Viral Biology and Immune Privilege in the Development of Extrahepatic Manifestations During Hepatitis E Virus Infection.

Hepatitis E virus (HEV) exhibits tropism toward hepatocytes and thus affects the liver; however, HEV may also affect other tissues, including the heart, kidneys, intestines, testicles, and central nervous system. To date, the pathophysiological links between HEV infection and extrahepatic manifestations have not yet been established. Considering that HEV infects multiple types of cells, the direct effects of virus replication in peripheral tissues represent a plausible explanation for extrahepatic manifestations. In addition, since the immune response is crucial in the development of the disease, the immune characteristics of affected tissues should be revisited to identify commonalities explaining the effects of the virus. This review summarizes the most recent advances in understanding the virus biology and immune-privileged status of specific tissues as major elements for HEV replication in diverse organs. These discoveries may open avenues to explain the multiple extrahepatic manifestations associated with HEV infection and ultimately to design effective strategies for infection control.

Autoantibody Diversity Is Augmented in Women with Breast Cancer and Is Related to the Stage of the Disease.

Breast cancer (BC) is the most frequent malignant neoplasia and leading cause of cancer mortality for women. A timely diagnosis of BC is crucial to ensure the best chances of survival. Among the various screening tools for BC, antibodies directed towards self-antigens or tumor-associated antigens (autoantibodies) have emerged as an alternative to image-based screening modalities. However, little attention has been paid to the global diversity of autoantibodies. This work aimed to analyze the diversity of autoantibodies reactive to antigens expressed by the BC cell line T47D in the sera of Mexican women with BC, benign breast pathology (BBP), or without breast pathology (WBP). We found that the diversity of antibodies in the sera was higher in the BC and BBP groups than in the WBP group. Likewise, the diversity changed with the progression of BC. Our results show and measure the complexity of the antibody response in breast health and disease.

COVID-19 in Latin America and the caribbean region: Symptoms and morbidities in the epidemiology of infection.

The COVID-19 pandemic has widespread economic and social effects on Latin America (LA) and the Caribbean (CA). This region, which has a high prevalence of chronic diseases, has been one of the most affected during the pandemic. Multiple symptoms and comorbidities are related to distinct COVID-19 outcomes. However, there has been no explanation as to why different patients present with different arrays of clinical presentations. Studies report that similar to comorbidities, each country in LA and the CA has its own particular health issues. Moreover, economic and social features have yet to be studied in detail to obtain a complete perspective of the disease in the region. Herein, the impact of demographic and economic characteristics in LA and the CA on COVID-19 are presented in combination with symptoms and comorbidities related to the disease as important aspects that can influence management and treatment.

Host membrane proteins in the HIV-induced membrane fusion: Role in pathogenesis and therapeutic potential of autoantibodies.

Host proteins such as receptors, adhesion and signaling molecules, promote virus-cell fusion, virus cell-cell transmission, and formation of multinucleated cells with outstanding properties. These events are implicated in virus dissemination and the induction of pathological effects such as the infection of the gut-associated lymphoid tissue, placenta infection, and neurological complications. Antibodies directed to the host membrane proteins are produced during the natural HIV infection and may contribute significantly to virus inhibition. Antibodies against the HIV receptor have been approved for therapy and others targeting additional host membrane proteins are currently under evaluation. This review emphasizes the relevance of the different pathways of HIV spreading between cells and of antibodies directed to host membrane components in the development of broad-range therapeutics against HIV.

Influence of adjuvants on the amount, specificity and functional activity of antibody response to human influenza vaccine in mice.

