that neurons exposed to the CATB antibody and SAPC antibody modulate similar proteins (TUBA1A and CYPA/PPIA) and unique proteins (LMNA and HSPH1 for the CATB antibody) or (CFL1 and PFN1 for the SAPC antibody). CATB, SAPC, or apoptosis-related proteins could become potential targets against HIV-induced neuronal degeneration.

Zika virus infection of the placenta alters extracellular matrix proteome.

Zika virus (ZIKV) infection has been associated with fetal abnormalities by compromising placental integrity, but the mechanisms by which this occurs are unknown. Flavivirus can deregulate the host proteome, especially extracellular matrix (ECM) proteins. We hypothesize that a deregulation of specific ECM proteins by ZIKV, affects placental integrity. Using twelve different placental samples collected during the 2016 ZIKV Puerto Rico epidemic, we compared the proteome of five ZIKV infected samples with four uninfected controls followed by validation of most significant proteins by immunohistochemistry. Quantitative proteomics was performed using tandem mass tag TMT10plex™ Isobaric Label Reagent Set followed by Q Exactive™ Hybrid Quadrupole Orbitrap Mass Spectrometry. Identification of proteins was performed using Proteome Discoverer 2.1. Proteins were compared based on the fold change and p value using Limma software. Significant proteins pathways were analyzed using Ingenuity Pathway (IPA). TMT analysis showed that ZIKV infected placentas had 94 reviewed differentially abundant proteins, 32 more abundant, and 62 less abundant. IPA analysis results indicate that 45 of the deregulated proteins are cellular components of the ECM and 16 play a role in its structure and organization. Among the most significant proteins in ZIKV positive placenta were fibronectin, bone marrow proteoglycan, and fibrinogen. Of these, fibrinogen was further validated by immunohistochemistry in 12 additional placenta samples and found significantly increased in ZIKV infected placentas. The upregulation of this protein in the placental tissue suggests that ZIKV infection is promoting the coagulation of placental tissue and restructuration of ECM potentially affecting the integrity of the tissue and facilitating dissemination of the virus from mother to the fetus.

Microwave & magnetic proteomics of macrophages from patients with HIV-associated cognitive impairment.

OBJECTIVE: HIV-infected monocytes can infiltrate the blood brain barrier as differentiated macrophages to the central nervous system, becoming the primary source of viral and cellular neurotoxins. The final outcome is HIV-associated cognitive impairment (HACI), which remain prevalent today, possibly due to the longer life-span of the patients treated with combined anti-retroviral therapy. Our main goal was to characterize the proteome of monocyte-derived macrophages (MDM) from HACI patients, and its association with their cognitive status, to find novel targets for therapy. METHODS: MDM were isolated from the peripheral blood of 14 HIV-seropositive women characterized for neurocognitive function, including: four normal cognition (NC), five asymptomatic (A), and five with cognitive impaired (CI). Proteins from macrophage lysates were isobaric-labeled with the microwave and magnetic (M2) sample preparation method followed by liquid chromatography-tandem mass spectrometry-based protein identification and quantification. Differences in protein abundance across groups classified by HACI status were determined using analysis of variance. RESULTS: A total of 2,519 proteins were identified with 2 or more peptides and 28 proteins were quantified as differentially expressed. Statistical analysis revealed increased abundance of 17 proteins in patients with HACI (p<0.05), including several enzymes associated to the glucose metabolism. Western blot confirmed increased expression of 6-Phosphogluconate dehydrogenase and L-Plastin in A and CI patients over NC and HIV seronegatives. CONCLUSIONS: This is the first quantitative proteomics study exploring the changes in protein abundance of macrophages isolated from patients with HACI. Further studies are warranted to determine if these proteins may be target candidates for therapy development against HACI.