Inhibition of HIV and virus replication by polysulphated polyxylan: HOE/BAY 946, a new antiviral compound.

Xylanpoly-(hydrogen sulphate) disodium salt with a molecular weight of about 6000 daltons (HOE/BAY 946) completely inhibited syncytium formation induced by the infection of T lymphocytes with HIV as well as viral replication at concentrations above 25 micrograms/ml. This dose was found to be inhibitory for several strains of HIV-1 and HIV-2. Low molecular weight fractions of the compound were less active against HIV, and high molecular derivatives were as active as HOE/BAY 946. A direct influence of the drug on the infectivity of the virus could not be demonstrated. The drug inhibited the reverse transcriptase of HIV. Treatment of permanently HIV-infected U937 cells resulted in a drastic reduction of virus particles released into the supernatant and points to an additional mode of action. A therapeutic effect of HOE/BAY 946 against retroviruses in vivo could be demonstrated in Friend leukaemia virus-infected mice. A clinical pilot study with the compound was started recently in Germany with AIDS patients who did not tolerate or refused to take zidovudine and with asymptomatic virus carriers.

Interaction of Escherichia coli and macrophages: alteration by treatment of bacteria with beta-lactam antibiotics.

Antibiotics are known to exert an influence on the host-parasite relationship either by impairment of immunocompetent cells or by alteration of the bacterium, such as changes of surface properties or the production of toxins. The main problem in investigating the effect of antibiotics on the surface properties of bacteria consists in morphological changes of bacteria (round cell or filament formation) after treatment e.g. with beta-lactam antibiotics. These changes of morphology lead to problems in the comparison of such bacterial forms with untreated organisms. Therefore, in this study outer membrane vesicles from bacteria were used as a model to investigate the effect of antibiotics on the surface properties of Escherichia coli with regard to the interaction with mouse peritoneal macrophages tested by chemiluminescence reaction. It could be shown that these membrane vesicles induce a luminol dependent chemiluminescence response. Treatment of E. coli with different beta-lactams lead to an increase of the stimulating properties. The relative effectiveness of certain antibiotics depended on the particular E. coli strain. Analysis of the different adhesions involved in the stimulation of macrophages revealed that only mannose-sensitive adhesins were increased after treatment with beta-lactam antibiotics. No stimulation of the membrane-bound NAD(P)H-oxidase could be found following the reaction with outer membrane vesicles. Even the treatment of bacteria with antibiotics did not evoke such a reaction.

Monoclonal antibodies detecting plasminogen activators on the membrane of leukemic lymphoid cells of T-cell origin.

The specificities of six monoclonal antibodies produced against plasminogen activator of the human Bowes melanoma cell line are described. They have been used to detect membrane-bound plasminogen activator on cultured human lymphoid cell lines and in neoplastic human lymphocytic and myeloid cells of leukemic patients. These studies indicate that only certain phenotypic subsets of the T-cell lineage derived from patients with chronic lymphocytic leukemia or with Szezary syndrome express plasminogen activator on their surface membrane.

Cultivation of Bowes melanoma cells in the Opticell system: influence of the addition of protein supplements to the serum-free medium on the production of plasminogen activator.

The influence of a protein-free and a protein-rich, supplemented serum-free medium on the production of plasminogen activator (t-PA) from Bowes melanoma cells was investigated in the Opticell culture system and compared to tissue culture flask cultures. In the presence of medium supplements metabolic activity and t-PA production were favoured in both systems. The addition of supplements was apparently more effective in the Opticell than in flask cultures, because t-PA activity obtained in the Opticell was 2-3 times higher in protein-rich medium, but 2 times lower in unsupplemented medium than in flasks. These results indicate that the protein content in a serum-free medium is important for product formation in the Opticell, and serum-free media which work at small scale in tissue culture flasks are not always suited for technical culture systems such as the Opticell but have to be adapted to them.

Influence of antibody and complement components on phagocytosis and chemiluminescence of macrophages.

Macrophages are known to release reactive oxygen species (O2-, 1O2, H2O2, OH.) in response to various membrane stimuli. However, our studies show that phagocytic stimulation of macrophages is not necessarily accompanied by a stimulation of the oxidative burst. Whereas IgG-opsonized erythrocytes were capable to induce phagocytosis and a chemiluminescence response, both being dependent on the number of IgG bound per erythrocyte, C3b-bearing erythrocytes were well ingested but failed to induce any chemiluminescence reaction. Furthermore, stimulation of macrophages, via the Fc-receptors, seems to alter their functional state in regard to the activation of a receptor, which enables them to recognize membrane lesions on the target erythrocyte. The presence of IgG and membrane lesions, e.g. the C5b-9-complex of complement, induced a marked increase in chemiluminescence compared with stimulation by IgG-bearing particles alone. The augmented response of macrophages was at least in part due to an additional release of H2O2, which was not liberated in response to IgG-bearing erythrocytes. This "lesion recognizing receptor" in the macrophage membrane could not be activated by stimulation of C3b-receptors, indicating its functional linkage to the Fc-receptors.

Chemiluminescence and superoxide generation by leukocytes stimulated by polyelectrolyte-opsonized bacteria. Role of histones, polyarginine, polylysine, polyhistidine, cytochalasins, and inflammatory exudates as modulators of oxygen burst.

Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.

Bone-marrow derived macrophages as targets for the replication of mouse hepatitis virus type 3.

Bone-marrow (BM) derived macrophages are sensitive target cells for replication of mouse hepatitis virus type 3 (MHV3). These cells can be grown in large numbers and the percentage of defined macrophages increased until day 10 when 100% of the cells represented macrophages. MHV3 replicated within these cells to high titers and caused the formation of multi-nucleated giant cells. This effect was seen with very low virus inocula in BM macrophages of C57BL/6 mice that are highly susceptible to in vivo infection with MHV3 whereas macrophages from resistant A/J mice did not show a cytopathic effect at these virus doses. 1000-fold higher virus doses, however, caused the cytopathic effect in macrophages of both C57BL/6 and A/J mice.

Chemotactic activity of bone marrow-derived macrophages changes with time in culture.

The number of bone marrow-derived macrophages able to respond to chemotactic stimuli varies with time in culture. Chemotactic response was optimal at 2 weeks and may depend on cell maturation or differentiation or both.

Lymphokine-induced secretion of plasminogen activator by murine macrophages.

Thioglycollate-stimulated macrophages are known to release a plasminogen activator (PA) into the medium. In this study it was investigated whether macrophages could be activated to release PA after exposure to lymphokines. Macrophage monolayers obtained by 24 h culture of proteose peptone-elicited murine exudate cells were incubated with lymphocyte culture supernatants. After 48 h the supernatants were replaced by serum-free medium and the macrophages were incubated for another 24-48 h. These supernatants were assayed for PA as measured by the lysis of 125I-labeled fibrin. The following results were obtained: (a) Supernatants of antigen or mitogen-stimulated spleen cells induced PA secretion by macrophages whereas control supernatants were ineffective. The same was found with supernatants of mitogen-stimulated lymph node cells. (b) PA secretion by macrophages seems to be induced by a rather narrow concentration range of lymphokines. (c) Lymphokine-induced PA secretion by macrophages is enhanced after phagocytosis of latex beads. The results show that PA secretion by activated macrophages can be considered as a parameter of immunoactivation.