Evaluating the Feasibility, Acceptance, and Beneficial Effects of Online Occupational Therapy for Post-COVID-19 Condition: Protocol for a Randomized Controlled Trial (ErgoLoCo Study).

BACKGROUND: Post-COVID-19 syndrome (PCS; also known as "long COVID") is a relatively novel disease comprising physical, psychological, and cognitive complaints persisting several weeks to months after acute infection with SARS-CoV-2. Approximately 10% of patients with COVID-19 are affected by long-term symptoms. However, effective treatment strategies are lacking. The ErgoLoCo (Occupational Therapy [Ergotherapie] for Long COVID) study was designed to develop and evaluate a novel occupational therapy (OT) concept of online delivery of therapy for long COVID. OBJECTIVE: The primary study objective is to assess the feasibility of the online OT intervention in PCS. Secondary aims include the evaluation of online OT concerning cognitive problems, occupational performance, and social participation. METHODS: This randomized controlled interventional pilot study involves parallel mixed methods process analyses and a realist evaluation approach. A total of 80 clients with PCS aged at least 16 years will be recruited into two interventional groups. The control cohort (watch and wait) comprises 80 clients with long COVID. Treatment is provided through teletherapy (n=40) or delivery of prerecorded videos (n=40) using the same standardized OT concept twice weekly over 12 weeks. Analyses of quantitative questionnaires and qualitative interviews based on the theoretical framework of acceptability will be performed to assess feasibility. Focus group meetings will be used to assess how acceptable and helpful the intervention was to the participating occupational therapists. Standardized tests will be used to assess the initial efficacy of the intervention on neurocognitive performance; limitations in mobility, self-care, and everyday activities; pain; disabilities; quality of life (QoL); social participation; and anxiety and depression in PCS, and the possible effects of online OT on these complaints. RESULTS: The German Ministry of Education and Research provided funding for this research in March 2022. Data collection took place from October 2022 to August 31, 2023. Data analysis will be completed by the end of April 2024. We anticipate publishing the results in the fall of 2024. CONCLUSIONS: Despite the enormous clinical need, effective and scalable treatment options for OT clients who have PCS remain scarce. The ErgoLoCo study will assess whether online-delivered OT is a feasible treatment approach in PCS. Furthermore, this study will assess the effect of the intervention on cognitive symptoms, QoL, and occupational performance and participation in everyday life. Particular emphasis will be placed on the experiences of clients and occupational therapists with digitally delivered OT. This study will pave the way for novel and effective treatment strategies in PCS. TRIAL REGISTRATION: German Clinical Trial Registry DRKS00029990; https://drks.de/search/de/trial/DRKS00029990. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/50230.

Upregulation of BST-2/Tetherin by HIV infection in vivo.

The interferon-inducible antiviral factor BST-2 prevents several enveloped viruses, including HIV, from escaping infected cells. The HIV protein Vpu antagonizes this host defense. Little is known about the expression of BST-2 during HIV infection in vivo and whether it can be modulated to the host's advantage. We studied the expression of BST-2 on blood cells from HIV-infected patients during the acute and chronic phases of disease as well as after antiretroviral treatment (ART). The expression of BST-2 was increased on mononuclear leukocytes, including CD4-positive T lymphocytes from HIV-positive patients, compared to that on cells of uninfected controls. The expression of BST-2 was highest during acute infection and decreased to levels similar to those of uninfected individuals after ART. Treatment of primary blood mononuclear cells in vitro with alpha interferon or with Toll-like receptor (TLR) agonists increased the expression of BST-2 to levels similar to those found during infection in vivo. The interferon-induced levels were sufficient to overcome the Vpu protein in vitro, reducing the release of wild-type HIV. These data show that BST-2 is upregulated during HIV infection, consistent with its role as an interferon-stimulated gene. The data further suggest that this upregulation is sufficient to saturate the activity of Vpu and inhibit wild-type HIV.

Healthcare Workers' Perceptions and Medically Approved COVID-19 Infection Risk: Understanding the Mental Health Dimension of the Pandemic. A German Hospital Case Study.

