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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 = .001) between virus production and the number of CD25+/HLA-DR+ T cells. HIV-1 infection of lymphoid tissue was associated with activation of both HIV-1–infected and uninfected (bystanders) T cells. In these tissues, apoptosis was selectively increased in T cells expressing CD25/HLA-DR and p24gag but not in cells expressing either of these markers alone. In the course of HIV-1 infection, there was a significant increase in the number of activated (CD25+/HLA-DR+) T cells both infected and uninfected (bystander). By inducing T cells to express particular markers of activation that create new targets for infection, HIV-1 generates in ex vivo lymphoid tissues a vicious destructive circle of activation and infection. In vivo, such self-perpetuating cycle could contribute to HIV-1 disease.

Immunoglobulin-secreting cells comprise both short-lived proliferating plasmablasts and long-lived nonproliferating plasma cells. To determine the phenotype and functional activity of Ig-secreting cells in human lymphoid tissue, we used a tonsillar organ culture model. A significant proportion of IgA and IgG secretion was shown to be mediated by long-lived, nonproliferating plasma cells that coexpressed high levels of CD27 and CD38. The presence of such cells was further corroborated by the finding of enhanced expression in the CD19(+) B-cell population of XBP-1, IRF-4, and particularly Blimp-1 genes involved in the differentiation of plasma cells. Intact tissue seemed to be necessary for optimal functional activity of plasma cells. A strong correlation was found between concentrations of interleukin-6 and IgA or IgG, but not IgM, in culture supernatants suggesting a role for interleukin-6 in the survival of long-lived plasma cells. Taken together, the present study demonstrates that human lymphoid tissue harbors a population of nonproliferating plasma cells that are dependent on an intact microenvironment for ongoing Ig secretion.

Gut-associated lymphoid tissue (GALT) has been identified as the primary target of HIV-1 infection. To investigate why GALT is especially vulnerable to HIV-1, and to determine whether the selective transmission of CCR5-using viral variants (R5) in vivo is the result of a greater susceptibility of GALT to this viral variant, we performed comparative studies of CXCR4-using (X4) and R5 HIV-1 infections of human lymphoid (tonsillar) and rectosigmoid tissues ex vivo under controlled laboratory conditions. We found that the relative level of R5 replication in rectosigmoid tissue is much greater than in tonsillar tissue. This difference is associated with the expression of the CCR5 co-receptor on approximately 70% of CD4 T cells in rectosigmoid tissue, whereas in tonsillar tissue it is expressed on fewer than 15% of CD4 T cells. Furthermore, tonsillar tissue responds to X4 HIV-1 infection by upregulating the secretion of CC-chemokines, providing a potential CCR5 blockade and further resistance to R5 infection, whereas gut tissue failed to increase such innate immune responses. Our results show that rectosigmoid tissue is more prone than tonsillar lymphoid tissue to R5 HIV-1 infection, primarily because of the high prevalence and availability of R5 cell targets and reduced chemokine blockade. The majority of CD4 T cells express CXCR4, however, and X4 HIV-1 readily replicates in both tissues, suggesting that although the differential expression of co-receptors contributes to the GALT vulnerability to R5 HIV-1, it alone cannot account for the selective R5 infection of the rectal mucosa in vivo.

A new class of anti-retrovirals, cyclotriazadisulfonamide (CADA) and its derivatives, specifically down-regulate CD4, the main receptor of HIV, and prevent HIV infection in vitro. In this work, several CADA derivatives, chemically labeled with a fluorescent dansyl group, were evaluated for their biological features and cellular uptake kinetics. We identified a derivative KKD-016 with antiviral and CD4 down-modulating capabilities similar to those of the parental compound CADA. By using flow cytometry, we demonstrated that the dose-dependent cellular uptake of this derivative correlated with CD4 down-modulation. The uptake and activity of the dansyl-labeled compounds were not dependent on the level of expression of CD4 at the cell surface. Removal of the CADA compounds from the cell culture medium resulted in their release from the cells followed by a complete restoration of CD4 expression. The inability of several fluorescent CADA derivatives to down-modulate CD4 was not associated with their lower cellular uptake and was not reversed by facilitating their cell penetration by a surfactant. These results prove the successful integration of the dansyl fluorophore into the chemical structure of a CD4 down-modulating anti-HIV compound, and show the feasibility of tracking a receptor and its down-modulator simultaneously. These fluorescent CADA analogs with reversible CD4 down-regulating potency can now be applied in further studies on receptor modulation, and in the exploration of their potentials as preventive and therapeutic anti-HIV drugs.

