Identification of oligopeptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non structural protein 8 (NSP8) and their similarities with type 1 angiotensin II receptor key sites.

Coronavirus disease 2019 is associated with clinical symptoms including severe inflammatory syndrome and a higher expression of angiotensin II. As a pro-inflammatory mediator, the physiologic effects of angiotensin II are mediated by a G-protein coupled receptor, termed AT1R. Following binding, AT1R initiates the process of signal desensitization necessary to maintain cellular homeostasis. At the cellular level, this function occurs via the G protein-dependent signaling and the phosphorylation. We describe amino acids similarities between SARS COV-2 nonstructural protein (NSP8) which is associated with intracellular membranes and AT1R key sites. Since abnormal activation of AT1R receptor leads to a number of physiological disorders, we hypothesize that SARS COV-2 might further interfere with the angiotensin II receptor functions.

Expression of sarcolectin in the human pituitary gland and amniotic fluid.

Sarcolectin (SCL) is a 55 kDa protein cross-reacting with a cytokeratin 7 monomer found in placental blood, sarcomas and various tissues. It blocks the synthesis of interferon-dependent secondary proteins, induces cell DNA activation and sensitizes cells to viral infection. SCL is a potent promoter of tissue growth. In the present report, we demonstrate that SCL is expressed in the human pituitary gland at the mRNA and protein levels. We show also its presence in human amniotic fluid in high titres while interferon titres is weak. These results allow to postulate a potential role of SCL as a growth factor participating in human foetal development.

Host factor pleiotrophin induces human immunodeficiency virus type 1 replication associated with inflammatory cytokine expression.

Pleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding growth factors; it displays mitogenic activity for a wide variety of cells. In a previous study, we reported that PTN induces the stimulation of expression of inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta and IL-6, in quiescent human peripheral blood mononuclear cells (PBMCs) through B-lymphocyte binding. These results emphasize the importance of PTN in the regulation of inflammatory processes. Moreover, using in vitro infection of PBMCs or using PBMCs from AIDS patients, we showed that PTN was sufficient to induce human immunodeficiency virus type 1 (HIV-1) replication. Moreover, neutralization of TNF-alpha, IL-1beta and IL-6 suppressed HIV replication in PTN-stimulated PBMCs. As these cytokines are potent upregulators of virus expression, these results should prove useful in investigating the role of PTN as a host factor in the regulation of pathological disorders in HIV-1 infection. Identification of this host factor could be important for understanding HIV disease and designating therapeutic approaches.

Enhanced endogenous type I interferon cell-driven survival and inhibition of spontaneous apoptosis by Riluzole.

Highly active antiretroviral therapy (HAART), although effective in improving the survival of HIV-1-infected individuals, has not been able to reconstitute the adaptive immune response. We have described the use of novel chemical agents to restore T-cell survival/proliferation by inducing cytokine production. Due to its cationic amphiphilic structure, these molecules appear to enhance immune restoration. In this study, we investigated the action of Riluzole (2-amino-6-trifuromethoxybenzothiazole) in HIV-1 infection. Riluzole is able to increase (effective dose from 1 to 1000 nM) the cell-survival of T cells from HIV-1-infected patients and inhibit spontaneous apoptosis. The immunomodulatory effect of riluzole-sensitized cells was ascribed to endogenous type I interferon (IFN) derived from monocytes. Riluzole might be used for restoring the cell survival of immunocompromised patients and eliminating latent infected cells upon HIV-1 reactivation.

Pleiotrophin induces expression of inflammatory cytokines in peripheral blood mononuclear cells.

Pleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding growth factor, which displays mitogenic activity for a wide variety of cells. Since PTN induces the proliferation of immune cells the mechanism of action was investigated. In the present study, we show for the first time that PTN induces the expression of inflammatory cytokines including TNF-alpha, IL-1beta and IL-6 in quiescent human peripheral blood mononuclear cells (PBMC). These results emphasize the importance of PTN in the regulation of inflammatory processes. Elucidation of the mechanisms by which a host factor such PTN regulates cytokines production will significantly advance our understanding of endothelium-immunity interactions.

Induction of human immunodeficiency virus (HIV-1) envelope specific cell-mediated immunity by a non-homologous synthetic peptide.

BACKGROUND: Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide. METHODOLOGY/PRINCIPAL FINDINGS: The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants. CONCLUSIONS/SIGNIFICANCE: For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.

Acute and selective inhibition of adipocyte glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase by interferon gamma.

Interferon gamma (IFN-gamma) was previously shown to promote fatty acid (FA) release from adipose tissue (AT). Net lipolysis is an equilibrium between triglyceride breakdown and FA re-esterification. The latter requires activated glyceroneogenesis for glycerol-3-phosphate synthesis and increased cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme in this pathway. We wondered whether glyceroneogenesis and PEPCK-C would be IFN-gamma targets. We injected mice with IFN-gamma, and exposed either AT explants and isolated adipocytes from humans and mice or 3T3-F442A adipocytes to IFN-gamma before monitoring expression of genes involved in lipid metabolism and the metabolic consequences. We show that IFN-gamma induces a large increase in FA release without affecting glycerol output and decreases [1-(14)C]-pyruvate incorporation into lipids, thus demonstrating that FA re-esterification is reduced due to diminished glyceroneogenesis. A series of mRNA encoding proteins involved in FA metabolism remained unaffected by IFN-gamma, while that of PEPCK-C was rapidly and drastically lowered. IFN-gamma effect opposed that of the beta-agonist isoproterenol and of 8-Br-cAMP. In IFN-gamma-treated mice, PEPCK-C gene expression was decreased in AT, but not in liver or kidney. Thus, IFN-gamma exerts a tissue-specific action in rodents and humans, having glyceroneogenesis and the PEPCK-C gene as selective targets to intensify FA release from adipocytes.

Differentiation-dependent expression of interferon gamma and toll-like receptor 9 in 3T3-F442A adipocytes.

3T3-F442A and BFC-1 cells are widely used for studying adipocyte differentiation and metabolism. Macrophage markers were previously reported in these cell lines. We examined whether 3T3-F442A and BFC-1 would produce interferon-gamma (IFN-gamma), the expression of which is a matter of debate in cells other than T-lymphocytes and natural killer cells, like macrophages or dendritic. IFN-gamma was absent from preadipocytes. However 3T3-F442A, but not BFC-1, presented a differentiation-dependent induction of IFN-gamma mRNA and protein. Immunofluorescence studies showed that IFN-gamma was located in mature adipocytes. IFN-gamma was retrieved in the culture medium. Then, we examined the expression of other markers of T-lymphocytes or macrophages, like the CD3/T-cell receptor complex or Toll-like receptors (TLR) -2 and -9, in these cells. Transcripts for the three subunits of CD3 were undetectable whatever the differentiation stage. In contrast, TLR-2 and -9 genes were expressed differentially during the differentiation process. TLR-2 mRNA was induced early then decreased while TLR9 transcript appeared at later days and increased in parallel to IFN-gamma. In contrast to what was expected from 3T3-F442A cells, IFN-gamma was absent from adipocytes isolated either from subcutaneous or periepidydimal mouse adipose tissue. However, TLR-2 and -9 mRNAs were present in both adipose depots although at various levels. Hence, we detect the presence of two markers of innate immunity, TLR-2 and -9, in in vivo-derived adipocytes and we demonstrate that differentiated 3T3-F442A cells selectively express IFN-gamma and TLR-9 in a manner that resembles what is occurring for natural killer dendritic cells.

Effects of sarcolectin (SCL) on human peripheral blood mononuclear cells.