It's been almost a century since immunologists started using adjuvants as tools to develop more effective vaccines. Despite the rising number of adjuvanted vaccines in the last decades, we still lack knowledge of the adjuvants' effects on antibody response. This study was aimed to test the effect of immunizing mice with the human Inactivated Influenza vaccine (IIV), either alone or combined with different widely used adjuvants on the specific antibody response induced. Differential levels of IgM and IgG subclasses were found with the different adjuvants tested. Higher levels of antibodies did not always correspond with a higher efficacy to interfere with the virus infectivity. Differences in neutralization properties are possibly mediated by the specificity of the repertoire of antibodies induced. The repertoire was studied using a phage display 7-mer peptide library to screen for epitopes/mimotopes recognized by serum pools from vaccinated mice. The selected phage clones included peptides that corresponded to conformational mimotopes since they have no homology with lineal sequences of the Influenza strains' proteins. Five peptides were identified as recognized by sera from mice immunized with the IIV vaccine alone, including peptides from the hemagglutinin stalk domain, and by sera from mice immunized with the vaccine plus the different adjuvants employed. Adjuvants elicited a more diverse repertoire of epitope-recognizing antibodies that recognized epitopes of the HA recombinant globular head. Mimotopes were theoretically located at the neutralizing antigenic sites of the globular head of Influenza A H1N1pdm09, Influenza A H3N2, and Influenza B hemagglutinin. This study illustrates how different adjuvants can modify the extent and quality of humoral immunity against the IIV vaccine and the effectiveness of vaccination.

Inhibition of HIV-1 envelope-dependent membrane fusion by serum antilymphocyte autoantibodies is associated with low plasma viral load.

The HIV-1 envelope protein (Env) mediates the membrane fusion process allowing virus entry to target cells and the efficiency to induce membrane fusion is an important determinant of HIV-1 pathogenicity. In addition to virus receptors, other adhesion/signaling molecules on infected and target cells and virus particles can enhance fusion. The presence of antilymphocyte autoantibodies (ALA) in HIV patients' serum suggests that they may contribute to the inhibition of Env-mediated membrane fusion. Here, sera from 38 HIV-1 infected treatment-naïve men and 30 healthy donors were analyzed for the presence of IgG and IgM able to bind to CD4-negative Jurkat cells. The use of CD4-negative cells precluded the binding of virus-antibody immune complexes, and allowed detection of ALA different from anti-CD4 antibodies. IgG and IgM antibodies binding to Jurkat CD4-negative cells was detected in 74% and 84% of HIV-positive sera, respectively. Then, the activity of sera on fusion of CD4+ with HIV Env+ Jurkat cells was determined before and after their adsorption on CD4-negative Jurkat cells to remove ALA. Sera inhibited fusion at variable extents, and inhibitory activity decreased in 58% of serum samples after adsorption, indicating that ALA contributed to fusion inhibition in these sera (herein called fusion inhibitory ALA). The contribution of ALA to fusion inhibition in individual sera was highly variable, with an average of 33%. IgG purified from a pool of HIV+ sera inhibited fusion of primary CD4 T lymphocytes with Jurkat Env+, and adsorption of IgG on CD4-negative Jurkat cells diminished the fusion inhibitory activity. Thus, the inhibitory activity of sera was related to IgG ALA. Our observations suggest that fusion inhibitory ALA other than anti-CD4 antibodies may contribute significantly to the inhibition of Env-mediated cell-cell fusion. Fusion inhibitory ALA, but not total ALA levels, associated with low plasma viral loads, suggesting that specific ALA may participate in virus containment by inhibiting virus-cell fusion in a significant fraction of HIV-infected patients.

Vaccination with human papillomavirus-18 E1 protein plus α-galactosyl-ceramide induces CD8+ cytotoxic response and impairs the growth of E1-expressing tumors.

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols.

Recovery from an acute systemic and central LPS-inflammation challenge is affected by mouse sex and genetic background.