Objective Intraamniotic inflammation/infection is the only mechanism of disease with persuasive evidence of causality for spontaneous preterm labor/delivery. Previous studies about the behavior of cytokines in preterm labor have been largely based on the analysis of the behavior of each protein independently. Emerging evidence indicates that the study of biologic networks can provide insight into the pathobiology of disease and improve biomarker discovery. The goal of this study was to characterize the inflammatory-related protein network in the amniotic fluid of patients with preterm labor. Study Design A retrospective cohort study was conducted that included women with singleton pregnancies who had spontaneous preterm labor and intact membranes (n = 135). These patients were classified according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and amniotic fluid concentration of interleukin (IL)-6 into the following groups: (1) those without intraamniotic inflammation (n = 85), (2) those with microbial-associated intraamniotic inflammation (n = 15), and (3) those with intraamniotic inflammation without detectable bacteria (n = 35). Amniotic fluid concentrations of 33 inflammatory-related proteins were determined with the use of a multiplex bead array assay. Results Patients with preterm labor and intact membranes who had microbial-associated intraamniotic inflammation had a higher amniotic fluid inflammatory-related protein concentration correlation than those without intraamniotic inflammation (113 perturbed correlations). IL-1β, IL-6, macrophage inflammatory protein (MIP)-1α, and IL-1α were the most connected nodes (highest degree) in this differential correlation network (degrees of 20, 16, 12, and 12, respectively). Patients with sterile intraamniotic inflammation had correlation patterns of inflammatory-related proteins, both increased and decreased, when compared to those without intraamniotic inflammation (50 perturbed correlations). IL-1α, MIP-1α, and IL-1β were the most connected nodes in this differential correlation network (degrees of 12, 10, and 7, respectively). There were more coordinated inflammatory-related protein concentrations in the amniotic fluid of women with microbial-associated intraamniotic inflammation than in those with sterile intraamniotic inflammation (60 perturbed correlations), with IL-4 and IL-33 having the largest number of perturbed correlations (degrees of 15 and 13, respectively). Conclusions We report for the first time an analysis of the inflammatory-related protein network in spontaneous preterm labor. Patients with preterm labor and microbial-associated intraamniotic inflammation had more coordinated amniotic fluid inflammatory-related proteins than either those with sterile intraamniotic inflammation or those without intraamniotic inflammation. The correlations were also stronger in patients with sterile intraamniotic inflammation than in those without intraamniotic inflammation. The findings herein could be of value in the development of biomarkers of preterm labor.

Critical events in HIV disease occur in lymphoid tissues that are often coinfected with other pathogens, in particular with herpesviruses, which may affect HIV. Here, we investigated infection by and evolution of R5 and X4 HIV-1 variants in human lymphoid tissues coinfected ex vivo with human herpesviruses (HHV) including HHV-6, HHV-7 and HCMV. We found that both HHV-6 and HHV-7 selectively suppress R5 replication, favoring X4 emergence as the dominant phenotype, and investigated the mechanisms of these phenomena. The selective pressure of HHV-7 on HIV-1 is mediated by down-modulation of CD4 in coinfected tissues, whereas HHV-6 mediates its effect on HIV-1 by dramatic upregulation of RANTES. In macaques coinfected with HHV-6 and SIV, HHV-6-resistant escape mutants are selected. These mutants are resistant to RANTES, or even dependent on this chemokine. HIV-1 infection does not affect the replication of HHV-6 but significantly changes the replication of CMV and HHV-7 depending on HIV-1 phenotype. Thus, human herpesviruses interact with HIV-1 in coinfected human lymphoid tissue via mechanisms that include modulation of HIV receptors and coreceptors, changing the cytokine/chemokine network, and competing for the same cell targets. Interactions between herpesviruses and HIV might affect the course of HIV disease by selecting for HIV-1 of a particular phenotype as well as for escape mutants. These findings support our hypothesis that interaction of HIV with other microbes in coinfected tissues can determine the pattern of HIV infection. Decipherment of the mechanisms of this phenomenon may significantly contribute to the development of efficient anti-HIV therapies.