Sarcolectin (SCL) is a tissue growth factor found in various human or animal tissues, functioning in balance with interferons (IFNs) that can inhibit growth and affect cell differentiation. Like somatotropin, SCL is found in the pituitary gland. In humans, the SCL gene is located on chromosome 12 (q12-q13) and expressed as a 55 kDa protein consisting of 469 amino-acids. After a single activation of peripheral blood mononuclear cells (PBMC) obtained from more than 30 individuals, highly significant cell proliferation was found to peak after 7 days in culture. The presence of adherent cells was necessary for cell proliferation. SCL induced over-expression of alpha-IL-2 receptor (CD25) leading to proliferation of CD3+/CD4+/CD45RO+ T cells. Thus in PBMC, SCL induced CD4+ T cell growth and expression of inflammatory cytokine genes, including TNF-alpha, IL-1beta, IL-6 and IL-8. IFNs are also produced following activation as a feedback response which is maintained for about 20 days.

Expression of macrophage-selective markers in human and rodent adipocytes.

CD14, CD68 and/or mouse F4/80 or human epidermal growth factor module-containing mucin-like receptor 1 (EMR1) are widely used as macrophage-specific markers. Since macrophages infiltrate several tissues during inflammatory processes, CD14, CD68 and EMR1-F4/80 have been employed to discriminate between tissue-containing macrophages, like adipose tissue (AT), and other cells. Using real-time PCR experiments, we show that isolated adipocytes from humans and mice AT express high levels of CD14 and CD68 mRNA, whereas EMR1-F4/80 is mainly present in the macrophage-containing stroma-vascular fraction. Furthermore, fibroblasts-like cells (adipoblasts), preadipocytes and adipocytes from the murine cell lines, 3T3-F442A and BFC-1, express CD14 and CD68 mRNA and protein as determined by fluorescence-activated cell sorter, but not F4/80 which, as expected, is strongly expressed in the macrophage cell line RAW264.7. These results reinforce the view that EMR1-F4/80 is the best macrophage marker to date and show that CD14 and CD68 are not macrophage-specific proteins.

T cell survival/proliferation reconstitution by trifluoperazine in human immunodeficiency virus-1 infection.

Recent findings support an indirect relationship between T cell depletion in HIV-1 infection and the rate of virus replication with implications for treatment strategies. We have initiated a new approach to recover immune function through the use of novel chemical agents. A cationic amphiphilic drug that binds to Ca(2+)-calmodulin at high concentrations, [10-[3-(4-methyl-1-piperazinyl)-propyl]-2- (trifluoromethyl)-(10)H-phenothiazine dihydrochloride] [denoted trifluroperazine dihydrochloride (Tfp); molecular weight 480.43] TFP was found at low concentrations (10(-6) to 10(-10) M) to help T cells from AIDS patients to restore proliferation in vitro. Here we show that the Tfp molecule can restore the cell survival of T lymphocytes from PBMCs derived from HIV-1-infected patients in vitro. Tfp enhances T cell proliferation and Th-cell responses by selectively inhibiting cell mortality and apoptosis. The restored antigen-specific response is associated with the synthesis of IL-2 and gamma-interferon. Even though this drug does not possess any detectable antiviral effect, it might be considered as a potential therapeutic agent in HIV-infected patients, to correct immune defects. Besides antiviral compounds, these data may facilitate immune reconstitution in patients with HIV infection and other immunosuppressive diseases.

The interferon antagonist sarcolectin in the progress of HIV-1 infection and in AIDS.

Sarcolectin (SCL) is a nonspecific stimulator of cellular DNA synthesis that was found in all animal sera tested to date. It inhibits the established interferon (IFN)-dependent antiviral state, restoring cells to their normal status. In this study, we examined the excretion/secretion of the IFN antagonist SCL in sera from healthy donors and in sera collected during different periods of human immunodeficiency virus type 1 (HIV-1) infection. We followed HIV-1-infected patients during all stages of development (seroconversion, initial and advanced phases of AIDS) and found a significant increase in SCL in sera of HIV-infected patients compared with seronegative subjects used as controls. This increase was established during seroconversion, and then the titers leveled off. In the final stage of the disease, the SCL titer increased again very significantly. We attribute this rapid rise to the virus-dependent destruction of T cells that can no longer be repaired. The high SCL level observed at this final stage, which is most predictive of the disease's progression, suggests that the action, rather than the production, of IFN is impaired.