[Cytomegalovirus Infection and Endothelial Function in Patients With Acute Myocardial Infarction].

PURPOSE: to study elucidate association of active cytomegalovirus (CMV) infection with endothelial dysfunction in patients with acute myocardial infarction (AMI). MATERIALS AND METHODS: The study included 42 volunteers without ischemic heart disease (IHD) and 63 patients with AMI. Blood samples for analysis of the deoxyribonucleic acid (DNA) of CMV in plasma by real-time polymerase chain reaction were taken in patients - before coronary angiography, in volunteers - at admission. In addition, in patients with AMI and volunteers without IHD, endothelial function was analyzed using endothelium-dependent vasodilatation (EDVD) test of the brachial artery. RESULTS: We showed that in patients with AMI, the concentration of CMV DNA in plasma was statistically significantly increased when compared with that in volunteers without IHD, which reflects active CMV infection - 1185.7 (0; 3003.0) vs. 0 (0; 910.8) copies of DNA / ml plasma (p=0.011). In comparison with volunteers without IHD, patients with AMI also more often had endothelial dysfunction - 11.5 (7.5, 15.2) % vs. 4.4 (0; 9.6) % of cases (p.

Juri Markovich Vasiliev - My Mentor and Friend.

This is an overview of the biography and scientific accomplishments of Dr. Juri Vasiliev, an outstanding Russian cell biologist. Ju. M. Vasiliev published seminal papers on carcinogenesis and cytoskeleton of normal and cancer cells. He founded a scientific school and mentored many students that are now occupying leading positions in different laboratories throughout the world.

Cytomegalovirus Infection in Cardiovascular Diseases.

Atherosclerosis underlies the development of many cardiovascular diseases that continue to hold a leading place among the causes of death in developed countries. The role of activated immune cells in atherosclerosis progression has been convincingly demonstrated, but the mechanism of their action remains poorly investigated. Since atherosclerosis is associated with chronic inflammatory response, involvement of viral and bacterial infections in atherogenesis has been examined. A special place among the infectious agents is held by human herpesviruses as the most common persistent viruses in human population coupled to chronic inflammation during atherosclerosis. We found that activation of cytomegalovirus (CMV, human herpesvirus 5) infection is associated with the emergence of acute coronary syndrome, which is in a good agreement with the data on productive CMV infection published elsewhere. In this review, we discuss the data obtained by us and other researchers regarding the role of cytomegalovirus infection and related potential mechanisms resulting in the expansion of atherosclerotic plaques during ischemic heart disease and stroke, including virus transfer to immune and endothelial cells via extracellular vesicles. In particular, the data presented in the review demonstrate that virus spreading in the vascular wall triggers immune system activation in atherosclerotic plaques and causes endothelial dysfunction. Moreover, productive CMV infection in patients with acute myocardial infarction correlates with the extent of endothelial dysfunction. The mechanisms described by us and other researchers may explain the role of CMV infection in atherosclerosis and development of ischemic heart disease.

Cytomegalovirus in Plasma of Acute Coronary Syndrome Patients.

The relationship between acute coronary syndrome (ACS) and local and systemic inflammation, including accumulation of macrophages in atherosclerotic plaques and upregulation of blood cytokines (e.g., C-reactive protein (CRP)), has been known for more than 100 years. The atherosclerosis-associated inflammatory response has been traditionally considered as an immune system reaction to low-density lipoproteins. At the same time, some data have indicated a potential involvement of cytomegalovirus (CMV) in the activation and progression of atherosclerosis-associated inflammation, leading to ACS. However, these data have been tangential and mainly concerned the relationship between a coronary artery disease (CAD) prognosis and the anti-CMV antibody titer. We assumed that ACS might be associated with CMV reactivation and virus release into the bloodstream. The study's aim was to test this assumption through a comparison of the plasma CMV DNA level in patients with various CAD forms and in healthy subjects. To our knowledge, no similar research has been undertaken yet. A total of 150 subjects (97 CAD patients and 53 healthy subjects) were examined. Real- time polymerase chain reaction (RT-PCR) was used to determine the number of plasma CMV DNA copies. We demonstrated that the number of plasma CMV genome copies in ACS patients was significantly higher than that in healthy subjects (p = 0.01). The CMV genome copy number was correlated with the plasma CRP level (p = 0.002). These findings indicate a potential relationship between CMV activation and atherosclerosis exacerbation that, in turn, leads to the development of unstable angina and acute myocardial infarction. Monitoring of the CMV plasma level in CAD patients may be helpful in the development of new therapeutic approaches to coronary atherosclerosis treatment.

