Cytokine production in an model of SARS-CoV-2 lung infection.

Introduction: The mechanisms of the SARS-CoV-2-triggered complex alterations in immune cell activation and production of cytokines in lung tissue remain poorly understood, in part because of the limited use of adequate tissue models that simulate the structure and cell composition of the lung . We developed a novel model of SARS-CoV-2 infection of lung explants, that maintains the intact tissue composition and the viral load for up to 7-10 days. Using this model, we studied cytokine production during SARS-CoV-2 infection.

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers surface chemokine receptors and integrins.

Syncytin 1 dependent horizontal transfer of marker genes from retrovirally transduced cells.

Retroviral transduction is routinely used to generate cell lines expressing exogenous non-viral genes. Here, we show that human cells transduced to stably express GFP transfer GFP gene to non-transduced cells. This horizontal gene transfer was mediated by a fraction of extracellular membrane vesicles that were released by the transduced cells.

Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine.

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS).

Extracellular vesicles and viruses: Are they close relatives?

Extracellular vesicles (EVs) released by various cells are small phospholipid membrane-enclosed entities that can carry miRNA. They are now central to research in many fields of biology because they seem to constitute a new system of cell-cell communication. Physical and chemical characteristics of many EVs, as well as their biogenesis pathways, resemble those of retroviruses.

HIV-1 expressing the envelopes of transmitted/founder or control/reference viruses have similar infection patterns of CD4 T-cells in human cervical tissue ex vivo.

Recently, it was found that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether the transmitted/founder (T/F) viruses have specific biological properties favoring sexual transmission, we inoculated human cervical tissue explants with isogenic HIV-1 viruses encoding Env sequences from T/F and control reference (C/R) HIV-1 variants as well as with full length T/F HIV-1 and compared their replication efficiencies, T cell depletion, and the activation status of infected cells. We found that all the HIV-1 variants were capable of transmitting infection to cervical tissue ex vivo and in this system preferentially replicate in activated CD4 T cells and deplete these cells.

Activation of T lymphocytes in atherosclerotic plaques.

Objective: To decipher the immunologic mechanisms of plaque maturation and rupture, it is necessary to analyze the phenotypes and distribution of individual lymphocytes that migrate to the plaques, as well as their activation at different stages of plaque formation.

Methods And Results: We developed a protocol to isolate plaque-residing immune cells and analyze their status using polychromatic flow cytometry. We found that the composition and phenotype of T lymphocytes in the plaques differs from that in blood.

CD4+ T cells, including Th17 and cycling subsets, are intact in the gut mucosa of HIV-1-infected long-term nonprogressors.

During acute human immunodeficiency virus (HIV) infection, there is a massive depletion of CD4(+) T cells in the gut mucosa that can be reversed to various degrees with antiretroviral therapy. Th17 cells have been implicated in mucosal immunity to extracellular bacteria, and preservation of this subset may support gut mucosal immune recovery. However, this possibility has not yet been evaluated in HIV-1-infected long-term nonprogressors (LTNPs), who maintain high CD4(+) T cell counts and suppress viral replication in the absence of antiretroviral therapy.

Selective transmission of R5 HIV-1 variants: where is the gatekeeper?

To enter target cells HIV-1 uses CD4 and a coreceptor. In vivo the coreceptor function is provided either by CCR5 (for R5) or CXCR4 (for X4 HIV-1). Although both R5 and X4 HIV-1 variants are present in body fluids (semen, blood, cervicovaginal and rectal secretions), R5 HIV-1 appears to transmit infection and dominates early stages of HIV disease.

Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000).

Evolution of SIV toward RANTES resistance in macaques rapidly progressing to AIDS upon coinfection with HHV-6A.

Background: Progression to AIDS is often associated with the evolution of HIV-1 toward increased virulence and/or pathogenicity. Evidence suggests that a virulence factor for HIV-1 is resistance to CCR5-binding chemokines, most notably RANTES, which are believed to play a role in HIV-1 control in vivo. HIV-1 can achieve RANTES resistance either by phenotypic switching from an exclusive CCR5 usage to an expanded coreceptor specificity, or by the acquisition of alternative modalities of CCR5 usage.

Measurement of human immunodeficiency virus type 1 preintegration transcription by using Rev-dependent Rev-CEM cells reveals a sizable transcribing DNA population comparable to that from proviral templates.

Preintegration transcription is an early process in human immunodeficiency virus type 1 infection and has been suggested to occur at a low level. The templates have also been suggested to represent a small population of nonintegrated viral DNA, particularly the two-long-terminal-repeat (2-LTR) circles. However, these determinations were made by either using PCR amplification of viral transcripts in bulk cell populations or utilizing the LTR-driving reporter cells that measure the synthesis of Tat.

Genetic variation in OAS1 is a risk factor for initial infection with West Nile virus in man.

West Nile virus (WNV) is a re-emerging pathogen that can cause fatal encephalitis. In mice, susceptibility to WNV has been reported to result from a single point mutation in oas1b, which encodes 2'-5' oligoadenylate synthetase 1b, a member of the type I interferon-regulated OAS gene family involved in viral RNA degradation. In man, the human ortholog of oas1b appears to be OAS1.

Upregulation of human cytomegalovirus by HIV type 1 in human lymphoid tissue ex vivo.

HIV-1 copathogens are believed to play a critical role in progression to AIDS. Human cytomegalovirus (HCMV) has a high prevalence in the general population and is a common copathogen in HIV-1-infected individuals. Important events in copathogen interactions with HIV-1 take place in lymphoid tissue where critical events in HIV-1 disease occur.

HIV-1 induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo.