During normal embryogenesis, embryonic stem cells (ESCs) reside in the context of complex three-dimensional tissue structures, in particular of extracellular matrices (ECMs), which determine cell migration, proliferation, and differentiation. Therefore, to study ESC differentiation in an in vivo-like microenvironment, three-dimensional culture systems are necessary. Here, we developed protocols for ESC cultures in three-dimensional systems consisting of collagen matrices (collagen gels and porous collagen sponges) to investigate the mechanisms of ESC differentiation as well as the formation of tissue-like structures. In collagen matrices, ESCs differentiate into neural, epithelial, and endothelial lineages. In this system, ESCs form various tissue-like structures. The abilities of ESCs to form such structures in two chemically similar but topologically different matrices are different. In particular, in collagen gels ESCs form gland-like circular structures, whereas in collagen sponges ESCs are scattered through the matrix and form aggregates. To mimic the in vivo situation further, we developed a protocol for co-cultures of ESCs with human dermal fibroblasts or keratinocytes in collagen matrixes. Co-culture with fibroblasts in collagen gel facilitates ESC differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule (NCAM), and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ESC differentiation into cells of an endothelial lineage expressing factor VIII. Thus, the developed protocols promote ESC differentiation into a particular lineage, accompanied by the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for directing ESC differentiation and the formation of organs and tissues.

During normal embryogenesis, embryonic stem cells (ESCs) reside in the context of complex three-dimensional tissue structures, in particular of extracellular matrices (ECMs), which determine cell migration, proliferation, and differentiation. Therefore, to study ESC differentiation in an in vivo-like microenvironment, three-dimensional culture systems are necessary. Here, we developed protocols for ESC cultures in three-dimensional systems consisting of collagen matrices (collagen gels and porous collagen sponges) to investigate the mechanisms of ESC differentiation as well as the formation of tissue-like structures. In collagen matrices, ESCs differentiate into neural, epithelial, and endothelial lineages. In this system, ESCs form various tissue-like structures. The abilities of ESCs to form such structures in two chemically similar but topologically different matrices are different. In particular, in collagen gels ESCs form gland-like circular structures, whereas in collagen sponges ESCs are scattered through the matrix and form aggregates. To mimic the in vivo situation further, we developed a protocol for co-cultures of ESCs with human dermal fibroblasts or keratinocytes in collagen matrixes. Co-culture with fibroblasts in collagen gel facilitates ESC differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule (NCAM), and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ESC differentiation into cells of an endothelial lineage expressing factor VIII. Thus, the developed protocols promote ESC differentiation into a particular lineage, accompanied by the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for directing ESC differentiation and the formation of organs and tissues.

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three‐dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland‐like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell–cell adhesion molecules, cell‐surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro‐environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro‐environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.

Understanding the mechanisms of HIV-1 transmission is crucial for the development of effective preventive microbicides and vaccine strategies, and remains one of the main goals of HIV research. Over the past decade, many studies have focused on trying to identify the 'gatekeeping' mechanism that restricts the transmission of CXCR4-utilizing HIV-1 more efficiently than CCR5-utilizing HIV-1. However, to date, no study has explained the almost perfect negative selection of the former in vivo. Here, we propose that there is no single gatekeeper and that, instead, the selective transmission of R5 HIV-1 depends on the superimposition of multiple imperfect gatekeepers.

Various pathogens enhance HIV replication and disease progression in coinfected individuals. However, we, and others, have provided examples of microbial interactions that suppress HIV-1 infection both in vivo and ex vivo. Here, we report on the mechanisms of interactions of HIV-1 with several such microbes in the context of ex vivo infected human lymphoid tissue. Blocks of human tonsils maintained ex vivo were coinfected with R5 or X4 HIV-1 and with another microbe, measles virus (MV), vaccinia, herpesvirus 6 (HHV-6), herpesvirus 7 (HHV-7), cytomegalovirus (CMV), or a parasite, Toxoplasma gondii (TG).

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. In contrast, the replication level hierarchy among sequential isolates and the levels of CCR5+CD4+T-cell depletion caused by the R5 isolates seemed to be controlled by viral factors. R5 viruses isolated from nonswitch virus patients depleted more target cells than R5 viruses isolated from switch virus patients. The high depletion of CCR5+cells by HIV-1 isolates from nonswitch virus patients may explain the steady decline of CD4+T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value reflecting whether, in an infected individual, X4 HIV-1 will eventually dominate.

Heterosexual transmission of human immunodeficiency virus remains the major route of transmission worldwide; thus, there is an urgent need for additional prevention strategies, particularly those that could be controlled by women. Using cellular and tissue explant models, we have evaluated the potential activity of thiocarboxanilide nonnucleoside analogue reverse transcriptase inhibitor UC-781 as a vaginal microbicide. We were able to demonstrate a potent dose-dependent effect against R5 and X4 infections of T cells. In human cervical explant cultures, UC-781 was not only able to inhibit direct infection of mucosal tissue but was able to prevent dissemination of virus by migratory cells. UC-781 formulated into a carbopol gel (0.1%) retained significant activity against both direct tissue infection and transinfection mediated by migratory cells. Furthermore, UC-781 demonstrated prolonged inhibitory effects able to prevent both localized and disseminated infections up to 6 days post compound treatment. Additional studies were carried out to determine the concentration of compound that might be required to block a primary infection within draining lymph nodes. While a greater dose of compound was required to inhibit both X4 and R5 infections of lymphoid versus cervical explants, this was equivalent to a 1:3,000 dilution of the 0.1% formulation. Furthermore, a 2-h exposure to the compound prevented infection of lymphoid tissue when challenged up to 2 days later. The prolonged protection observed following pretreatment of both genital and lymphoid tissues with UC-781 suggests that this class of inhibitors may have unique advantages over other classes of potential microbicide candidates.

Objective: To investigate, in human lymphoid tissue infected with HIV-1 ex vivo, the immunostimulatory and HIV inhibitory properties of pertussis toxin B oligomer (PTX-B) and of the genetically modified non-toxic PT-9K/129G. Methods: Human tonsils from uninfected donors were infected ex vivo with R5 or X4 HIV-1 in the presence or absence of PTX-B. Virus replication was evaluated in culture supernatants; cells emigrated from tissue blocks were immunostained for lymphocytic and activation markers. HIV DNA and cell proliferation were evaluated with real-time PCR and [H-3]thymidine incorporation, respectively. Results: Both PTX-B and PT-9K/129G inhibited HIV-1 replication. These compounds activated and stimulated the proliferation of emigrated cells, most of which were CD4 T lymphocytes. Cells emigrated from infected tissues did not produce detectable virus in unstimulated or in PTX-B- or PT-9K/129G-stimulated cultures whereas robust virus production was triggered by phytohemagglutinin (PHA) or interleukin-2 (IL-2). Analysis of HIV DNA content indicated that infected cells were present among emigrated cells and that their number greatly increased following IL-2 stimulation, whereas it remained constant in the presence of PTX-13 or PT-9K/129G. Conclusions: PTX-13 and PT-9K/129G inhibit both R5 and X4 HIV-1 replication in human lymphoid tissue ex vivo. In contrast to PHA and IL-2, they promote the proliferation of CD4 T lymphocytes emigrated from tissue, including HIV-infected cells, without triggering virus replication. Therefore, these emigrated CD4 T cells represent a novel model of a latent inducible HIV reservoir. Thus, PTX-B and the clinically approved PT-9K/129G are potential antiretroviral agents endowed with immunostimulatory capacity. (c) 2005 Lippincott Williams & Wilkins.

Borrelia burgdorferi sensu stricto is an etiological agent of Lyme disease. The lack of an adequate ex vivo system for human tissue infection is an obstacle to fully understanding the molecular mechanisms of invasion of tissue by B. burgdorferi and its adaptation within the human host. Here, we report on the development of such a system. We inoculated blocks of human tonsillar tissue with B. burgdorferi spirochetes, cultured them in a low-shear rotating wall vessel (RWV) bioreactor, and analyzed them using light and electron microscopy, nested polymerase chain reaction (PCR), and quantitative real-time PCR. Also, we evaluated the expression of the outer surface proteins (Osps) OspA and OspC by use of quantitative Western blotting. Light and electron microscopic analysis revealed multiple spirochetes localized extracellularly within the tissue, and their identity was confirmed by PCR. Quantification of spirochetes inside the RWV-cultured tonsillar tissue demonstrated that the number of B. burgdorferi exceeded the initial inoculum by an order of magnitude, indicating that spirochetes replicated in the tissue. Electron microscopic analysis showed that some spirochetes were arranged in cystic structures and that invading spirochetes differentially expressed surface proteins; both of these features have been described for infected tissues in vivo. The system we have developed can be used to study B. burgdorferi pathogenesis under controlled conditions ex vivo, in particular to explore the gene activation responsible for the adaptation of B. burgdorferi to human tissue that leads to Lyme disease.