Introduction: This study analyses how healthcare workers (HCWs) perceived risks, protection and preventive measures during the COVID-19 pandemic in relation to medically approved risks and organizational measures. The aim is to explore "blind spots" of pandemic protection and make mental health needs of HCWs visible. Methods: We have chosen an "optimal-case" scenario of a high-income country with a well-resourced hospital sector and low HCW infection rate at the organizational level to explore governance gaps in HCW protection. A German multi-method hospital study at Hannover Medical School served as empirical case; document analysis, expert information and survey data (n = 1,163) were collected as part of a clinical study into SARS-CoV-2 serology testing during the second wave of the pandemic (November 2020-February 2021). Selected survey items included perceptions of risks, protection and preventive measures. Descriptive statistical analysis and regression were undertaken for gender, profession and COVID-19 patient care. Results: The results reveal a low risk of 1% medically approved infections among participants, but a much higher mean personal risk estimate of 15%. The majority (68.4%) expressed "some" to "very strong" fear of acquiring infection at the workplace. Individual protective behavior and compliance with protective workplace measures were estimated as very high. Yet only about half of the respondents felt strongly protected by the employer; 12% even perceived "no" or "little" protection. Gender and contact with COVID-19 patients had no significant effect on the estimations of infection risks and protective workplace behavior, but nursing was correlated with higher levels of personal risk estimations and fear of infection. Conclusions: A strong mismatch between low medically approved risk and personal risk perceptions of HCWs brings stressors and threats into view, that may be preventable through better information, training/education and risk communication and through investment in mental health and inclusion in pandemic preparedness plans.

Effects of Pre-Existing Mental Conditions on Fatigue and Psychological Symptoms Post-COVID-19.

BACKGROUND: Patients who are post-COVID-19 will require more treatment soon. Therefore, it is important to understand the root cause of their psychological and somatic conditions. Previous studies showed contradictory results on the influence of pre-existing mental conditions. The present study examines the influence of these pre-existing conditions and their pre-treatment on the severity of post-COVID-19 symptoms. METHODS: This analysis employs questionnaire data from a large study sample in Germany. Overall, 801 participants were included. All participants rated their health status on a scale from 0 to 100. Fatigue, depression, and anxiety were measured using the FAS, PHQ-9, and GAD-7 scales. RESULTS: All pre-pandemic values showed no significant differences between the groups. The current health status was rated similarly by the recovered patients (µ = 80.5 ± 17.0) and the control group (µ = 81.2 ± 18.0) but significantly worse by acutely infected (µ = 59.0 ± 21.5) and post-COVID-19 patients (µ = 54.2 ± 21.1). Fatigue, depression, and anxiety were similar for recovered patients and the control group. By contrast, there were significant differences between the control and the post-COVID-19 groups concerning fatigue (45.9% vs. 93.1%), depression (19.3% vs. 53.8%), and anxiety (19.3% vs. 22.3%). CONCLUSION: Fatigue and psychological conditions of post-COVID-19 patients are not associated with pre-existing conditions.

ß-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release.

BACKGROUND: The cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. ß-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 receptor for proteasomal degradation. Recently, it has been suggested that ß-TrCP is also critically involved in Vpu's ability to overcome the CD317-mediated virion release block. RESULTS: To test this model, we analyzed the consequences of several experimental strategies to interfere with the Vpu-ß-TrCP protein-protein interaction. Under these conditions, we studied effects of Vpu on expression and localization of CD317 and CD4, as well as on its ability to promote HIV-1 release. Our results demonstrate a strict requirement for Vpu's di-serine motif for degradation of CD4 and also CD317, reduction of cell surface exposure of CD317, and HIV-1 release enhancement. We further show a critical role of ß-TrCP2, but not of the structurally related ß-TrCP1 isoform, for Vpu-mediated degradation of both receptors. Most importantly, Vpu remained active in downregulating CD317 from the cell surface and in overcoming the HIV-1 release restriction in ß-TrCP-depleted cells. CONCLUSIONS: These results demonstrate that ß-TrCP is not strictly required for Vpu's ability to counteract the CD317-imposed virion release block and support the relevance of cell surface down-modulation of the restriction factor as a central mechanism of Vpu antagonism. Moreover, we propose the existence of a critical, yet to be identified cellular factor that interacts with Vpu via its di-serine motif to alter the trafficking of the restriction factor.

