1. Transmission of HIV associated with human seminal leucocytes Transmission of HIV mainly occurs through unprotected sexual intercourse in which seminal HIV is deposited in rectal or vaginal mucosa. In semen, HIV exists as free-floating viral particles and is also associated with seminal cells, such as monocytes and lymphocytes. While free HIV can transmit infection both in ex vivo models and in non-human primates, the ability of cell-associated HIV to transmit infection remains a matter of debate. In model experiments in human tissues ex vivo we investigated whether T-cell-associated HIV is capable of transmitting infection to human lymphoid tissue ex vivo. To suppress all the other potential sources of viruses other than cell-associated HIV, we irradiated monocytes and lymphocytes. We found that the amount of attached virus to monocytes and lymphocytes was not statistically different. In spite of similar amounts of HIV attached to monocytes and lymphocytes, transmission of this virus to human lymphoid tissues was dramatically different: while monocytes readily transfer infection to human tissue, lymphocytes did not. In summary, our results show that non-infected monocytes but not lymphocytes present in semen of HIV-infected men may adsorb virus in HIV and are able to transfer infection to mucosal tissues. This pattern of transmission may contribute to the spread of infection, in particular at the stages of the disease at which viral load is low. 2. Cytomegalovirus and destabilization of atherosclerotic plaques It is well established that immunoactivation is associated with the destabilization of atherosclerotic plaques. Although herpesviruses have been considered to be associated with atherosclerosis, the data supporting such an association are controversial and, in most cases, are based on serological evidence or on the presence of cell-associated HHV DNA, which do not report about actual viral replication. To evaluate productive infection: rather than focusing on HHV replication in particular cells, we evaluated the presence of HHV DNA of all eight types of herpesviruses using quantitative real-time PCR in blood samples from 71 patients with acute coronary syndrome (ACS), from 26 patients with stable coronary artery disease (SCAD), and from 53 healthy volunteers, as well as in atherosclerotic plaques of 22 patients with peripheral artery disease who underwent endarterectomy. This analysis revealed all the HHVs in plasma of healthy controls and patients with ACS. HHV-5 (cytomegalovirus, CMV) was the only HHV whose amount was higher in ACS patients than in the control group. We found that for CMV the DNA copy numbers in plasma positively correlated with ACS. There was an enrichment of intermediate-differentiation (CD27+/-CD28+), but not late-differentiated (CD27-CD28-) T effector memory (Tem) cells compared to healthy controls. In summary, we found that CMV is strongly associated with the atherosclerotic process and, in particular, with ACS. Future studies should shed more light on the relation of ACS in particular and cardio-vascular diseases in general to CMV and possibly other HHVs, which may result in new treatment strategies. 3. Extracellular vesicles in human pathologies The composition of EVs reflects properties of the cells that release them and can be indicators of pathological alterations and may play a role in the development of these pathologies. We refined methods for EVs isolation and tested this hypothesis in two models: (a) We compared EVs in blood of healthy volunteers with those in blood of patients with acute coronary syndrome, and (b) We investigated the effect of EVs released by normal retinal cells and retinal cells treated with pro-inflammatory cytokines on lymphocytes and monocytes. The latter model of retinal cells culture simulates important aspect of uveitis. (a) EVs in patients with ACS. Cells in norm or pathology release EVs into blood where they are pooled. With the current bulk analysis individual features of the released EVs are lost. Here, we analyzed the antigenic composition of individual EVs isolated from blood of healthy volunteers and from blood of patients with acute forms of coronary artery disease, in particular myocardial infarction. Plasma EVs were captured with 15-nm magnetic nanoparticles coupled to antibodies against cellular proteins (CD31, CD41a, CD63, CD62E, CD105, CD146) and we evaluated the presence of these antigens on EVs isolated from blood. The total amounts of EVs were higher in the ACS patients than in the controls. For all captured fractions, the differences in the EV amounts were restricted to EVs expressing CD41a+, a platelet marker. The increase in the numbers of EVs in the ACS patients, predominantly of platelet origin, probably reflects platelet activation and may indicate disease progression. Future studies should reveal whether the increase in the number of platelet-derived EVs may constitute one of the early indicators of the disease and may lead to the development of new diagnostic tools. (b) EVs released by human retinal cells Retinal pigment epithelium (RPE) cells are critical to the normal function of overlying photoreceptors. Normal RPE cells possess immunosuppressive properties. RPE cells may become damaged during inflammatory eye diseases. We investigated if EVs released from RPE cells, either in homeostatic or inflammatory environments, alter T-cell responses and monocyte phenotypes in vitro. In particular, we compared the size, concentration, and immunomodulatory function of EVs released by RPE stimulated with the inflammatory cytokines IL-1beta, IFN-gamma, and TNF-alpha with those released by nonstimulated RPE. Flow analysis revealed an approximately 3-fold increase in the concentration of EVs released from cytokine-stimulated RPE cell line ARPE-19 compared with nonstimulated controls. Extracellular vesicles from both resting and cytokine-stimulated RPE inhibited the proliferation of T cells in PBMC cultures. Importantly, RPE-derived EVs did not induce T-cell death. Human monocytes exposed to EVs released by nonstimulated RPE cells resulted in an enrichment of the CD14++CD16+ intermediate monocyte population without affecting monocyte viability. Thus, ARPE-19 cells may use EVs to mediate immunosuppressive properties, representing a mechanism by which RPE maintains an immunosuppressive environment to protect neighboring RPE and photoreceptors from inflammatory damage. Incubation of human monocytes with EVs released by cytokine-stimulated ARPE-19 cells led to monocyte cell death and upregulation of several proinflammatory cytokines. We hypothesize that the EVs released by RPE cells exposed to inflammatory conditions are modified to express proapoptotic molecules that may reduce the number of infiltrating immune cells in an attempt to abrogate immune-mediated damage to surrounding RPE cells and photoreceptors. In summary, our results indicate that under resting conditions, RPE constitutively release EVs that inhibit T-cell proliferation and render monocytes immunosuppressive without affecting their survival. Under inflammatory conditions, RPE release EVs that inhibit T-cell proliferation without affecting T-cell viability but that induce monocyte cell death, perhaps to dampen an actively destructive immune response. In conclusion, EVs secretion may be an important mechanism used by RPE cells to exert their known immunomodulatory effects.
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