Identification of the main ubiquitination site in human erythroid α-spectrin

AbstractErythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid α-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of α-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in α-spectrin and may be present in several other proteins

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells

The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total HIV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA–positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART

Loss of CD103+ DCs and Mucosal IL-17+ and IL-22+ Lymphocytes is Associated with Mucosal Damage in SIV Infection

HIV/SIV disease progression is associated with multifocal damage to the GI tract epithelial barrier that correlates with microbial translocation and persistent pathological immune activation but the underlying mechanisms remain unclear. Investigating alterations in mucosal immunity during SIV infection, we found that damage to the colonic epithelial barrier was associated with loss of multiple lineages of IL-17-producing lymphocytes, cells that microarray analysis showed express genes important for enterocyte homeostasis, including IL-22. IL-22-producing lymphocytes were also lost after SIV infection. Potentially explaining coordinate loss of these distinct populations, we also observed loss of CD103+ DCs after SIV infection which associated with loss of IL-17 and IL-22-producing lymphocytes. CD103+ DCs expressed genes associated with promotion of IL-17/IL-22+ cells, and co-culture of CD103+ DCs and naïve T-cells led to increased IL17A and RORc expression in differentiating T-cells. These results reveal complex interactions between mucosal immune cell subsets providing potential mechanistic insights into mechanisms of mucosal immune dysregulation during HIV/SIV infection, and offer hints for development of novel therapeutic strategies to address this aspect of AIDS virus pathogenesis

Mechanisms of action and antiproliferative properties of Brassica oleracea juice in human breast cancer cell lines

none7noCruciferous vegetables are an important source of compounds that may be useful for chemoprevention. In this study, we evaluated the antiproliferative activity of juice obtained from leaves of several varieties of Brassica oleracea on both estrogen receptor (ER)-positive (ER; MCF-7 and BT474) and ER-negative (ER; MDA-MB-231 and BT20) human breast cancer cell lines. The effect of juice on cell proliferation was evaluated on DNA synthesis and on cell cycle–related proteins. Juice markedly reduced DNA synthesis, evaluated by [3H]thymidine incorporation, starting from low concentrations (final concentration 5–15 mL/L), and this activity was independent of ER. All cauliflower varieties tested suppressed cell proliferation in a dose-dependent manner. Cell growth inhibition was accompanied by significant cell death at the higher juice concentrations, although no evidence of apoptosis was found. Interestingly, the juice displayed a preferential activity against breast cancer cells compared with other mammalian cell lines investigated (ECV304, VERO, Hep2, 3T3, and MCF-10A) (P 0.01). At the molecular level, the inhibition of proliferation was associated with significantly reduced CDK6 expression and an increased level of p27 in ER cells but not in ER cells, whereas a common feature in all cell lines was significantly decreased retinoblastoma protein phosphorylation. These results suggest that the edible part of Brassica oleracea contains substances that can markedly inhibit the growth of both ER and ER human breast cancer cells, although through different mechanisms. These results suggest that the widely available cruciferous vegetables are potential chemopreventive agents. JopenBrandi, Giorgio; Schiavano, GIUDITTA FIORELLA; Zaffaroni, N; De Marco, C; Paiardini, M; Cervasi, B; Magnani, MauroBrandi, G; Schiavano, Gf; Zaffaroni, N; De Marco, C; Paiardini, M; Cervasi, B; Magnani, M

Sooty Mangabey Genome Sequence Provides Insight into AIDS Resistance in a Natural SIV Host

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS

CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication

Damaged Intestinal Epithelial Integrity Linked to Microbial Translocation in Pathogenic Simian Immunodeficiency Virus Infections

The chronic phase of HIV infection is marked by pathological activation of the immune system, the extent of which better predicts disease progression than either plasma viral load or CD4+ T cell count. Recently, translocation of microbial products from the gastrointestinal tract has been proposed as an underlying cause of this immune activation, based on indirect evidence including the detection of microbial products and specific immune responses in the plasma of chronically HIV-infected humans or SIV-infected Asian macaques. We analyzed tissues from SIV-infected rhesus macaques (RMs) to provide direct in situ evidence for translocation of microbial constituents from the lumen of the intestine into the lamina propria and to draining and peripheral lymph nodes and liver, accompanied by local immune responses in affected tissues. In chronically SIV-infected RMs this translocation is associated with breakdown of the integrity of the epithelial barrier of the gastrointestinal (GI) tract and apparent inability of lamina propria macrophages to effectively phagocytose translocated microbial constituents. By contrast, in the chronic phase of SIV infection in sooty mangabeys, we found no evidence of epithelial barrier breakdown, no increased microbial translocation and no pathological immune activation. Because immune activation is characteristic of the chronic phase of progressive HIV/SIV infections, these findings suggest that increased microbial translocation from the GI tract, in excess of capacity to clear the translocated microbial constituents, helps drive pathological immune activation. Novel therapeutic approaches to inhibit microbial translocation and/or attenuate chronic immune activation in HIV-infected individuals may complement treatments aimed at direct suppression of viral replication

Author Correction to: SARS-CoV-2 interaction with Siglec-1 mediates trans-infection by dendritic cells

Correction to: https://doi.org/10.1038/s41423-021-00794-6; http://hdl.handle.net/10261/257710In the version of this correspondence initially published, one of the SARS-CoV-2 variants used in Fig. 1B, which was originally described in the article as the SARS-CoV-2 variant ‘B.1.1.248.2 Gamma’, is actually the ‘P.2 Zeta’ SARS-CoV-2 variant of interest. The GISAID accession ID EPI_ISL_1831696 provided is correct, but it belongs to the Zeta variant. The results and conclusions are not affected by this unintentional inaccuracy.Peer reviewe

Siglec-1 on dendritic cells mediates SARS-CoV-2 trans-infection of target cells while on macrophages triggers proinflammatory responses

COVID-19 pandemic is not yet under control by vaccination, and effective antivirals are critical for preparedness. Here we report that macrophages and dendritic cells, key antigen presenting myeloid cells (APCs), are largely resistant to SARS-CoV-2 infection. APCs effectively captured viruses within cellular compartments that lead to antigen degradation. Macrophages sense SARS-CoV-2 and released higher levels of cytokines, including those related to cytokine storm in severe COVID-19. The sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) present on APCs, which interacts with sialylated gangliosides on membranes of retroviruses or filoviruses, also binds SARS-CoV-2 via GM1. Blockage of Siglec-1 receptors by monoclonal antibodies reduces SARS-CoV-2 uptake and transfer to susceptible target cells. APCs expressing Siglec-1 and carrying SARS-CoV-2 are found in pulmonary tissues of non-human primates. Single cell analysis reveals the in vivo induction of cytokines in those macrophages. Targeting Siglec-1 could offer cross-protection against SARS-CoV-2 and other enveloped viruses that exploit APCs for viral dissemination, including those yet to come in future outbreaks.The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). J.M-P. is supported by grant PID2019-109870RB-I00 from the Spanish Ministry of Science and Innovation and in part also by Grifols. CR lab is funded by RTI2018-094445-B100 (MCIU/AEI/FEDER, UE). The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). The NHP study was primarily supported by YNPRC Coronavirus Pilot Research Project Program grant to M.Pa. under award P51 OD11132, Emergent Venture Fast grant program to MPa under awards #2206 and #2144, and William and Lula Pitts Foundation (to MPa).N