We demonstrate mechanisms by which HIV-1 appears to facilitate its own infection in ex vivo-infected human lymphoid tissue. In this system, HIV-1 readily infects various CD4+ T cells, but productive viral infection was supported predominantly by activated T cells expressing either CD25 or HLA-DR or both (CD25/HLA-DR) but not other activation markers: There was a strong positive correlation (r=0.64, P=.

Abnormal activation and cytokine spectra in lymph nodes of people chronically infected with HIV-1.

There is growing recognition that HIV-1 infection leads to an activation of the immune system that includes perturbations of cytokine expression, redistribution of lymphocyte subpopulations, cell dysfunction, and cell death. Here, we explored the relationships between HIV-1 infection and immune activation in chronically HIV-1-infected human lymph nodes. In addition to CD4 T-cell depletion, we found increased effector T-cell frequencies associated with profound up-regulation of an activation marker CD38 in naive, central memory, and effector CD4(+) and CD8(+) T cells.

Evolution of CXCR4-using human immunodeficiency virus type 1 SF162 is associated with two unique envelope mutations.

CCR5-using human immunodeficiency virus type 1 (HIV-1) isolates typically gain CXCR4 use via multiple mutations in V3 and often V1/V2 regions of envelope, and patterns of mutations are distinct for each isolate. Here, we report that multiple CXCR4-using variants of a parental CCR5-using HIV-1 isolate, SF162, obtained by either target cell selection or CCR5 inhibition have a common mutation pattern characterized by the same two V3 mutations and that these mutations preexisted in some of the SF162 stocks. These results imply that SF162 has a single pathway for acquiring CXCR4 use and that prolonged culture is sufficient to select for R5X4 variants.

Viral interactions in human lymphoid tissue: Human herpesvirus 7 suppresses the replication of CCR5-tropic human immunodeficiency virus type 1 via CD4 modulation.

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens that affect the clinical course of HIV disease. Here, we identified another virus, human herpesvirus 7 (HHV-7) that interferes with HIV type 1 (HIV-1) replication in human lymphoid tissue, where critical events of HIV disease occur. Like the closely related HHV-6, HHV-7 suppresses the replication of CCR5-tropic (R5) HIV-1 in coinfected blocks of human lymphoid tissue.

Differential pathogenesis of primary CCR5-using human immunodeficiency virus type 1 isolates in ex vivo human lymphoid tissue.

In the course of human immunodeficiency virus (HIV) disease, CCR5-utilizing HIV type 1 (HIV-1) variants (R5), which typically transmit infection and dominate its early stages, persist in approximately half of the infected individuals (nonswitch virus patients), while in the other half (switch virus patients), viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. Here, we used a system of ex vivo tonsillar tissue to compare the pathogeneses of sequential primary R5 HIV-1 isolates from patients in these two categories. The absolute replicative capacities of HIV-1 isolates seemed to be controlled by tissue factors.

Inhibition of HIV-1 replication in human lymphoid tissues ex vivo by measles virus.

Human immunodeficiency virus (HIV) type 1 replication and disease progression are enhanced by various pathogens in coinfected individuals. However, acute infection with measles virus (MV) has been found to suppress HIV-1 replication in coinfected children. We investigated the mechanisms of this phenomenon using human lymphoid tissues coinfected ex vivo with HIV-1 and MV.

Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1.

We investigated whether CD4+ T cells that do not produce HIV-1 are killed in HIV-infected human lymphoid tissue. Tissue blocks were inoculated with high amount of doxycycline-dependent HIV-rtTA. Doxycycline triggered productive infection and loss of CD4+ T cells in these tissues, whereas without doxycycline, neither productive infection nor CD4+ T cell depletion was detected in spite of the massive presence of virions in the tissue and of viral DNA in the cells.

Dual role of prostratin in inhibition of infection and reactivation of human immunodeficiency virus from latency in primary blood lymphocytes and lymphoid tissue.

To design strategies to purge latent reservoirs of human immunodeficiency virus type 1 (HIV-1), we investigated mechanisms by which a non-tumor-promoting phorbol ester, prostratin, inhibits infection of CD4(+) T lymphocytes and at the same time reactivates virus from latency. CD4(+) T lymphocytes from primary blood mononuclear cells (PBMC) and in blocks of human lymphoid tissue were stimulated with prostratin and infected with HIV-1 to investigate the effects of prostratin on cellular susceptibility to the virus. The capacity of prostratin to reactivate HIV from latency was tested in CD4(+) T cells harboring preintegrated and integrated latent provirus.

Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo.

Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo.

Bystander CD4+ T lymphocytes survive in HIV-infected human lymphoid tissue.

HIV infection is associated with depletion of CD4(+) T cells. The mechanisms of this phenomenon remain to be understood. In particular, it remains controversial whether and to what extent uninfected ("bystander") CD4(+) T cells die in HIV-infected individuals.

Segregation of R5 and X4 HIV-1 variants to memory T cell subsets differentially expressing CD62L in ex vivo infected human lymphoid tissue.

Objective: The mechanisms of HIV-triggered immunodeficiency were examined by determining the segregation of R5 and X4 HIV-1 variants into memory T cell subsets expressing differentially a homing receptor, CD62L-selectin, in human lymphoid tissue.

Methods: Subpopulations of CD3 and intracellular p24 gag-positive cells in human lymphoid tissue infected ex vivo with X4 HIV-1 variant NL4-3 and R5 HIV-1 variant AD8 were analysed for expression of the T cell memory markers CD45RO and CD45RA, the T cell homing receptor for lymphoid tissue CD62L, and the HIV-1 coreceptors CCR5 and CXCR4.

Results: Memory CD4 T cells were the predominant targets for productive infection of lymphoid tissue ex vivo with both R5 and X4 HIV-1.