Ralph van Furth

Monocyte adherence to human vascular endothelium

An acute inflammatory response requires that circulating monocytes bind to and migrate across the endothelium of the vessel wall to gain access to inflamed sites. Various mediators of inflammation ‐ cytokines and chemoattractants ‐ have been shown to initiate and regulate the margination and extravasation of monocytes. This review summarizes evidence that the mechanism underlying the initial adhesion of monocytes to normal and cytokine‐stimulated endothelial cells and their subsequent transendothelial migration are successive events in monocyte‐endothelial cell interaction. Special emphasis is given to the current knowledge of the contribution of adhesion molecules belonging to the family of 13i‐ and 132‐integrins, the immunoglobulin supergene family, and the selectins to such cellular interaction. The sequence of events that allow monocytes to attach to, migrate over and finally pass the endothelium is discussed in detail.

Antibody response after influenza vaccination in HIV-infected individuals: a consecutive 3-year study ☆

In a consecutive 3-year study the antibody response after immunization with influenza vaccine of a cohort of HIV-infected adults was studied. The haemagglutination-inhibiting (HAI) antibody titres after vaccination correlated with the number of CD4(+) T lymphocytes (p<0.001), the prevaccination antibody titres (p<0.001), and the proliferative response to anti-CD3 (p<0.001). Severely impaired antibody responses were observed in HIV-infected individuals with CD4(+) T-lymphocyte counts < or =100x10(6)/l. Significantly higher prevaccination antibody titres were observed in healthy controls in the 2nd or 3rd year of vaccination, but not in HIV-infected individuals. Annually repeated vaccination of HIV-infected individuals did not lead to higher postvaccination antibody titres. Annual vaccination of HIV-infected individuals with CD4(+) T-lymphocyte counts exceeding 100x10(6)/l seems to be worthwhile, although it may not be expected to render the same level of protection against influenza as in non-infected individuals.

Ubiquicidin, a novel murine microbicidal protein present in the cytosolic fraction of macrophages.

Previously we have identified and characterized three murine microbicidal proteins purified from the granule fraction of cells from the murine macrophage cell line RAW264.7. During these studies evidence was obtained for the presence of an additional antimicrobial protein in the cytosolic fraction of RAW264.7 cells that had been activated with interferon‐γ (IFN‐γ). In this study we have purified this protein, designated ubiquicidin, to apparent homogeneity and demonstrated that it is a cationic, small (M r 6654) protein. Ubiquicidin displayed marked antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium. Using a gel overlay procedure evidence was obtained that the protein also displays activity against Escherichia coli, Staphylococcus aureus, and an avirulent strain of Yersinia enterocolitica. Amino‐terminal amino acid sequencing and mass spectrometry analysis of purified ubiquicidin indicated that it is most likely identical to the ribosomal protein S30. This protein is produced by posttranslational processing of the Fau protein, a 133‐amino‐acid fusion protein consisting of S30 linked to an unusual peptide with significant homology to ubiquitin. The fau gene has been reported to be expressed in a variety of tissues in humans and various animal species. The presence of ubiquicidin in the cytosol of macrophages may serve to restrict the intracellular growth of microorganisms. In addition, because macrophage disintegration will likely lead to release of ubiquicidin into the extracellular environment, it may contribute to host defense after macrophage death. J. Leukoc. Biol. 66: 423–428; 1999.

Enhanced antibody response to pneumococcal polysaccharide vaccine after prior immunization with conjugate pneumococcal vaccine in HIV-infected adults.

The antibody production by HIV-infected adults after two vaccinations with conjugated pneumococcal vaccine (CPV) and consecutive vaccination with polysaccharide pneumococcal vaccine (PPV) was studied. Thirty days after the second CPV, the geometric mean antibody concentrations (GMC) against pneumococcal polysaccharide serotypes (PPS) 6B, 14 and 19F were significantly lower in the group HIV-infected individuals with <200x10(6)/l CD4(+) T lymphocytes (group 1) than in the group with >/=200x10(6)/l CD4(+) T lymphocytes (group 2) and healthy controls. Thirty days after PPV vaccination the GMC against PPS 6B, 14, 19F and 23F in group 1, and against 6B and 19F in group 2, were significantly lower compared with healthy controls. Both in HIV-infected and in healthy individuals who received CPV and PPV the postvaccination GMC against PPS 14, 19F and 23F were higher compared with historical controls who were not previously immunized with CPV but only received PPV. We conclude that the antibody response to CPV is impaired in HIV-infected individuals. Higher antibody concentrations were achieved in HIV-infected and healthy individuals after sequential vaccination with CPV and PPV compared with PPV vaccination alone.

