Lymphatic endothelial cells derived from metastatic and non-metastatic lymph nodes of human colorectal cancer reveal phenotypic differences in culture.

Colorectal cancer is one of the most frequent causes of death in Western countries. Most patients develop metastasis traveling through the lymphatic system, and regional lymph node metastasis is considered a marker for dissemination, increased stage, and worse prognosis. Despite rapid advances in tumor biology, the processes that underpin lymphatic invasion and lymph node metastasis remain poorly understood. The aim of this study was to establish an easy protocol for isolation of pure tumor lymphatic endothelial cells derived from lymph nodes to study differences compared with normal endothelial cells of uninvolved tissue from the same patients. Cells were isolated with very high purity via magnetic cell sorting and express the specific lymphatic markers Prox-1 and Lyve-1. They show differences in expression of adhesion molecules, chemokines, and growth factor secretion, and capability to form capillaries when seeded on basal membrane, thereby, revealing important differences between the two cell type. These cultures may provide a promising platform for the comparative analysis of both cell types at the molecular and biological level and to optimize treatment strategies.

Generation of CD28- cells from long-term-stimulated CD8+CD28+ T cells: a possible mechanism accounting for the increased number of CD8+CD28- T cells in HIV-1-infected patients.

According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28-. The CD28dim T cells were found to derive from mitogenic stimulated CD28-T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright, cells showed a CD28dim expression before further evolution to a stable CD28-phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28- T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bright T cell clones. A high percentage of CD28dim and CD28- cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28- T cells in HIV-infected patients.

Isolation, purification, and heterogeneity of human lymphatic endothelial cells from different tissues.

Relatively few attempts have been made in the past to isolate and expand lymphatic endothelial cells (LECs). Recently this task has become feasible thanks to the identification of new lymphatic markers such as Podoplanin, Lyve-1, Prox-1 and D2-40. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1-coated beads followed by purification with monoclonal antibody D2-40, we were able to purify and in vitro expand human derived LECs from tissues such as lymph node, spleen, thymus, palatine tonsil and iliac lymphatic vessels. The isolated LECs were expanded on collagen type 1 and fibronectin coated flasks for up to 8-10 passages and then analyzed for phenotypic and functional properties. LECs were able to form a capillary like network, when seeded on Cultrex BME, indicating their capability to form lymphatic vessels in vitro. Comparative studies were performed, and we found that specific lymphatic and vascular markers were differentially expressed by LECs prepared from different sources, clearly demonstrating the phenotypic heterogeneity of LECs from different organs and different segments of the lymphatic vasculature. We here propose a new technique to make available ready sources of abundant well-characterized human LECs to examine normal profiles and behavior to compare with abnormal conditions.

Adult human heart microvascular endothelial cells are permissive for non-lytic infection by human cytomegalovirus.

OBJECTIVE: Human cytomegalovirus (CMV) infection has been linked to chronic heart disease. The mechanism of CMV dissemination to the heart remains unknown. CMV antigens and nucleic acid sequences have been detected in endothelial cells (ECs) in vivo, and ECs are fully permissive hosts to CMV replication in vitro. This report examines the characteristics of CMV replication in primary cultures of human heart microvascular ECs (HHMECs). METHODS: Capillary ECs were isolated from heart tissue biopsies of six patients at the time of heart surgery. HHMECs were infected with CMV and viral antigens were detected by immunofluorescence assay using monoclonal antibodies as specific reagents. Cytokine and chemokine release in the supernatant of sham- and CMV-infected cells was quantitated by ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyse expression of mRNA for adhesion molecules. RESULTS: CMV was found to productively infect HHMECs without cytolytic effects. Infected cultures released high levels of pro-inflammatory chemokines and enhanced their adhesion molecule expression. CONCLUSIONS: Our data provide new insights into the mechanism of CMV dissemination to the heart, signalling the need for further investigation of the pathogenetic role of this virus in cardiac disorders.

Development of horse polyclonal antiserum inhibiting all in vitro biological functions of human IFN-gamma.

Following a standard immunization protocol with recombinant human interferon-gamma (IFN-gamma), a horse polyclonal antiserum was obtained and evaluated for its ability to interfere with multiple IFN-gamma activities in vitro. Data obtained show that polyclonal horse antiserum neutralizes the antiproliferative activity of IFN-gamma, inhibits the binding of IFN-gamma to cellular receptors, and can up-regulate HLA-DR antigen expression and interfere with its antiviral activity. The broad neutralizing capacity of horse polyclonal antiserum has been assessed on cell lines which differ as to origin and sensitivity to IFN-gamma. Moreover, we observed that this antiserum could inhibit the binding of radiolabeled IFN-gamma to its cellular receptor, its subsequent internalization into the target cell, and its antiviral activity. As it is able to inhibit all the biological activities of IFN-gamma, this antiserum might provide new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.

Gas chromatographic assay of cellular fatty acids and alcohols for the identification of Mycobacterium species.