Analysis of Extracellular Vesicles Using Magnetic Nanoparticles in Blood of Patients with Acute Coronary Syndrome.

Extracellular vesicles (EVs) are released from various cell types and play an important role in intercellular interactions. In our study, we investigated abundance of individual EVs in patients with acute forms of ischemic heart disease. Previously, we developed an approach for individual analysis of EVs conjugated with magnetic nanoparticles (MNPs), which was applied in the current study for analyzing phenotypic composition of EVs (by staining for markers CD31, CD41a, and CD63). EVs were isolated using fluorescently labeled MNPs containing anti-CD31, CD41a, or CD63 antibodies and analyzed by combining fluorescently labeled anti-CD41a and CD63, CD31 and CD63, or CD41a and CD31 antibodies, respectively. EVs were analyzed in 30 individuals: 17 healthy volunteers and 13 patients with acute coronary syndrome (ACS). Six and seven ACS patients were with acute myocardial infarction and unstable angina, respectively. It was found that patients with ACS and healthy volunteers contained a dominant subset of EVs expressing surface CD41a antigen, suggesting that they originated from platelets. In addition, the total number of EVs isolated using either of the surface markers examined in our study was higher in patients with ACS compared to healthy volunteers. The subgroup of patients with acute myocardial infarction was found to contain significantly higher number of blood EVs compared to the control group. Moreover, increased number of EVs in patients with ACS is mainly due to the increased number of EVs in the subset of EVs bearing CD41a. By analyzing individual EVs, we found that plasma of patients with ACS, particularly upon developing of myocardial infarction, contained dominant platelet-derived EVs fraction, which may reflect activation of platelets in such patients.

[Immunological Analysis of Human Atherosclerotic Plaques in ex vivo Culture System].

