In situ polymerase chain reaction-based localization studies support role of human herpesvirus-8 as the cause of two AIDS-related neoplasms: Kaposi's sarcoma and body cavity lymphoma.

Several lines of investigation point to a new herpesvirus, human herpesvirus-8 (HHV-8), as the cause of two different neoplasms seen in AIDS patients-Kaposi's sarcoma (KS) and body cavity B cell lymphoma. If this virus is the etiological agent, rather than another opportunistic infectious agent, it should be present in the earliest detectable clinical lesions on a temporal basis, and localize to specific target cells in a spatial pattern consistent with tumorigenic pathways. In this study, we take advantage of the clinical accessibility to biopsy early (patch stage) skin lesions of KS to address the temporal issue, combined with in situ PCR and dual immunostaining using a marker identifying malignant cells, to address the spatial localization issue. 21 different tissue samples were subjected to PCR analysis and in situ PCR with and without simultaneous immunostaining. In normal skin from healthy individuals, no HHV-8 DNA was detected by PCR or in situ PCR. However, in all PCR-positive tissues, distinct and specific in situ PCR staining was observed. In four different patch stage KS lesions, in situ PCR staining localized to nuclei of endothelial cells and perivascular spindle-shaped tumor cells. Later stage KS lesions (plaques and nodules) revealed additional positive cells, including epidermal keratinocytes (four of five), and eccrine epithelia (two of four). These patterns were nonrestricted to skin, as pulmonary KS also revealed HHV-8-specific infection of endothelial cells and KS tumor cells, as well as epithelioid pneumocytes (two of two). In body cavity B cell lymphoma by dual staining, HHV-8 was present in malignant tumor cells (EMA immunostained positive) and not in reactive lymphocytes. These results reveal an early temporal onset and nonrandom tissue and cellular distribution pattern for HHV-8 infection that is consistent with a causal link between this DNA virus and two AIDS-related neoplasms.

C5a-induced expression of P-selectin in endothelial cells.

Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.

The alternative complement pathway promotes IgM antibody-dependent and -independent adherence of Bacteroides to polymorphonuclear leukocytes through CR3 and CR1.

Natural immunoglobulin M (IgM) antibodies act synergistically with the alternative complement pathway to promote opsonization and adherence of Bacteroides thetaiotaomicron and Bacteroides fragilis to polymorphonuclear leukocytes (PMNs). This study characterizes the PMN receptors involved in adherence of Bacteroides opsonized by the alternative pathway and determines the effect of natural IgM antibodies on receptor involvement and on the molecular form of C3 deposited on the bacteria. A model system consisting of the six isolated proteins of the alternative pathway with or without supplemental isolated normal IgM was used for opsonization, and results were compared to those obtained with serum. The results demonstrate that the alternative pathway promotes adherence of Bacteroides to PMNs through complement receptors 1 and 3 (CR3), and epitopes in CR3 besides those mediating iC3b binding do not participate. The results also demonstrate that natural IgM antibodies do not influence the character of the ligands or receptors involved in this interaction.

Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes.

Bladder cancer, the fourth most common noncutaneous malignancy in the United States, is characterized by high recurrence rate, with a subset of these cancers progressing to a deadly muscle invasive form of disease. Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA, thus potentially modulating signaling pathways in recipient cells. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has been implicated in the initiation of metastasis for cancer progression. We investigated the ability of bladder cancer-shed exosomes to induce EMT in urothelial cells. Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media or from patient urine or bladder barbotage samples. Exosomes were then added to the urothelial cells and EMT was assessed. Urothelial cells treated with bladder cancer exosomes showed an increased expression in several mesenchymal markers, including α-smooth muscle actin, S100A4 and snail, as compared with phosphate-buffered saline (PBS)-treated cells. Moreover, treatment of urothelial cells with bladder cancer exosomes resulted in decreased expression of epithelial markers E-cadherin and ß-catenin, as compared with the control, PBS-treated cells. Bladder cancer exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pretreatment. We further showed that exosomes isolated from patient urine and bladder barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. In conclusion, the research presented here represents both a new insight into the role of exosomes in transition of bladder cancer into invasive disease, as well as an introduction to a new platform for exosome research in urothelial cells.