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. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.

Responses to cholesterol depletion are often taken as evidence of a role for lipid rafts in cell function. Here, we show that depletion of cell cholesterol has global effects on cell and plasma membrane architecture and function. The lateral mobility of membrane proteins is reduced when cell cholesterol is chronically or acutely depleted. The change in mobility is a consequence of the reorganization of the cell actin. Binding of a GFP-tagged pleckstrin homology domain specific for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to the plasma membrane is reduced after cholesterol depletion. This result implies that the reorganization of cytoskeleton depends on the loss or redistribution of plasma membrane PI(4,5)P2. Consistent with this observation, agents that sequester plasma membrane PI(4,5)P2 mimic the effects of cholesterol depletion on actin organization and on lateral mobility.

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. We address this question using a system of human lymphoid tissue ex vivo. Tissue blocks were inoculated with HIV-1. After productive infection was established, they were treated with the reverse transcriptase inhibitor nevirapine to protect from infection those CD4(+) T cells that had not yet been infected. These CD4(+) T cells residing in HIV-infected tissue are by definition bystanders. Our results demonstrate that after nevirapine application the number of bystander CD4(+) T cells is conserved. Thus, in the context of HIV-infected human lymphoid tissue, productive HIV infection kills infected cells but is not sufficient to cause the death of a significant number of uninfected CD4(+) T cells.

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. R5 HIV-1 predominantly infected CD62L-negative memory T cells, which selectively express CCR5. In contrast, X4 HIV-1 variants predominantly infected CD62L + memory T cells, although CXCR4 coreceptor was equally expressed by memory T cells of both CD62L-positive and CD62L-negative subsets. A high proportion of X4 HIV-1, but not of R5 HIV-1, productively infected T cells, displayed a CD45RA+CD45RO+ phenotype. Conclusion: The selective expression of the CCR5 coreceptor by CD62L-negative terminally differentiated memory T cells correlates with the preferential productive infection of these cells with the R5 HIV-1 variant. The predominance of X4 HIV-1 variants in less-differentiated memory T cells may be related to their recent activation state, as suggested by the coexpression of both CD45RA and CD45RO molecules on their surface. Differential homing of CD62L-positive and CD62L-negative cells suggests different routes of dissemination of X4 and R5 viruses.

The mechanisms of male-to-female HIV-1 transmission mediated by semen are not well-understood. To decipher these mechanisms, we developed a system of cervico-vaginal tissue ex vivo to which HIV-1 is transmitted from semen under controlled laboratory conditions. Semen samples obtained from HIV-1-infected and control men were analyzed for the presence of 21 cytokines, and HIV-1 transmission was simulated ex vivo by deposition on cervico-vaginal explants of virus suspended in semen or in PBS enriched with particular cytokines. In HIV-1-infected men, the cytokine spectrum was significantly changed, resulting in the establishment of new correlations and the strengthening of pre-existing correlations between different cytokines: HIV-1 infection increases the number of such correlations from 21 to 72. These changes in semen were local and different from ones in blood from the same individuals. One of the most upregulated seminal cytokines was IL-7, which enhanced replication of HIV-1 in cervico-vaginal tissue. This enhancement was associated with the suppression of apoptosis as evaluated from the expression of apoptotic markers, a decrease in the depletion of infected cells as evaluated from flow cytometry, and an increase in cell cycling as evaluated from Ki67 staining. In conclusion, HIV-1 establishes new strong correlations between various cytokines, thus imposing a high rigidity on their network that may contribute to the impaired capacity of the immune system to respond to microbial challenges. Seminal cytokines, in particular IL-7, may be key determinants of HIV-1 transmission from men to their uninfected female partners through vaginal intercourse and may become a new target for preventive and therapeutic strategies.

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important virulence factor. Nef has several functions, including down-modulation of CD4 and class I major histocompatibility complex cell surface expression, enhancement of virion infectivity, and stimulation of viral replication in peripheral blood mononuclear cells. Nef also increases HIV-1 replication in human lymphoid tissue (HLT) ex vivo . We analyzed recombinant and primary nef alleles with highly divergent activity in different in vitro assays to clarify which of these Nef activities are functionally linked. Our results demonstrate that Nef activity in CD4 down-regulation correlates significantly with the efficiency of HIV-1 replication and with the severity of CD4 + T-cell depletion in HLT. In conclusion, HIV-1 Nef variants with increased activity in CD4 down-modulation would cause severe depletion of CD4 + T cells in lymphoid tissues and accelerate AIDS progression.

We sought to determine the relationship between virus-mediated CD4 + T-lymphocyte cytopathicity and viral coreceptor preference among various human immunodeficiency virus type 1 (HIV-1) subtypes in an ex vivo-infected human lymphoid tissue model. Our data show that all R5 HIV-1 infections resulted in mild depletion of CD4 + T lymphocytes, whereas all X4 HIV-1 infections caused severe depletion of CD4 + T lymphocytes regardless of their subtype origin. Thus, at least for the viruses within subtypes A, B, C, and E that were tested, coreceptor specificity is a critical factor that determines the ability of HIV-1 to deplete CD4 + T cells in human lymphoid tissue infected ex vivo.

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-1Ba-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 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (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.

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.

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4 + T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4 + lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.

Philippe Van de Perre and colleagues 1 argue that herpes simplex virus (HSV) 2 and HIV infections are synergistic, since suppression of HSV2 decreases HIV load, while Rex G Cheng and Douglas F Nixon 2 insist that interactions between HSV2 and HIV-1 are more complex and that HSV2 coinfection might, in certain cases, be associated with elevated CD4+ T-cell counts. 3 Both groups provide arguments on the basis of their own work and selected results of various clinical trials. 4‐7 Although there has been some criticism of the design and interpretation of results of some of these trials, 8‐10 all of them have one thing in common: they use aciclovir or its prodrug valaciclovir to suppress HSV2 replication and to investigate whether such suppression affects HIV replication and infection.

In this issue of Blood, Ballana and colleagues show that integrins that mediate macrophage adhesion are important for HIV replication and inhibition of these integrins suppresses HIV replication at the transcriptional level.

To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes.Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays.We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants.The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

The human chemokine receptors CCR5 and CXCR4 have emerged as the predominant cofactors, along with CD4, for cellular entry of HIV-1in vivowhereas the contribution of other chemokine receptors to HIV disease has not been yet determined. CCR5-specific (R5) viruses predominate during primary HIV-1 infection whereas viruses with specificity for CXCR4 (R5/X4 or X4 viruses) often emerge in late stages of HIV disease. The evolution of X4 viruses is associated with a rapid decline in CD4+ T cells, although a causative relationship between viral tropism and CD4+ T cell depletion has not yet been proven. To rigorously test this relationship, we assessed CD4+ T cell depletion in suspensions of human peripheral blood mononuclear cells and in explants of human lymphoid tissue on exposure to paired viruses that are genetically identical (isogenic) except for select envelope determinants specifying reciprocal tropism for CXCR4 or CCR5. In both systems, X4 HIV-1 massively depleted CD4+ lymphocytes whereas matched R5 viruses depleted such cells only mildly despite comparable viral replication kinetics. These findings demonstrate that the coreceptor specificities of HIV-1 are a causal factor in CD4+ T cell depletionex vivoand strongly support the hypothesis that the evolution of viral envelope leading to usage of CXCR4in vivoaccelerates loss of CD4+ T cells, causing immunodeficiency.

Dramatic progress has been made recently in identifying both viral and cellular molecules responsible for binding and fusion of HIV-1 to target cells. In vivo, HIV-1 infection is transmitted by viruses that recognize chemokine receptor CCR5, while viruses isolated at later stages of HIV disease often recognize another chemokine receptor, CXCR4. It is still not understood how this molecular tropism of HIV-1 is translated into the virus' ability to compromise normal cell functions, which results in impairment of lymphoid tissue and causes AIDS. Here, we discuss how the new molecular findings might relate to HIV pathogenesis in cells and tissues.

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.

Both cellular and humoral immunodeficiency develop in vivo after prolonged infection with HIV-1, but the mechanisms are unclear1. Initial infection with HIV-1 is transmitted by macrophage (M)-tropic/non-syncytia-inducing (NSI) viruses2,3, which hyperactivate the immune system4,5, and, in one view, cause immunodeficiency by “exhaustion”4,6 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 suppo rts productive infection with both M- and T-tropic HIV-1 isolates when cultured ex vivo10,11. 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.

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.