Ten mycobacterial species obtained from 141 cultures isolated from clinical specimens were studied. The cultures were grown on solid medium and then analysed-after saponification, methylation, extraction with organic solvent and washing of the organic phase--by capillary gas-liquid chromatography for fatty acid and secondary alcohol composition. The absence of secondary alcohols was characteristic of M. genavense, M. tuberculosis and the following Mycobacterium species with specific branched-chain fatty acids allowing their direct identification: M. gordonae, M. kansasii and M. marinum. The presence of secondary alcohols was characteristic of M. avium, M. phlei, M. scrofulaceum, M. terrae and M. xenopi. In the case of M. xenopi direct identification was made possible by the presence of a specific alcohol.

Isolation and chemoantibiotic resistance of Ureaplasma urealyticum in HIV-1 infected patients.

The presence of Ureaplasma urealyticum was evaluated on 1912 vaginal and urethral swabs from HIV-1 seronegative (HIV-) inpatients (210) and outpatients (503) suffering from acute urethritis or vaginitis; asymptomatic HIV- outpatients (201); and asymptomatic HIV-1 seropositive (HIV+) inpatients (120). The study reported an increased frequency of Ureaplasma urealyticum isolates in asymptomatic HIV+ compared to asymptomatic HIV- subjects. As expected, the frequency of Ureaplasma urealyticum isolates increased in symptomatic HIV- subjects. Strains of Ureaplasma urealyticum resistant to ciprofloxacin, tetracycline and minocycline were more frequently isolated in HIV+ (34.1%) than HIV- (3.8%) subjects; on the other hand, only 1 out of 704 (0.1%) strains isolated from outpatients was resistant to ciprofloxacin. We found no association in HIV+ patients between Ureaplasma urealyticum infection and CD4 count or HIV-1 p24 antigenemia.

Increased frequency of detection of Ureaplasma urealyticum and Mycoplasma genitalium in AIDS patients without urethral symptoms.

The roles of Mycoplasma genitalium and Ureaplasma urealyticum in nongonococcal urethritis are not yet well established. The aim of this study was to determine the presence of these microorganisms in the urethral tracts of 187 human immunodeficiency virus type 1 (HIV-1)-infected male patients with no clinical signs of urethritis. The results indicate that the prevalence of M. genitalium and U. urealyticum was higher in AIDS patients than in asymptomatic, HIV-1-infected patients and in healthy individuals. The high rate of mycoplasmas and ureaplasmas detected in AIDS patients, in the absence of urethritis, argues against major roles in causing disease at the urethral mucosal level for these microorganisms.

CD11b expression identifies CD8+CD28+ T lymphocytes with phenotype and function of both naive/memory and effector cells.

A previously unreported CD8(+)CD28(+)CD11b(+) T cell subset occurs in healthy individuals and expands in patients suffering from primary viral infections. In functional terms, these cells share the features of naive/memory CD8(+)CD28(+)CD11b(-) and terminally differentiated effector CD8(+)CD28(-)CD11b(+) subpopulations. Like CD28(-) cells, CD28(+)CD11b(+) lymphocytes have the ability to produce IFN-gamma, to express perforin granules in vivo, and to exert a potent cytolytic activity. Moreover, these cells can respond to chemotactic stimuli and can efficiently cross the endothelial barrier. In contrast, like their CD11b(-) counterpart, they still produce IL-2 and retain the ability to proliferate following mitogenic stimuli. The same CD28(+)CD11b(+) subpopulation detected in vivo could be generated by culturing naive CD28(+)CD11b(-) cells in the presence of mitogenic stimuli following the acquisition of a CD45RO(+) memory phenotype. Considering both phenotypic and functional properties, we argue that this subset may therefore constitute an intermediate phenotype in the process of CD8(+) T cell differentiation and that the CD11b marker expression can distinguish between memory- and effector-type T cells in the human CD8(+)CD28(+) T cell subset.

Expansion of rare CD8+ CD28- CD11b- T cells with impaired effector functions in HIV-1-infected patients.

The decline in the number of CD4+ T cells in HIV-1-infected patients is known to be related to the increased number of CD8+CD28- T cells. In this paper, we show that CD8+CD28- T cells from HIV-positive patients have an impaired capability to interact with human endothelial cells. This is due to the dramatic expansion, within this subset, of rare CD11b- cells lacking cell-cell adhesion functions. In 50 HIV-positive patients, 19.5% +/- 6.5% of all T cells were CD8+CD28-CD11b-, whereas only 0.8% +/- 0.4% of all T cells from healthy donors showed this uncommon phenotype. The percentage of circulating CD8+CD28-CD11b- T cells was strongly related to the percentage of CD4+ T cells (r = -0.82). This population is peculiar in terms of HIV infection and was found to possess some characteristics associated with effector functions but its cytotoxic properties were impaired. The percentage of target cells lysed by CD8+CD28-CD11b- was significantly lower than that of cells lysed by its CD11b- counterpart (p <.05) both at low (5:1) or at relatively high (20:1) effector/target ratios. CD8+CD28-CD11b- T cells, which lack the ability to interact with endothelial cells, are likely to accumulate and persist in circulation. The biologic properties of CD8+CD28-CD11b- T cells suggest that these cells might be endstage or aberrant differentiated effector cells. Lack of cell-cell adhesion and impaired cytolytic functions favor the hypothesis of a role for CD8+CD28-CD11b- T cells in the development of immunodeficiency.