CD317/tetherin is enriched in the HIV-1 envelope and downregulated from the plasma membrane upon virus infection.

CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.

Antagonism of CD317 restriction of human immunodeficiency virus type 1 (HIV-1) particle release and depletion of CD317 are separable activities of HIV-1 Vpu.

Vpu antagonizes human immunodeficiency virus type 1 (HIV-1) particle release inhibition by CD317/BST-2/Tetherin. Whether this Vpu activity strictly requires cellular depletion of the restriction factor is unclear. Here, we characterized CD317 variants with mutations in putative sorting or ubiquitination motifs. All mutants still potently impaired release of Vpu-defective HIV-1 and remained sensitive to Vpu-mediated release enhancement. Importantly, this virological antagonism correlated with surface downregulation of CD317 mutants by Vpu, while intracellular pools of these mutants, which were consistently depleted of the wild-type protein, were highly variable or even enhanced. Thus, Vpu can efficiently antagonize virion tethering in the absence of CD317 degradation.

Determinants in HIV-1 Nef for enhancement of virus replication and depletion of CD4+ T lymphocytes in human lymphoid tissue ex vivo.

BACKGROUND: HIV-1 Nef critically contributes to AIDS in part by augmenting virus titers in infected individuals. Analyzing which of Nef's activities contribute to HIV pathogenesis has been hampered by the lack of a cell culture model in which Nef exerts pronounced effects on HIV replication. The human lymphoid aggregate culture (HLAC) from tonsil maintains the cell populations and cytokine milieu found in vivo, supports a productive infection without exogenous stimulation, and Nef contributes to efficient HIV-1 replication as well as CD4+ T cell depletion in this experimental ex vivo-model. RESULTS: To identify determinants in Nef that mediate these activities, we infected HLAC with a panel of isogenic HIV-1NL4-3 strains that encode for well-characterized mutants of HIV-1SF2 Nef. Determination of HIV-1 replication revealed that enhancement of the virus spread by Nef is governed by a complex set of protein interaction surfaces. In contrast, increased CD4+ T lymphocyte depletion depended on only two protein interaction surfaces in Nef that mediate either downregulation of cell surface CD4 or interaction with the NAKC signalosome. Consistently, in HLAC from 9 out of 14 donors, Nef enhanced CD4+ T cell depletion in the absence of a significant effect on virus replication. Moreover, our results suggest that this Nef-dependent enhancement in depletion occurred predominately in uninfected bystander CD4+ T cells. CONCLUSION: Our findings suggest that Nef facilitates depletion of CD4+ T lymphocytes in HIV-1-infected lymphoid tissue ex vivo by increasing the pool of productively infected cells and by sensitizing bystander cells for killing. This ability might contribute to Nef's pathogenic potential in vivo.

A Flow Cytometric Method to Determine Transfection Efficiency.

Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive and robust method that can optimize transfection efficiency based not only on expression of the target protein of interest but also on the uptake of the nucleic acids, can be an important tool in molecular biology. Herein, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency while overcoming limitations of prior established methods that quantify transfection efficiency.

Dysfunctional high-density lipoprotein from HIV+ individuals promotes monocyte-derived foam cell formation in vitro.

OBJECTIVE: The role of high-density lipoprotein (HDL) function in HIV-related atherosclerotic cardiovascular disease (CVD) is unclear. HDLs isolated from HIV [HIV(+)HDL] and HIV-uninfected individuals [HIV(-)HDL] were assessed for HDL function and ability to promote monocyte-derived foam cell formation (MDFCF; a key event in HIV-related CVD) ex vivo. DESIGN/METHODS: Using an established in-vitro model of atherogenesis and plasma samples from an established cross-sectional study of virologically suppressed HIV men on stable effective antiretroviral therapy and with low CVD risk (median age: 42 years; n = 10), we explored the impact of native HDL [HIV(+)HDL] on MDFCF. In this exploratory study, we selected HIV(+)HDL known to be dysfunctional based on two independent measures of impaired HDL function: antioxidant (high HDLox) ability of HDL to release apolipoprotein A-I (ApoA-I) (low HDL-ApoA-I exchange). Five healthy men matched by age and race to the HIV group were included. Given that oxidation of HDL leads to abnormal HDL function, we also compared proatherogenic effects of HIV(+)HDL vs. chemically derived HDLox. The ex-vivo atherogenesis assay was performed using lipoproteins (purchased or isolated from plasma using ultracentrifugation) and monocytes purified via negative selection from healthy donors. RESULTS: HIV(+)HDL known to have reduced antioxidant function and rate of HDL/ApoAI exchange promoted MDFCF to a greater extent than HDL (33.0 vs. 26.2% foam cells; P = 0.015). HDL oxidized in vitro also enhanced foam cell formation as compared with nonoxidized HDL (P < 0.01). CONCLUSION: Dysfunctional HDL in virologically suppressed HIV individuals may potentiate atherosclerosis in HIV infection by promoting MDFCF.The role of HDL function in HIV-related atherosclerotic CVD is unclear. HDL isolated from HIV [HIV(+)HDL] and HIV-uninfected individuals [HIV(-)HDL] were assessed for HDL function and ability to promote foam cell formation ex vivo. HIV(+)HDL known to have reduced antioxidant function and rate of HDL/ApoA1 exchange promoted MDFCF to a greater extent than HDL(-)HDL (33.0 vs. 26.2% foam cells.Subject codes: Inflammation, Lipids and Cholesterol, Vascular Biology, Oxidant Stress, Atherosclerosis.

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells.

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.

Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef.

The HIV-1 protein Nef inhibits antigen presentation by class I major histocompatibility complex (MHC-I). We determined the mechanism of this activity by solving the crystal structure of a protein complex comprising Nef, the MHC-I cytoplasmic domain (MHC-I CD) and the µ1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-µ1 interface, which encompasses the cargo-recognition site of µ1 and the proline-rich strand of Nef. The Nef C terminus induces a previously unobserved conformational change in µ1, whereas the N terminus binds the Nef core to position it optimally for complex formation. Positively charged patches on µ1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity.

HIV-1 antagonism of CD317 is species specific and involves Vpu-mediated proteasomal degradation of the restriction factor.

Mammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/HM1.24/tetherin) is a restriction factor that blocks release of human immunodeficiency virus type 1 (HIV-1) from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1 release but are resistant to Vpu. Interspecies chimeras reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism involve all three major structural domains of CD317. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in human cells, and the species-specific resistance to this suppression may guide the development of small animal models of HIV infection.

Specific and distinct determinants mediate membrane binding and lipid raft incorporation of HIV-1(SF2) Nef.

Membrane association is believed to be a prerequisite for the biological activity of the HIV-1 pathogenicity factor Nef. Attachment to cellular membranes as well as incorporation into detergent-insoluble microdomains (lipid rafts) require the N-terminal myristoylation of Nef. However, this modification is not sufficient for sustained membrane association and a specific raft-targeting signal for Nef has not yet been identified. Using live cell confocal microscopy and membrane fractionation analyses, we found that the N-terminal anchor domain (aa 1-61) is necessary and sufficient for efficient membrane binding of Nef from HIV-1(SF2). Within this domain, highly conserved lysine and arginine residues significantly contributed to Nef's membrane association and localization. Plasma membrane localization of Nef was also governed by an additional membrane-targeting motif between residues 40 and 61. Importantly, two lysines at positions 4 and 7 were not essential for the overall membrane association but critically contributed to Nef's incorporation into lipid raft domains. Cell surface receptor downmodulation was largely unaffected by mutations of all N-terminal basic residues, while the association of Nef with Pak2 kinase activity and its ability to augment virion infectivity correlated with its lysine-mediated raft incorporation. In contrast, all basic residues were required for efficient HIV-1 replication in primary human T lymphocytes but did not contribute to the incorporation of Nef into HIV-1 virions. Together, these results unravel that Nef's membrane association is governed by a complex pattern of signature motifs that differentially contribute to individual Nef activities. The identification of a critical raft targeting determinant and the functional characterization of a membrane-bound, non-raft-associated Nef variant indicate raft incorporation as a regulatory mechanism that determines the biological activity of distinct subpopulations of Nef in HIV-infected cells.