THE AIM OF THE STUDY: to analyze the dynamics of lymphocytic composition of human atherosclerotic plaques in ex vivo culture system. MATERIALS AND METHODS: The study included 15 atherosclerotic plaques obtained from patients who underwent carotid endarterectomy. Plaques were cultured as ring-shaped explants on collagen rafts in culture medium of special composition in CO2 incubator according to the previously developed technique. On day 0, and also on the 4th and 19th days of culture we extracted cells from plaque explants and analyzed B- and T-lymphocytic content of the tissue, as well as the percentage of CD16+ natural killer (NK) cells, using multichromatic flow cytometry. For this purpose we digested the explants with an original enzymatic cocktail, which allows preservation of cell surface markers, and we stained extracted cells with fluorescence-labelled monoclonal antibodies against CD45, CD3, CD19, CD4, CD8, CD16. In addition, we estimated the amount of interleukin 2 (IL-2) and interferon-gamma (IFN-)-producing T-cells by means of flow cytometry. RESULTS: After 4 days of culture the amount of lymphocytes in plaques explants decreased, however live lymphocytes were still preserved (2619.3 [1680.4, 3478.2] cells/100mg tissue). Viable lymphocytes population included T cells (2123.4 [484.9; 3181.2] cells/100 mg tissue), B cells (5.6 [3.4, 27.9] cell/100 mg tissue) and CD16+ NK cells (10.6 [1.8, 23.7] cell/100mg tissue). On the 4th day of culture T cells were presented by CD4+CD8- (797 [475.5, 1000.7] cells/100mg tissue, 37.5 [32.1; 46.3]%) and CD4-CD8+ (686.2 [423.6; 1158.4] cells/100 mg tissue, 45.6 [38.1; 47.9]%) populations. The percentage of CD4+CD8- T cell population decreased compared to the 1st day of culture, and this decrease correlated with the increase in CD4-CD8- T cells content (p<0.05). Additionally, after 4 days of culture we found in tissue explants both CD8+ (17.5[13.3;19.9]%) and CD8- (9.9 [6.4; 14]%) IFN--producing T-cells, however, their percentage, as well as the percentage of IL-2-producing T cells tended to decrease. After 19 days of culture explants of atherosclerotic plaques also contained lymphocytes (2830.1 [2350.3, 5900.2] cells/100mg tissue). Lymphocytes population included T cells (2594.5 [2035.7, 5306.7] cells/100mg tissue), presented by CD4+CD8- (1016.8 [671.2, 2201.7] cells/100mg tissue, 42.3 [34.3; 47.8]%) and CD4-CD8+ (1534.3 [813.8; 2207.2] cells/100mg tissue, 50.8 [45.6; 56.5]%) subsets, B cells (31 [18.3; 64.4] cell/100 mg tissue) and CD16+ NK cells (44.9 [33.4; 138.9] cells/100 mg of tissue). DISCUSSION: An ex vivo model of human atherosclerotic plaque culture that we previously developed enables to preserve viability of various lymphocyte subsets for up to 19 days. We also found that cultured tissue explants retain T cells that can maintain T-helper-1-dependent immune response, which demonstrates inflammation in atherosclerotic plaques. Our results allow to perform experiments on immunological mechanisms of atherogenesis and to develop new approaches for treatment of atherosclerosis, devoted to the suppression of local inflammatory processes in atherosclerotic plaques. CONCLUSIONS: An ex vivo model of human atherosclerotic plaque preserves CD4+CD8- and CD4-CD8+ T cells, B cells, and CD16+ NK cells for a long time. Moreover, after 4 days of culture tissue explants also retain IFN-++ T cells.

5-Arylaminouracil Derivatives as Potential Dual-Action Agents.

Several 5-aminouracil derivatives that have previously been shown to inhibit Mycobacterium tuberculosis growth at concentrations of 5-40 µg/mL are demonstrated to act also as noncompetitive non-nucleoside inhibitors of HIV-1 reverse transcriptase without causing toxicity in vitro (MT-4 cells) and ex vivo (human tonsillar tissue).

Candidate microbicides block HIV-1 infection of human immature Langerhans cells within epithelial tissue explants.

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1(Ba-L) infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08-4.77%). HIV-1-infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1-1 microg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1(Ba-L) (an R5 HIV-1 strain) more efficiently infected LC-T cell cocultures when compared with HIV-1(IIIB) (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.

HIV-infected human Langerhans cells transmit infection to human lymphoid tissue ex vivo.

OBJECTIVES: To create a novel ex vivo model for early biologic events involved in sexual transmission of HIV and to demonstrate that Langerhans cells (LC), the purported initial mucosal target cells for HIV, play a critical role in this process. METHODS: Epidermal cells containing LC were isolated from normal-appearing skin of healthy volunteers and exposed to a panel of primary and laboratory-adapted R5- and X4-HIV isolates, washed and applied to the surfaces of allogeneic tonsil tissue blocks. Viral replication was followed by measuring HIV p24 protein in culture supernatants by ELISA. RESULTS: Both R5- and X4-HIV isolates could be transmitted by LC and established high levels of infection in lymphoid tissue (p24 > 10 ng/ml). Depletion of LC within epidermal cell suspensions abrogated the ability of HIV-exposed suspensions to transmit virus to tonsil histocultures. CONCLUSIONS: Using a novel ex vivo model, human LC are shown for the first time to be the major epidermal cell type that is involved in transmission of HIV infection to human lymphoid tissue. Importantly, this system could prove useful in further understanding LC trafficking and other early biological events involved in primary HIV infection.

CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue.

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.

CD4(+) T-lymphocyte depletion in human lymphoid tissue ex vivo is not induced by noninfectious human immunodeficiency virus type 1 virions.

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4(+) T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4(+) T-cell depletion, consistent with mechanisms requiring productive infection.

Blockade of CC chemokine receptor 5 (CCR5)-tropic human immunodeficiency virus-1 replication in human lymphoid tissue by CC chemokines.

The CC chemokines MIP-1alpha, MIP-1beta, and RANTES suppress replication of certain HIV-1 strains in cultured PBMC and T cell lines by blocking interaction of gp120 with CC chemokine receptor 5 (CCR5). However, the same chemokines can enhance HIV-1 replication in cultured macrophages. The net effect of chemokines on HIV-1 infection in intact lymphoid tissue, the major reservoir of HIV-1 in vivo, is unknown and unpredictable since the tissue contains both T lymphocytes and macrophages. Here we show that exogenous MIP-1alpha, MIP-1beta, and RANTES markedly suppressed replication of CCR5-tropic HIV-1 strains in blocks of human lymphoid tissue infected ex vivo. Moreover, endogenous MIP-1alpha, MIP-1beta, and RANTES were upregulated in tissues infected ex vivo with CXC chemokine receptor 4-tropic but not CCR5-tropic HIV-1. Such an upregulation may contribute to the virus phenotype shift in the course of HIV disease in vivo.

Dependence of neutrophil activation on cell density and adhesion.

Upon an increasing cell density human neutrophils develop more cell-to-cell contacts in conjunction with an increase in the pHi. These changes are accompanied by decreased superoxide formation after adherence, and a decrease in the total amount of 5-lipoxygenase products after various stimuli. Among the various arachidonate metabolites, leukotriene formation remained almost constant but the yield in 5-HETE decreased. This drop in could account for the decrease in total 5-lipoxygenase products observed when the cell density increased. We conclude that cellular signalling can be affected by an increase of cell-cell interactions. Whether the increase in cellular pH is a cause or consequence of such contact inhibition has yet be answered.

Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency.

Both cellular and humoral immunodeficiency develop in vivo after prolonged infection with HIV-1, but the mechanisms are unclear. Initial infection with HIV-1 is transmitted by macrophage (M)-tropic/non-syncytia-inducing (NSI) viruses, which hyperactivate the immune system, and, in one view, cause immunodeficiency by "exhaustion" of lymphoid tissue. An alternative hypothesis is that immunodeficiency is caused by the replacement of M-tropic viruses by T cell (T)-tropic/syncytia-inducing (SI) viruses, which are known to be highly cytopathic in vitro and emerge late in infected individuals around the time of transition to AIDS (refs. 1, 7-9). To test these two possibilities, we have developed an ex vivo model of humoral immunity to recall antigens using human lymphoid tissue. This tissue supports productive infection with both M- and T-tropic HIV-1 isolates when cultured ex vivo. We found that specific immune responses were enhanced by productive infection of the tissue with M-tropic/NSI HIV-1 isolates, but were blocked by T-tropic/SI HIV-1 isolates. The mechanism involves specific irreversible effect on B-cell activity. Our results support the hypothesis that the phenotype switch to T-tropic viruses is a key determinant of acquired humoral immunodeficiency in patients infected with HIV.

Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor.

The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Experimental HIV infection of human lymphoid tissue: correlation of CD4+ T cell depletion and virus syncytium-inducing/non-syncytium-inducing phenotype in histocultures inoculated with laboratory strains and patient isolates of HIV type 1.

It is now apparent that HIV infection leads to a gradual collapse of a complex system of lymphoid tissue. This collapse tends to be associated with a change in virus tropism from macrophages to T lymphocytes, and a change in phenotype from nonsyncytium inducing (NSI) to syncytium inducing (SI). An experimental system is required to study the role of this change in HIV pathogenesis in lymphoid tissue. Here we describe such a system. Histocultures of human lymphoid tissue preserve their general cytoarchitecture, including a network of follicular dendritic cells (FDCs). Histocultures of tonsils, adenoids, or lymph nodes support productive infection with various laboratory and primary isolates of HIV-1 of different tropism and phenotype and exhibit isolate-dependent CD4+ T lymphocyte depletion. A strong correlation between the extent of CD4+ T cell depletion and the SI/NSI phenotype of the isolates is demonstrated. AZT was used as a model drug to inhibit viral replication and CD4+ T cell depletion in lymphoid histocultures. HIV pathogenesis and the effect of antivirals can now be studied in human lymphoid tissue under controlled conditions in vitro.

Regulation of intracellular pH by phospholipase A2 and protein kinase C upon neutrophil adhesion to solid substrata.

Adhesion to solid substrata has been shown to increase intracellular pH (pH(i)) of fibroblasts and of other cells (FEBS Lett. (1988) 234, 449-450; Proc. Natl. Acad. Sci. USA (1989) 86, 4525-4529; J. Biol. Chem. (1990) 265, 1327-1332; Exp. Cell Res. (1992) 200, 211-214; FEBS Lett. (1995) 374, 17-20). We have found that the inhibitors of PLA2, 4-bromophenacyl bromide and manoalide, completely blocked the increase of pH(i) and spreading of neutrophils upon adhesion to solid substrata. Inhibition of phospholipase C with neomycin or removal of extracellular Ca2+ affects neither neutrophil spreading nor their pH(i). Inhibition of PKC with H-7 or staurosporin increased pH(i). PMA, an activator of PKC, dramatically decreased pH(i) but did not impair the spreading of neutrophils. The effect of arachidonic acid, a product of PLA2 activity, on neutrophil pH(i) and spreading was similar to that of PMA. H-7, an inhibitor of PKC, partially blocked the effect of arachidonic acid (AA) on pH(i). BW755C, an inhibitor of AA metabolism by cyclooxygenase or lipoxygenase, affected neither the pH(i) nor cell spreading. We propose that the increase of pH(i) upon neutrophil adhesion is mediated by PLA2 activity, while PKC decreased pH(i). AA produced by PLA2 activates PKC, thus forming a feedback regulation of pH(i).

Regulation of intracellular pH by cell-cell adhesive interactions.

As was shown in our previous work, the intracellular pH (pHi) of cultured human fibroblasts depends on cell density. The pHi is low in single cells, higher in cells, forming small groups and maximal in a sparse monolayer. On the other hand, the pHi is low in areas of confluent monolayers. In the present work, we show that the effects of inhibitors of various pH-controlling mechanisms as well as inhibitors of key enzymes in signal transduction pathways depend on the local cell density. We have found that N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, known as inhibitors of V-type H+ ATPase, inhibit the elevation of pHi induced by cell-cell contact interactions; meanwhile Cd2+ ions, which inhibit H+ conductive pathway, cause an increase of pHi in a confluent monolayer. Our data revealed also that the Na+/H+ antiporter does not play an essential role in the pHi regulation by intercellular contacts. Inhibitors of phospholipase A2 (4-bromophenacyl-bromide), phospholipase C (neomycin) and protein kinase C (H-7) dramatically change the way the pHi is modulated by local cell density. It is suggested that cell-cell interactions regulate cell activities via modulation of pHi, which is under positive control from phospholipase A2 and under negative control from protein kinase C.

Syncytium formation in cultured human lymphoid tissue: fusion of implanted HIV glycoprotein 120/41-expressing cells with native CD4+ cells.

While glycoprotein gp120/41 clearly causes HIV-infected cells to form syncytia in monolayers and in suspension, there is unfortunately scant knowledge on syncytium formation in tissues. We implanted gp120/41-expressing cells labeled with fluorescent particles inside blocks of human lymphoid tissue kept in long-term histoculture. Observed by confocal microscopy, together with immunohistochemical and morphological analysis of implanted cells, more than one-third of these gp120/41-expressing cells fused with native CD4+ cells of the host tissue, yielding small (three to five nuclei) syncytia. Such widespread fusion of gp120/41-expressing cells in tissue in vitro, together with the finding of increased virulence of syncytium-inducing isolates of HIV, support the hypothesis that syncytium formation within lymph tissue of HIV-infected individuals contributes to AIDS pathogenesis. This system and the methods developed may provide a way to study HIV-infected cells inside the very tissue whose destruction may prevent immune system repopulation.