Antibodies against pneumococcal polysaccharides after vaccination in HIV-infected individuals: 5-year follow-up of antibody concentrations.

We studied the production of IgG antibodies against eight pneumococcal polysaccharide serotypes (PPS) 1, 4, 6B, 9V, 14, 18C, 19F and 23F after vaccination of 50 HIV-infected adults with 23-valent Pneumovax((R))23 and the course of the antibodies against four PPS during the following years. Mean antibody concentrations against PPS 18C, 19F and 23F were sigificantly lower in the patients with CD4(+)-lymphocyte counts <200x10(6)/l than in healthy controls; mean antibody concentrations against PPS 1, 4, 9V, 6B and 14 were similar in HIV-infected individuals and controls. Although it has been assumed that polysaccharides induce a T-cell-independent immune response, our results indicate that some PPS are T-cell-independent type 2 antigens. The rates of decline of mean antibody concentrations in HIV-infected individuals and in healthy controls were similar during 5 y after vaccination. However, as a consequence of the low postvaccination antibody concentrations against several PPS, within 3 y after vaccination most HIV-infected individuals had antibody concentrations below the level which is assumed to be required for protection.

Growth Characteristics of Cultured Human Macrovascular Venous and Arterial and Microvascular Endothelial Cells

The morphological and growth characteristics of human macrovascular endothelial cells (ECs) from venous and arterial umbilical cord vessels and microvascular ECs from foreskin were compared during cultivation. By means of time-lapse microcinematography and phase-contrast microscopy, differences in cell morphology and migratory activity between the different types of ECs were found. Growth characteristics were dependent on the type of EC, the nature of the substrates on which the ECs were grown and the presence of growth factors. For all types of ECs optimal growth and formation of a monolayer were observed when the ECs were cultured on fibronectin or gelatin substrates in the presence of EC growth factor and heparin. Under these conditions confluent cultures of macrovascular ECs reached maximal cell densities of 1,400-1,900 ECs/mm2, whereas microvascular ECs reached maximal cell densities of about 700-900 ECs/mm2. The cell cycle times calculated from the population-doubling time and the stathmokinetic index, respectively, amounted to 63 and 83 h for microvascular ECs, 33 and 35 h for venous macrovascular ECs, and 29 and 35 h for arterial macrovascular ECs.

Impaired antibody response after immunization of HIV-infected individuals with the polysaccharide vaccine against Salmonella typhi (Typhim-Vi®)

Infections with Salmonella species, including Salmonella typhi, are more frequently observed in HIV-infected individuals than in healthy individuals. HIV-infected individuals were vaccinated with polysaccharide vaccine against Salmonella typhi (Typhim-Vi) which is assumed to be a T-cell-independent antigen. We found that the antibody response in patients with < 200 x 10(6)/l CD4+ T lymphocytes was significantly lower compared with patients with > or = 200 x 10(6)/l CD4+ T lymphocytes and healthy controls. The antibody response after vaccination with the polysaccharide salmonella Vi-antigen was correlated with the number of CD4+ T lymphocytes and therefore Typhim-Vi can be considered to be a T-cell-independent type 2 antigen. The results of this study indicate that after vaccination the proportion of HIV-infected individuals with protective antibody concentrations against Salmonella typhi will be lower than in healthy controls.

Morphological, cytochemical, functional, and proliferative characteristics of four murine macrophage-like cell lines.

The aim of the present study was to obtain objective data on the morphology and quantitative information about other characteristics of murine macrophage-like cell lines J774.1, PU5-1.8, WEHI-3, and P388-D1, and to compare the findings with those in resident and exudate macrophages collected directly from mice. Fetal fibroblasts were included to serve as controls. Evaluation of the morphological data showed that the cell lines J774.1 and WEHI-3 are almost identical in most respects, that the cells of P388-D1 differ widely from both of the former lines, and that the morphometric parameters of cell line PU5-1.8 occupy an intermediate position. The cells of the P388-D1 line show the most similarity to resident and exudate macrophages, and cell lines J774.1 and WEHI-3 the least. Fetal fibroblasts had divergent values for all morphometric parameters. Good correspondence was found when the quantitative data obtained by morphometric analysis of the cells in question were compared with the morphological pictures. No gross differences as to cytochemical characteristics were found between the cells of the four cell lines, except for 5-nucleotidase activity. The occurrence of IgG receptors and the ingestion of EIgG were also similar, but the percentage of cells with C3b receptors was much lower in two of the cell lines (WEHI-3 and P388-D1) and the level of EIgMC ingestion was very much higher in one (J774.1) compared with both the other cell lines and the resident and exudate macrophages. The ingestion of opsonized bacteria and latex varied widely within and between the cell lines. Quantitative data on the binding of monoclonal antibodies by the cells of the macrophage cell lines and the resident and exudate macrophages showed a wide variation. The doubling time of the cell lines is on average 1 day; distinct differences were found between these lines with respect to the lag-time of proliferation after replating. Cluster analysis and statistical analysis of morphological and other characteristics gave insight into the degree of resemblance between the cells of the four cell lines on the one hand and the resident and exudate macrophages on the other.

Kinetics of Phagocytosis and Intracellular Killing of Staphytococcus aureus and Escherichia coli by Human Monocytes

Tie kinetic patterns of the phagocytosis and intracellular killing of Staphytococcus and Echerichia coli by monocytes were investigated separately to acquire more insight into the total process, i.e. from the ingestion to the death of the micro‐organisms. Phagocytosis proved to be dependent on: (1) both the bacteria‐to‐monocyte ratio and the monocyte concentration; a concentration of at least 5 × 105 monocytes/ml proved necessary for the measurement of ingestion, whereas the rate of ingestion was found to be proportional to the number of extracellular bacteria until a maximum rate is reached, (2) the serum concentration in the incubation medium, which influenced both the rate of phagocytosis and the maximum number of bacteria taken up by one monocyte, and (3) the temperature, the highest rate of phagocytosis being reached at 37–41°C The intracellular killing proved to be dependent on: (1) the number of bacteria ingested; the rate of killing was proportional to the number of ingested bacteria until a maximum rate was reached; (2) the temperature, since a maximum rate of killing is only reached at 37–41°C: at tower and higher temperatures the rate of killing is lower, in the latter case due to inactivation of extracellular stimuli. These separate data on the ingestion and killing processes made it possible to compute the theoretical numbers of extracellular, viable intracellular, and total intracellular bacteria for a model system consisting of 5×106 monocytes, 5×106 bacteria, and 10% serum. These calculated values are in agreement with the experimental data.

Protection and humoral immune responses against Bordetella pertussis infection in mice immunized with acellular or cellular pertussis immunogens

In the present study, protection against Bordetella pertussis infection and humoral immunological responses in mice has been assessed upon immunization with custom-made acellular pertussis vaccines (ACVs) and whole-cell pertussis vaccine (WCV). Mice were immunized, next intranasally infected with B. pertussis and during 14 days the number of bacteria in the trachea and lungs and the level of serum antibodies were determined. ACV contained five immunogens, filamentous hemagglutinin, pertactin, fimbriae serotypes 2 and 3, and chemically detoxified pertussis toxin (PMC-5), or three immunogens, filamentous hemagglutinin, pertactin, and genetically detoxified (BC-3) or chemically detoxified pertussis toxin (SKB-3). Immunization with a high or low dose of ACV or WCV resulted in significant protection against B. pertussis, with differences in the degree of protection between the vaccines. The lowest protection was found with a low dose of SKB-3 and WCV. The pattern of cytokine production by spleen cells of immunized, non-infected, mice indicated that T-helper 1 cells are activated by vaccination with WCV, and T-helper 1 and T-helper 2 cells are involved in the immune response upon vaccination with ACVs. Each vaccine stimulated the production of IgG, but not IgA, antibodies. In mice immunized with ACV, elimination of B. pertussis from trachea and lungs correlated significantly with the titre of IgG1, but not IgG2a, antibodies.