Killing of sarcoma cells by proapoptotic Bcl-X(S): role of the BH3 domain and regulation by Bcl-X(L).

Kaposi's sarcoma (KS) is the most common tumor affecting AIDS patients with over 20% of these patients afflicted by this disease. Previous studies have demonstrated that KS tumor cells predominantly express the prosurvival protein Bcl-X(L) compared with Bcl-2. In the current study, we have used an adenoviral vector that expresses Bcl-X(S), a functional inhibitor of Bcl-X(L), to study the significance of Bcl-X(L) expression in the KS cell line (SLK) or KS primary cultures. The results demonstrate that 75% to 80% of SLK or KS primary cells were killed by the Bcl-X(S) containing adenovirus whereas KS cells infected with control adenovirus showed no significant cell death or growth inhibition. Overexpression of Bcl-X(L), but not Bcl-2, in SLK cells attenuated apoptosis induced by adenovirus Bcl-X(S). Immunoprecipitation experiments revealed that adenoviral Bcl-X(S) associated with Bcl-X(L), but not with Bcl-2. Mutational analysis showed that the alpha 2 helical region of Bcl-X(S) containing the BH3 motif was critical for killing activity and interaction with Bcl-X(L). These results suggest that Bcl-X(S) is a direct killer and Bcl-X(L) may act by interacting with and sequestering Bcl-X(S.) These studies also suggest that targeting Bcl-X(L) may be of therapeutic benefit for the treatment of tumors that are characterized by inappropriate expression of Bcl-X(L).

Molecular and pathologic characterization of an AIDS-related body cavity-based lymphoma, including ultrastructural demonstration of human herpesvirus-8: a case report.

Body cavity-based lymphoma, also known as primary effusion lymphoma, is a newly recognized acquired immunodeficiency syndrome (AIDS)-related lymphoma that has been linked to the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). To date, direct visualization of the virus in a clinical sample has not been demonstrated. We have performed an extensive clinical, histologic, immunophenotypic, ultrastructural, and molecular genetic correlative study on multiple tissue samples obtained premortem and at autopsy from an patient with AIDS with Kaposi's sarcoma and body cavity-based lymphomas. We demonstrate the presence of human herpesvirus-8 in a primary clinical sample at the ultrastructural and molecular level, as well as document multiple lymphomatous tumor masses at autopsy.

Phenotype and proliferation characteristics of cultured spindle-shaped cells obtained from normal human skin and lesions of dermatofibroma, Kaposi's sarcoma, and dermatofibrosarcoma protuberans: a comparison with fibroblast and endothelial cells of the dermis.