Genetic and sexual factors influence the prevalence and the pathogenesis of many inflammatory disorders. In this study their relevance on the peripheral and central inflammatory status induced by a peripheral injection of lipopolysaccharide (LPS) was evaluated. BALB/c and CD-1 male and female mice were intraperitoneally injected with LPS. Spleens and brains were collected 2 and 72 hours later to study the levels of IL-6, TNF-α and IL-1ß. Percentage of microglia and astrocytes was determined in the cortex and hippocampus. Locomotor activity was registered before and during the 72 hours after LPS-treatment. Two hours after LPS-injection, a peripheral increase of the three cytokines was found. In brains, LPS increased TNF-α only in males with higher levels in CD-1 than BALB/c. IL-1ß increased only in CD-1 males. IL-6 increased in both strains with lower levels in BALB/c females. Peripheral and central levels of cytokines decline 72 hrs after LPS-treatment whilst a significantly increase of Iba-1 expression was detected. A dramatic drop of the locomotor activity was observed immediately after LPS injection. Our results show that acute systemic administration of LPS leads to peripheral and central increase of pro-inflammatory cytokines and microglia activation, in a strain and sex dependent manner.

FRET in the Analysis of In Vitro Cell-Cell Fusion by Flow Cytometry.

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.

The network of antigen-antibody reactions in adult women with breast cancer or benign breast pathology or without breast pathology.

The Immunoglobulin G (IgG) antibody response to different protein antigens of the mammary ductal carcinoma by adult women affected by Breast Cancer (BC) distinguishes at least 103 proteins that differ in their molecular weights (MW). The IgG producing cell clones (nodes) coexist with each other in each individual organism and share energy resources among themselves, as well as factors that control the level of expression and Specificity of their IgG antibodies. So, it can be proposed that among them there is a Network of interconnections (links) unveiled by the antigens, which specifically react with the IgG antibodies produced by the clones. This Network possibly regulates IgG antibodies' activity and effectiveness. We describe the Network of nodes and links that exists between the different antigens and their respective IgG producing cell clones against the extracted protein antigens from the cells of the T47D Cell-Line, in 50 women with BC, 50 women with Benign Breast Pathology (BBP) and 50 women without breast pathology (H). We have found that women with BBP have the highest number of Links, followed by the H group and, lastly, the women with BC, a finding which suggests that cancer interferes with the Connectivity between the IgG producing cell clones and blocks the expression of 322 links in women with BBP and 32 links in women with H. It is also plausible that the largest number of links in the women with BBP indicates the Network's state of arousal that provides protection against BC. On the other hand, there were many missing links in the BC group of women; the clone which lost more links in the BC group was the hub 24, which point to some of the antigens of T47D as potentially useful as vaccines, as the immune system of women with BBP is well aware of them.

Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro.

Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell-cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4(+) cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4(+) cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env(+) cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4(+) cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell-cell fusion can be performed by flow cytometry.

Role of innate immunity against human papillomavirus (HPV) infections and effect of adjuvants in promoting specific immune response.

During the early stages of human papillomavirus (HPV) infections, the innate immune system creates a pro-inflammatory microenvironment by recruiting innate immune cells to eliminate the infected cells, initiating an effective acquired immune response. However, HPV exhibits a wide range of strategies for evading immune-surveillance, generating an anti-inflammatory microenvironment. The administration of new adjuvants, such as TLR (Toll-like receptors) agonists and alpha-galactosylceramide, has been demonstrated to reverse the anti-inflammatory microenvironment by down-regulating a number of adhesion molecules and chemo-attractants and activating keratinocytes, dendritic (DC), Langerhans (LC), natural killer (NK) or natural killer T (NKT) cells; thus, promoting a strong specific cytotoxic T cell response. Therefore, these adjuvants show promise for the treatment of HPV generated lesions and may be useful to elucidate the unknown roles of immune cells in the natural history of HPV infection. This review focuses on HPV immune evasion mechanisms and on the proposed response of the innate immune system, suggesting a role for the surrounding pro-inflammatory microenvironment and the NK and NKT cells in the clearance of HPV infections.

Quantitative and phenotypic analyses of lymphocyte-monocyte heterokaryons induced by the HIV envelope proteins: Significant loss of lymphoid markers.