Aim: It is generally thought, that myocardial infarction (MI) is the one of the main causes of death in developed countries. Vulnerable atherosclerotic plaque more likely can rupture and lead to myocardial infarction. The causes of plaques' maturation and rupture may be associated with inflammation and local and/or systemic immune activation. But it's still unknown, if there any infectious agent in plaques that may lead to such an activation. We have investigated if the presence of human herpes viruses DNA in vessels of different group of patients is associated with plaque instability. Methods: We included 30 patients hospitalized with MI and who died from MI or it's complications. Coronary arteries samples were taken during autopsy. Macroscopic observations of the tissues allowed their classification in 3 groups: 1) clean samples, without macroscopic signs on atherosclerosis, 2) non-ruptured plaques; 3) ruptured plaques, usually MI-causative vulnerable plaques with thrombus or ulceration. Also we analyzed samples obtained from 10 patients who dyed from coronary-artery–disease unrelated causes. In these samples there was no significant evidence of atherosclerosis. Tissue's DNA was extracted and HHVs DNA were measured by quantitative real-time-PCR (Q-PCR). HHVs genome copy numbers were normalized by cellular genomes and are reported as HHVs copies of per million cells. Results: Q-PCR results showed that in MI patients, more than 93% of samples contained at least one HHVs. HHV-1&2, HHV-3, HHV-5, and HHV-8 were found in more than 80% of the samples. HHV-4, HHV-6 and HHV-7 were the less frequent and were found in 49% to 55% of the samples. Analysis of the distribution of HHV-s between the three sample groups did not reveal any significant difference. However, in samples from patients with non MI-related cause of death, DNA of HHV1&2, HHV5, HHV8 was found in all samples, HHV6 in 90%, HHV3 and HHV7 in 80% and HHV4 in 70% of samples. Conclusion: We found that all HHVs are present in artery samples of people, who either died from MI or other causes. The higher viral DNA load in arterial tissues did not correlate with the presence of plaque or the stability thereof. Interestingly, HHV-8, the virus associated with Kaposi's sarcoma, a disease usually developed by immunosuppressed patients, was abundant in this cohort of non-immunosuppressed patients.

Aim: In our previous work we found that T-lymphocytes phenotypes in atherosclerotic plaques are different from those found in blood. T cells are more activated in plaque than in blood as judged by the expression of CD25, CD38, and HLA-DR. The purpose of this work was to evaluate the presence of herpesviruses (HHV) in these plaques and investigate whether their presence correlates with activation of resident lymphocytes. Methods: 27 atherosclerotic plaques from patients who underwent either carotid endarterectomy (22) or aortofemoral bypass grafting (5). The degree of carotid artery stenosis varied from 70% to 90% (median 80.0%, IQR 72.5–90). The phenotype of lymphocytes was analyzed using polychromatic flow cytometry, and HHVs DNA amount in plaques was measured by Real Time PCR. Results: We found a correlation between T lymphocytes activation and the presence of certain HHVs in atherosclerotic plaques. The fraction of CD4+ T cells expressing the late activation marker HLA-DR positively correlates with HHV-1 & -2 viral load (Spearman's r=0.438, p=0.0381). Also, there was a negative correlation between the fraction of naive CD8 T cells and HHV-7 viral load (Spearman's r= -0.7381, p=0.0366). And the viral load of HHV-4 correlated with the presence of late-differentiated effector memory CD4 T cells (Spearman's r=0.6364, p=0.0353) and effector memory CD8 T cells (Spearman's r=0.6636, p=0.026). Conclusions: The activation of T-lymphocytes in atherosclerotic plaque is correlated with the presence of different HHV types (1, 4 and 7). The presence of HHVs in atherosclerotic plaques may be an important factor of lymphocyte activation thus affecting plaques' maturation and rupture. This is the first study, which quantitatively associates activation of atherosclerotic plaques' resident lymphocytes with the presence of certain HHVs in theses plaques. The finding of this association may lead to the development of new targets for atherosclerosis preventive therapy and warrants further investigation.

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).

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.

In three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, them implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.

We investigated the effect of arachidonic acid on the intercellular communication of a rat mammary tumor cell line. This polyunsaturated fatty acid reversibly reduced the intracellular pH and raised the concentration of cytoplasmic free Ca 2+ . However, neither low pH nor elevated [Ca 2+ ] j influenced gapjunctional coupling. Measurements with the double whole cell patch-clamp technique imply that arachidonic acid influences gap-junctional permeability via a direct lipid-protein interaction.