Normal human dermis contains mesenchymal cells that are generally referred to as fibroblasts. However the relationships between fibroblasts and endothelial cells with respect to the types of spindle-shaped cells that are present in cultures obtained from tumor bearing-skin is unclear. To explore the potential heterogeneity amongst dermal-derived cells that grow in culture with a spindle-shaped morphology, we compared the immunophenotype and growth characteristics of several types of cells. Besides dermal fibroblasts and microvascular endothelial cells derived from normal adult skin, we also studied large vessel-derived endothelial cells, and spindle-shaped cells derived from three different tumor-bearing dermal-based neoplasms. Kaposi's sarcoma (KS), dermatofibroma (DF), and dermatofibrosarcoma protuberans (DFSP). A broad panel of eight different antibodies were used to immunophenotype the multi-passaged cultured cells. Spindle-shaped cells from all three neoplasms could be distinguished from the normal skin derived fibroblasts by their constitutive expression of factor XIIIa, and the gamma-interferon induced expression of VCAM-1. All seven types of cultured cells stained positive for s-actin and proline-4-hydroxylase, and none of the cells expressed CD34. Both large and small-vessel derived endothelial cells expressed factor VIII, ELAM-1, and VCAM-1. Using two different types of growth media, significant differences were also observed amongst these cultured cell types. Spindle-shaped cells from DFSP did not grow in DMEM containing 10% fetal bovine serum (DMEM-FBS); but they proliferated in KS cell growth medium (KSGM). Spindle-shaped cells from DF grew best in KSGM, but not in DMEM-FBS. KS tumor cells grew well in KSGM, but not in DMEM-FBS. Fibroblasts proliferated in DMEM-FBS, but failed to grow in KSGM; and even when pre-treated with conditioned medium from a transformed KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that even though dermal-derived cells can have an identical spindle-shape by light microscopy, significant heterogeneity can be defined amongst such cells from normal and tumor-bearing human skin. Having established culture conditions to propagate these different cell types and phenotypic criteria to distinguish them from one another, will provide new research opportunities to explore the function and ontogeny of the diverse mesenchymal cells that take on a spindle-shaped morphology in culture.

Injection of human herpesvirus-8 in human skin engrafted on SCID mice induces Kaposi's sarcoma-like lesions.

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) has been implicated in the development of Kaposi's sarcoma (KS) and several B-cell lymphoproliferative diseases. Serologic and molecular genetic association data has implicated HHV-8 as the causal agent of KS, but its role in the development of KS lesions is not understood. To examine the etiology of KS, HHV-8 was injected into normal human skin transplanted onto SCID mice. Injection of HHV-8 induced lesion formation that is morphologically and phenotypically consistent with KS, including the presence of angiogenesis and spindle-shaped cells latently infected with HHV-8. These findings suggest that HHV-8 is indeed the etiologic agent of KS, and that the virus plays an important role in initiation of this disease.

Comparative effect of C3a and C5a on adhesion molecule expression on neutrophils and endothelial cells.

Complement activation is known to enhance neutrophil binding to human umbilical vein endothelial cells (HUVECs). Recently, we have shown that recombinant human C5a upregulates P-selectin in HUVECs. Unstimulated human neutrophil binding is also increased on C5a stimulated HUVECs. We demonstrate in this report that C5a upregulates CD11b/CD18 in human neutrophils. Also shown is that synthetic C3a57-77 and an analog 15 amino acid C3a peptide (C3a15) neither upregulate CD11b/CD18 nor do the C3a peptides increase P-selectin, ICAM-1 or E-selectin in HUVECs. Thus C5a and not C3a is responsible for early (approximately 30 minutes) neutrophil adhesion to endothelial cells after complement activation.

Caspase inhibitor blocks human immunodeficiency virus 1-induced T-cell death without enhancement of HIV-1 replication and dimethyl sulfoxide increases HIV-1 replication without influencing T-cell survival.