Cells infected with the human immunodeficiency virus (HIV) can fuse with CD4(+) cells leading to the formation of multinucleated cells. The presence of multinucleated cells infected with HIV in tissues of patients has been documented, although their cellular composition and role in AIDS pathogenesis is still under study. Here, we present evidence of in vitro heterotypic lymphocyte-monocyte fusion in cocultures of lymphocytic Jurkat T cells expressing the HIV-1 gp120/gp41 glycoproteins (Env) and CD4(+) monocytic THP-1 cells. Using a previously characterized method that involves differential labeling of fusion partners with fluorescent probes and flow cytometry analysis after coculture, up to 20% of double fluorescent cells were detected in 48h. This double fluorescent cell population was produced by heterotypic lymphocyte-monocyte fusion as it was not observed when Jurkat T cells expressing a mutant non-fusogenic Env protein were used. Heterokaryon formation was inhibited by an anti-CD4 monoclonal antibody and the HIV-fusion inhibitor peptide T-20. About 68% of heterokaryons remained alive and non-apoptotic after 2days of coculture. In heterokaryons, CD4 was barely detectable and the expression of the CD3 and CD28 lymphoid markers was greatly reduced, whereas the expression of CD32 and the intracellular antigen CD68, both markers of monocytic cells, remained unchanged. In contrast with unfused T cells, heterokaryons only expressed very low levels of the lymphoid activation marker CD25 following treatment with PMA plus ionomycin. These studies point to the possible generation of lymphocyte-monocyte heterokaryons with a myeloid phenotype during HIV infection, with unknown consequences for AIDS pathogenesis.

Decreased CD4 and wide-ranging expression of other immune receptors after HIV-envelope-mediated formation of syncytia in vitro.

In human HIV infection, multinucleated cells (syncytia) are formed by fusion of HIV-infected cells with CD4+ cells. In order to examine possible functional implications of syncytia formation for the immune response, the expression of important surface molecules by T-cell syncytia and surrounding cells that remain unfused (bystander cells) was analyzed in cocultures of HIV-Env- and CD4-expressing E6 Jurkat T cells. Fusion partners were differentially labeled with lipophilic probes, and syncytia and bystander cells were identified by flow cytometry. The cellular phenotype and response to activation stimulus after fusion were analyzed with antibodies coupled to third-party fluorochromes. Cocultured unfused E6 cells showed a marked decrease in CD4 expression, suggesting the selective recruitment of cells strongly expressing CD4 into syncytia. However, the incorporated CD4 was not detected in the syncytia, whereas the range of expression of CD28, ICAM-1, CXCR4 and CD3 was wider than that of unfused cells. Limited expression of CD4 in the bystander unfused population, as well as in the newly formed syncytia, would result in limitation of further viral entry and a failure to identify these cells, and it could partially contribute to functional impairment and a decrease in the number of CD4+ T cells in AIDS. Most of the syncytia were viable and expressed CD25 and IL-2 in response to activation by phorbol myristate acetate (PMA) and ionomicyn. Thus, syncytia populations harboring widely heterogeneous levels of receptors would constitute a potential source of anomalous immune function.

HIV-envelope-dependent cell-cell fusion: quantitative studies.

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.

Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.

Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

Taenia crassiceps cysticercosis: immune response in susceptible and resistant BALB/c mouse substrains.

Taenia crassiceps can naturally and experimentally infect rodents in which they reproduce by budding. Differences in the susceptibility to T. crassiceps cysticercosis were found between two BALB/c substrains: BALB/cAnN (susceptible) and BALB/cJ (resistant). In chimeric mice, resistance was transferred to susceptible mice with bone marrow cells from the resistant mice, which argues in favor of an immune mediation of the resistant phenotype. To further explore the immune response that could underlie these differences in susceptibility, the specific cellular immune response elicited by the parasite was explored in both substrains. An increased proliferative response and IL-2 levels were induced by cysticercal antigens only in splenocytes from resistant mice. A decrease in the percentage of CD4(+) (11.1%), CD8(+) (17.5%) was found in splenocytes from susceptible BALB/cAnN mice. A study of the TCRV beta repertoire revealed a significant decrease in V beta 2 in both CD4(+) and CD8(+) splenocytes only in the susceptible BALB/cAnN strain.

Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.

BACKGROUND: In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. METHODS: We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. RESULTS: Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). CONCLUSIONS: The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion.