Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-b; high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.
The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10nm) to the chromosomal (>200nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using Partial Wave Spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20-200nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nano-architecture. Therefore, we developed a live cell PWS technique which allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real-time. In this work, we employ live cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live cell DNA binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Since biological function is tightly paired with structure, live cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.Significance StatementChromatin is one of the most critical structures within the cell because it houses most genetic information. Its structure is well understood at the nucleosomal (<20nm) and chromosomal (>200nm) levels, however, due to the lack of quantitative imaging modalities to study this organization, little is known about the higher-order structure between these length scales in live cells. We present a label-free technique, live cell Partial Wave Spectroscopic (PWS) microscopy with sensitivity to structures between 20-200nm that can quantify the nano-architecture in live cells. With this technique, we can detect DNA fragmentation and expand on the link between metabolic function and higher-order chromatin structure. Live cell PWS allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in live cells.
3520 Background: Anal cancer affects over 8,000 patients per year in the United States and the incidence is increasing. A significant risk factor for anal cancer and precancerous lesions is human papilloma virus (HPV), with up to 90% of cases being associated with HPV infection. Another emerging risk factor is HIV co-infection. In the present study, we further address if CD4 count is a significant factor for driving higher-grade HPV-mediated anal squamous lesions in HIV/HPV co-infection patients with a single institution large cohort. Methods: A retrospective cohort of HPV-positive patients with anal biopsies was obtained from 2002-2020. Information on the grade of their anal lesion, HIV status, and CD4 count (cells/mm3) were collected. In patients with HIV, the most recent CD4 count up to one year prior to or one month after their biopsy was utilized in our analysis. Lesions were grouped into low grade squamous intraepithelial lesions (LSIL) and higher grade squamous intraepithelial lesions (HSIL), carcinoma in situ (CIS), or squamous cell carcinoma (SCC). The Center for Disease Control CD4 count levels to define HIV status were utilized in our sub-analyses. The distribution of lesion grades was compared between HIV-negative and -positive patients, and between HIV-negative and three subgroups of HIV-positive patients using the Fisher’s exact test. Results: Our cohort comprised of 3,354 total HPV-positive patients. 2,036 of these patients were HIV-negative and 1318 were HIV-positive. The proportion of higher grade lesions was significantly higher in HIV/HPV co-infected patients regardless of CD4 count (Table). The full cohort of HIV-positive patients had lower rates of LSIL (60.8% vs. 74.0%) and higher rates of higher-grade lesions (39.2% vs. 26.0%) (p<0.001) compared to HIV-negative patients. The distribution of lesion grades was also significantly different between HIV-negative patients and all HIV-positive patient subgroups, with all subgroups having higher rates of higher-grade lesions than HIV-negative patients (all p<0.001). Conclusions: Our data show that HIV-HPV co-infection is a risk factor for higher grade anal lesions, regardless of CD4 status. This suggests that CD4 count is not the only factor responsible for the increased risk of higher-grade anal lesions, as the groups of HIV-positive patients with CD4 counts between 200-499 and above 499 still had a higher rate of higher-grade lesions than HIV-negative patients. Further research into other HIV-related immunologic changes that increase risk for higher-grade HPV-driven anal lesions is warranted.[Table: see text]
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