OBJECTIVES: To determine the relationship, if any, between reagents that modulate survival of T-cells and replication of human immunodeficiency virus 1 (HIV-1) and to determine the effects of the solvent dimethyl sulfoxide (DMSO) and drugs such as cyclosporin A and all-trans retinoic acid on HIV-1 replication. DESIGN: To first establish the direct effects of solvent alone (ie, DMSO) at various concentrations on HIV-1 replication, followed by the ability of various compounds such as the caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethylketone (z-VAD-fmk), cyclosporin A, and all-trans retinoic acid on HIV-1 replication. Next, to determine if HIV-1 induces T-cell apoptosis using TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assays and DNA fragmentation and poly-(ADP-ribose)-polymerase (PARP) cleavage, and then to examine how the various compounds influence T-cell survival after HIV-1 exposure. METHODS: The human T-cell line, CEM cells, were exposed to HIV(IIIB) and viral replication monitored using reverse transcription assays at 3, 6, and 9 days following infection. Cells were pretreated with various compounds dissolved in DMSO over a wide range of concentrations, and DMSO itself was also examined. T-cell death and apoptosis were assessed using TUNEL staining to detect 3'-OH DNA strand breaks and agarose gel electrophoresis to detect DNA fragmentation (laddering). Furthermore, PARP cleavage implicated in the apoptotic process was also examined. RESULTS: At very low levels, such as 0.002%, DMSO itself appears to enhance HIV-1 replication at 6 and 9 days after infection. At low levels of cyclosporin A, such as 0.01 microgram/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 microgram/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 microgram/mL did not influence HIV-1 replication. In addition, a discrepancy was noted in that HIV-1-infected T-cells were TUNEL positive, indicating DNA strand breaks; however, more complete DNA fragmentation was not detected nor was PARP cleavage identified. The induction of TUNEL positivity was blocked by the caspase inhibitor z-VAD-fmk but not by DMSO or cyclosporin A. Even though z-VAD-fmk blocked the appearance of TUNEL-positive T-cells, there was not a consistently observed increase in HIV-1 replication. CONCLUSION: Low levels of DMSO and cyclosporin A can enhance HIV-1 replication in CEM cells. At higher levels, cyclosporin A inhibits HIV-1 replication with no significant effects by all-trans retinoic acid. No evidence for classic apoptosis was detected in CEM cells after HIV-1 infection, although DNA strand breaks may be present as revealed by TUNEL positivity. There was no correlation between levels of HIV-1 replication and T-cell survival or death. The mechanism of T-cell death after HIV-1 infection requires further study, and investigators who add compounds dissolved in DMSO must include controls to carefully examine the direct effects of even trace levels of this solvent on HIV-1 replication.

Induction of human immunodeficiency virus 1 replication by human herpesvirus 8.

BACKGROUND: Human immunodeficiency virus 1 (HIV-1)-infected individuals are commonly infected with herpesviruses, including cytomegalovirus, herpes simplex virus, varicella-zoster virus, and human herpesvirus 8 (HHV-8, also known as Kaposi sarcoma-associated herpesvirus [KSHV]). Previous studies have demonstrated that coinfection with herpesviruses can modulate HIV-1 replication. This can occur either through direct interaction between the 2 viruses or through secondary effects resulting from the release of cellular factors in response to infection. OBJECTIVE: To investigate HIV-1 replication in the presence and absence of HHV-8. DESIGN AND METHODS: HIV-1 replication was analyzed following culture of HIV-1-infected CD4(+) T cells in the presence of HHV-8 infected B-cell lines or control, uninfected B-cell lines. To confirm and extend the results of these in vitro studies, HIV-1-infected T cells were injected into human skin transplanted onto severe combined immunodeficient mice. The human skin was also injected with purified HHV-8 or phosphate-buffered saline as a control and HIV replication measured in biopsy specimens taken 5 to 8 days later. RESULTS AND CONCLUSIONS: The results demonstrated a significant increase in HIV-1 replication in the presence of HHV-8 in both the in vitro and in vivo model systems. Although the mechanism responsible for HHV-8 induction of HIV-1 replication remains to be identified, the results indicate that these 2 viruses may interact at the molecular level in coinfected patients, resulting in increased HIV-1 viral load.

Cultured Kaposi's sarcoma tumor cells fail to stimulate T cell proliferation.

Prior to the AIDS epidemic, Kaposi's sarcoma (KS) was a rare neoplasm. However, in the context of immunosuppression, cutaneous KS lesions more frequently develop and express various surface molecules recognized by T cells such as intercellular adhesion molecule-1 (ICAM-1; CD54) and HLA-DR. The KS tumor cells are thought to arise locally from endothelial cells via a transdifferentiation process. To determine if KS tumor cells can stimulate resting T cell proliferation, we asked whether the tumor cells express the critically important T cell costimulatory molecules B7-1 (CD-80) and B7-2 (CD-86). In contrast to cytokine-activated endothelial cells, which were induced to express B7-1, but not B7-2 and could function in bacteria-derived superantigen-driven T cell proliferation, four different KS tumor cell lines failed to express either B7-1 or B7-2 and were unable to stimulate allogeneic T cell proliferation upon addition of bacteria-derived superantigen. These results suggest that KS tumor cells behave differently in their response to cytokines compared with endothelial cells and may be able to evade the local immune response by not expressing costimulatory molecules necessary for T cell proliferation.

