Interaction in vitro between cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17–23; López-Balderas et al., 2007, Virus Res. 123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

Fusion of CD4+ cells by HIV-1 envelope proteins (Env) is a mechanism of virus spread and cell damage. Production of antibodies able to influence cell-cell fusion in vivo may affect the course of the infection. The effect of sera from 49 HIV-1-positive patients was tested on an in vitro fusion assay using Env-expressing and normal Jurkat T cells labelled with DiI and DiO dyes, and flow cytometry for quantification of cell-cell fusion. Sera varied in their activity on fusion: 69?4 % inhibited, 24?5 % had no effect and 6?1 % enhanced cell fusion. Fusion activity correlated positively with the CD4 + T-cell count and inversely with the viral load. Removal of IgG or IgM from sera reduced or eliminated inhibition and enhancing activities, respectively. Antibodies with inhibitory activity predominate in early and intermediate stages of infection, whereas loss of inhibition or enhancement of fusion correlates with progression to AIDS.Human immunodeficiency virus type 1 (HIV-1) frequently induces cell-cell fusion with syncytia formation in cultures of CD4 + T cells. Syncytia formation result from the interaction between infected cells expressing the virus envelope glycoprotein (Env) on their surface and neighbouring uninfected CD4+ cells (Lifson et al., 1986;Sodroski et al., 1986). Cell-cell fusion may have an important pathogenic role in vivo. HIV-1-infected giant multinucleated cells are frequently detected in the lymph nodes of asymptomatic and AIDS patients, and have been proposed as virus reservoirs (Budka 1986; Frankel et al., 1996;Koenig et al., 1986). Syncytium-inducing viruses (usually requiring CXCR4 as a co-receptor) associate with an increased rate of CD4 + T-lymphocyte depletion in patients and progression to AIDS (Blaak et al., 2000; Connor et al., 1993;Miedema et al., 1994). Peptides that interfere with virus-cell fusion decrease viral loads in humans (Kilby et al., 1998;Lazzarin et al., 2003). Furthermore, the membrane fusing ability of Env from a simian-human immunodeficiency virus chimera was determinant for CD4 + T-cell depletion in macaques (Etemad-Moghadam et al., 2001). Cell fusion is a significant cause of cell death in vitro, mainly by apoptosis triggered by aberrant initiation of mitosis (Castedo et al., 2002; Ferri et al., 2000;Laurent-Crawford et al., 1991). The magnitude of CD4 + cell-cell fusion events can be significant in lymphoid tissues, where there is close contact between cells (Amendola et al., 1996). On the other hand, cell-to-cell HIV-1 transmission without formation of syncytia, likely involving localized fusion events at cell contact points was proposed as a mechanism of transmission between individuals, and an important route of viral spread (Gupta et al., 1989;Phillips, 1994;Sato et al., 1992). Recently, it was shown that virus strains highly efficient in cell-to-cell transmission and with a syncitium-inducing phenotype are selected in an in vitro model of the rapid T-cell turnover known to occur during HIV-1 infection (Gummurulu et al., 2000).Inhibition of pathogenic cell-cell ...

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Very early on COVID-19 pandemic outbreak, it was noted that the some of the virus-induced clinical conditions resembled features of toxaemia caused by the toxic shock syndrome toxin type 1, which is a soluble superantigen produced by Staphylococcus aureus. Among all SARS proteins, the ORF8 protein from SARS-2 virus is significantly different from other known SARS-like coronaviruses, and therefore could exhibit unique pathogenic properties. We assess if ORF8 protein bears super antigenic features using in silico tools. We show that ORF8 has properties of an extracellular soluble protein and shares a significant degree of amino acid sequence identity with toxic shock syndrome toxin. Besides, docking and binding affinity analyses between monomeric and homodimeric ORF-8 with Vβ 2.1 and TRBV11-2 reveal strong interaction and high binding affinity. ORF8-TRBV11-2 strong interaction can contribute to the observed clonal expansion of that chain during COVID-19-associated multisystem inflammatory syndrome. Taken together, the evidence presented here supports the hypothesis that ORF8 protein from SARS-2 bears super antigenic properties.

Cell-cell fusion is a frequent event in nature leading to modification of cell fate. In this chapter, we describe a flow cytometric procedure for the quantitative assessment of in vitro cell-cell fusion events that allows the discrimination of fused from aggregated cells. The assay is based on the differential labeling of fusion partners with lipophilic fluorescent probes DiI (red) and DiO (green). Double fluorescent fused cells can be detected after coculturing by means of a flow cytometer equipped with a 488 nm laser. Fusion events can be distinguished from cell aggregates by the enhancement of the DiI red fluorescence intensity due to resonance energy transfer between the two probes occurring in the fused but not in the aggregated cell population.

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The use of monocytes, macrophages, dendritic or Kupffer cells, to test drug metabolism, has serious limitations because these cells express lower levels of cytochrome P450 enzymes than PHH. The best available option, to replace PHH, have been tumor cell lines such as HepaRG, as well as immortalized hepatocytes from adult or fetal sources. Monocyte-derived NeoHep cells are novel and easily accessible cells, which express many drug metabolizing enzymes at levels comparable to PHH. These cells allow drug evaluation under a diverse genetic background. While these cells are in the early stages of evaluation and do need to be examined more thoroughly, they constitute a promising new tool for in vitro drug testing.