Expression of costimulatory molecules CD80 and/or CD86 by a Kaposi's sarcoma tumor cell line induces differential T-cell activation and proliferation.

During physiological stimulation of resting T-cells, at least two activation signals by antigen presenting cells are required. Besides the first antigen-specific signal, the second costimulatory signal involves CD80 and CD86 expressed by the antigen presenting cell. These costimulatory molecules have been suggested to be of clinical relevance in many different autoimmune and malignant disease processes. We previously observed that tumor cells in Kaposi's sarcoma (a common AIDS-related cutaneous neoplasm) completely lack both CD80 and CD86, and these tumor cells fail to stimulate T-cell proliferation. In this study, using a Kaposi's sarcoma tumor cell line designated SLK, various stable transfected cell lines were produced. Tumor cells that were either singly positive for either CD80 or CD86, as well as a double-positive cell line, were examined for their ability to induce T-cell activation, T-cell proliferation, and cytokine production profiles. Compared to the parental double-negative tumor cell line, the CD80-positive cells, but not the CD86-positive tumor cells, induced significant T-cell activation and proliferation. Tumor cells expressing both CD80 and CD86 also induced T-cell activation. After stimulation by the transfected tumor cells, T-cells produced a Th-1 type cytokine production profile with increased IL-2 and IFN-gamma levels. These results demonstrate that Kaposi's sarcoma tumor cells lacking co-stimulatory molecules cannot induce T-cell activation; however, if they express CD80, they can induce peripheral blood T-cell proliferation, and there is a differential response as expression of CD86 did not have the same immunostimulatory effect.

Apoptosis in keratinocytes is not dependent on induction of differentiation.

During embryological development and throughout life, regulation of the thickness of skin is likely to involve modulation of keratinocyte proliferation, differentiation, and cell death. One major mechanism of cell death is apoptosis; but the precise relationship between apoptosis and differentiation has not been well-defined. In this report, we demonstrate that when cultured undifferentiated keratinocytes have their adhesive interactions interrupted by either enzymatic treatment (ie, trypsin) and suspension in a semi-solid methyl cellulose medium, or exposure to antibodies against beta 1 integrins and E-cadherin, induction of differentiation occurs (expression of involucrin), as well as apoptosis (positive terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP-biotin nick end labeling (TUNEL) assay and DNA fragmentation). However, these events are not directly interdependent processes, as determined by two-color immunofluorescence staining. Thus, apoptosis can occur without evidence of differentiation and vice versa. The process o apoptosis in keratinocytes was dissected at the molecular level and found to be correlated with increased expression of Bax and decreased levels of Bcl-XL, with no role for either Bcl-2 or Bcl-XS. We conclude that keratinocytes do not need to undergo differentiation before undergoing apoptosis.

Induction of HHV-8 lytic cycle replication by inflammatory cytokines produced by HIV-1-infected T cells.

Human herpesvirus 8 (HHV-8) is a gamma2-herpesvirus consistently identified in Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. Although HHV-8 infection appears to be necessary, it may not be sufficient for development of KS without the involvement of other cofactors. One potentially important cofactor is HIV-1. HIV-1-infected cells produce HIV-1-related proteins and cytokines, both of which have been shown to promote growth of KS cells in vitro. Though HIV-1 is not absolutely necessary for KS development, KS is the most frequent neoplasm in AIDS patients, and AIDS-KS is recognized as a particularly aggressive form of the disease. To determine whether HIV-1 could participate in the pathogenesis of KS by modulating HHV-8 replication (rather than by inducing immunodeficiency), HIV-1-infected T cells were cocultured with the HHV-8-infected cell line, BCBL-1. The results demonstrate soluble factors produced by or in response to HIV-1-infected T cells induced HHV-8 replication, as determined by production of lytic phase mRNA transcripts, viral proteins, and detection of progeny virions. By focusing on cytokines produced in the coculture system, several cytokines known to be important in growth and proliferation of KS cells in vitro, particularly Oncostatin M, hepatocyte growth factor/scatter factor, and interferon-gamma, were found to induce HHV-8 lytic replication when added individually to BCBL-1 cells. These results suggest specific cytokines can play an important role in the initiation and progression of KS through reactivation of HHV-8. Thus, HIV-1 may participate more directly than previously recognized in KS by promoting HHV-8 replication and, hence, increasing local HHV-8 viral load.

Geographically distinct HHV-8 DNA sequences in Saudi Arabian Iatrogenic Kaposi's sarcoma lesions.

A new member of the gamma-herpesvirus family, HHV-8 (also known as Kaposi's sarcoma (KS)-associated herpesvirus), has been linked to KS and body cavity-based lymphoma. Other members of this family, eg, Epstein-Barr virus, were originally thought to have only one strain, but subsequent analysis revealed different strains correlating to cellular patterns of infectivity and geographical location. To determine whether multiple strains of HHV-8 exist, we compared DNA sequences among KS and body cavity-based lymphoma-derived HHV-8 and examined differences in HHV-8 subgroups between American and Saudi Arabian iatrogenic KS patients. Samples were analyzed by polymerase chain reaction using multiple primer sets to five different open reading frames from HHV-8, and DNA sequencing was performed. HHV-8 DNA was present in all of our KS and body cavity-based lymphoma samples by polymerase chain reaction. HHV-8 DNA was detected in each body cavity-based lymphoma sample using a majority of the primers, whereas only two primer sets consistently amplified HHV-8 DNA derived from KS lesions. DNA sequencing within open reading frames 26 and 27 indicate the existence of at least three variants of HHV-8, with the majority of iatrogenic KS patients in Saudi Arabia containing unique nucleotide changes that may define a distinct, previously unidentified subgroup we term SA, whereas those from America were of Group A or B. Thus, although the sequencing data within open reading frames 26 and 27 did not permit discrimination between patients with lymphoma versus KS disease processes, HHV-8 derived from Saudi Arabian KS lesions were shown to have a distinct nucleotide sequence not seen in any of the other clinical samples examined.

Compartmentalized roles for leukocytic adhesion molecules in lung inflammatory injury.

By using the model of acute injury caused by intrapulmonary deposition of IgG immune complexes, blocking mAb to CD11a, CD11b, L-selectin, and intercellular adhesion molecule-1 (ICAM-1) were administered either i.v. or intratracheally (i.t.). The effects of these interventions were assessed according to lung injury, lung content of myeloperoxidase (MPO), TNF-alpha, and cellular content in bronchoalveolar lavage (BAL) fluids, and up-regulation of pulmonary vascular ICAM-1. In animals treated i.v. with Abs to CD11a, L-selectin, or ICAM-1 lung injury was significantly attenuated in parallel with reduced lung content of MPO. Under similar conditions, treatment with anti-CD11b had no effect. However, when the same mAb were administered i.t., anti-CD11a and anti-L-selectin were without protective effects, whereas i.t. administered anti-CD11b and anti-ICAM-1 were each highly protective. The protective effects of anti-CD11b were related to profound reductions in BAL levels of TNF-alpha, pulmonary vascular up-regulation of ICAM-1, and lung content of MPO. The protective effects of i.t.-administered anti-ICAM-1 were not associated with reduced BAL levels of TNF-alpha. Protective effects of mAb were also reflected in reductions of retrievable neutrophils in BAL fluids. mAb to rat CD11b and CD18 but not to rat CD11a suppressed in vitro production of TNF-alpha by immune complex-stimulated rat alveolar macrophages. The mAb did not reduce NO2-/NO3- generation in stimulated macrophages but all mAb (except anti-ICAM-1) reduced O2- responses in macrophages. These data suggest a compartmentalized role for adhesion molecules in lung inflammatory injury after intraalveolar deposition of IgG immune complexes, with CD11a, L-selectin, and ICAM-1 being important in the vascular compartment for neutrophil recruitment, whereas in the alveolar compartment CD11b and ICAM-1 (but not CD11a and L-selectin) seem to play key roles.

Kaposi's sarcoma tumor cells preferentially express Bcl-xL.

Several recently identified proteins such as Bcl-2 and Bcl-x have been found to regulate programmed cell death (i.e., apoptosis). In this report, we examined the levels of expression of proteins that can either prevent apoptosis (i.e., Bcl-2 or the long form of Bcl-x, designated Bcl-x1) or promote apoptosis (i.e., Bax or the short form of Bcl-x, designated Bcl-xs) in proliferating benign and malignant endothelial cells (ECs). In normal skin with quiescent ECs, no detection by immunohistochemical staining was observed for Bcl-xL, Bcl-xs, or Bcl-2. However, in diseased skin samples that feature a prominent angiogenic response such as in psoriasis or pyogenic granulomas, the proliferating ECs markedly overexpressed Bcl-xL, with little to no Bcl-2. In an acquired-immune-deficiency-syndrome-related neoplasm, Kaposi's sarcoma, the spindle-shaped tumor cells also overexpressed Bcl-xL compared with Bcl-2. These in vivo studies were extended in vitro using cultured ECs and Kaposi's sarcoma tumor cells that were examined by flow cytometry and immunoblot analysis. Both cultured ECs and Kaposi's sarcoma tumor cells express significantly higher levels of Bcl-xL than Bcl-2. Such overexpression of cell survival gene products may contribute to prolonging the longevity of EC-derived cells in several different benign and neoplastic skin disorders that are characterized by a prominent angiogenic tissue response.

Propagation of a human herpesvirus from AIDS-associated Kaposi's sarcoma.

BACKGROUND: Although unique DNA sequences related to gammaherpesviruses have been found in Kaposi's sarcoma lesions, it is uncertain whether this DNA encodes a virus that is able to reproduce. METHODS: We isolated and propagated a filterable agent whose DNA sequences were found to be identical to those of the Kaposi's sarcoma-associated herpesvirus (KSHV). We obtained early-passage spindle cells from skin lesions of patients with the acquired immunodeficiency syndrome (AIDS) who had Kaposi's sarcoma and cultured them with cells of the human embryonal-kidney epithelial-cell line 293. We characterized the virus according to its effects on cellular morphology and viral replication and its appearance on electron microscopy. RESULTS: KSHV was cytotoxic to 293 cells and was detected by the polymerase chain reaction (PCR) in infected cells but not uninfected ones. Cytotoxicity and positive PCR signals were consistently maintained with viral titers of 1 million per milliliter, for about 20 serial infections of 293 cells. The viral copy number was relatively low (1 to 10 copies per cell). Viral replication was confirmed by Southern blot analysis of DNA isolated from the enriched nuclear fraction of infected cells and by a semiquantitative PCR using dilutions of the lysates of infected cells to detect the 233-bp viral DNA fragment originally described in association with Kaposi's lesions. Electron microscopy revealed herpesvirus-like particles in about 1 percent of cells from infected cultures, as compared with none in cells from uninfected cultures. CONCLUSIONS: A herpesvirus with DNA sequences identical to those of KSHV can be propagated from skin lesions of patients with AIDS-associated Kaposi's sarcoma.