Batimastat reduces Mycobacterium tuberculosis-induced apoptosis in macrophages.

In this study, we report evidences that Mycobacterium tuberculosis (MTB)-induced apoptosis in macrophages is reduced by a broad-spectrum hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, Batimastat (BB-94). In particular, we show that BB-94 administration to MTB-infected macrophages inhibits apoptosis and the downmodulation of membrane CD14 expression. Moreover, the addition of broad spectrum matrix metalloproteinase inhibitor to cell culture, during MTB infection, decreases the release of soluble TNF-alpha and leads to a simultaneous increase of membrane TNF-alpha. These results show that MTB-induced apoptosis in macrophages is reduced by a MMP inhibitor and most probably is related to TNF-alpha release. This identifies BB-94 as a simultaneous anti-apoptotic and anti-inflammatory molecule during MTB infection.

Inhibition of contact hypersensitivity reaction to picryl chloride: effect of small molecular weight peptidomimetic compounds possessing inhibitory activity against metalloproteinases.

We have studied the effect of small molecular weight inhibitors of snake venom metalloproteinases (SVMP) and matrix metalloproteinases (MMPs) on the induction and effector phase of the contact hypersensitivity reaction (CHR) in a mouse model. Identification of nonsteroid small molecules is very important for the development of new anti-inflammatory drugs. The compounds that we tested were synthetically modified tripeptides (peptidomimetic compounds) POL-257, POL-509, POL-443, POL-491, and POL-647, with structures based on natural occurring peptides in snake venom. A well-known hydroxamate-based inhibitor of the MMPs, Batimastat (BB-94), was also used. We have shown that these peptidomimetics possess in vitro inhibitory activity against the MMP-2 (gelatinase-A), MMP-9 (gelatinase-B), and MMP-3 (stromelysin). They also inhibit metalloproteinases purified from the venom of Crotalus adamanteus and C. atrox snakes, which are very similar to the so-called A Desintegrine, A Metalloproteinase (ADAMs) enzymes. When injected intraperitonealy before the topical application of the contact sensitizer (picryl chloride) or before the challenge, these compounds significantly inhibited the development of CHR. BB-94 at doses 0.4 and 4 mg/kg before the sensitization or before the challenge almost completely abrogated the reaction. POL-257 and POL-443 were among the most active peptidomimetics tested. They inhibited the inflammatory reaction up to 70-80%, when applied either immediately before sensitization or before challenge. POL-509, a methylated derivative of POL-257, inhibited the CHR to 40-50% when administered at either challenge or sensitization. However, when applied 24 h before the challenge, it completely abrogated the inflammatory reaction. The results show that these small molecular weight peptidomimetic compounds, as well as BB-94, are able to significantly inhibit the CHR. This finding opens possibilities for using metalloproteinase inhibitors in the treatment of allergic contact dermatitis and other inflammatory diseases.

Technological aspect of the production of live dysentery vaccines for oral administration.

An experimental technology for the production of live freeze-dried vaccines prepared from attenuated Shigella flexneri 2a and Shigella sonnei I strains was developed. It is based on the cultivation of bacterial strains in a fermentor under conditions which ensure high yields. The strains grow in S-form, their antigenic structure is preserved and they remain non-virulent. The attenuating markers are stable. The freeze-dried vaccines retain good immunogenicity when applied intra-intestinally to rats.

Potential Shigella flexneri 2a and Shigella sonnei I live vaccinal strains. Characterization of immunogenicity in animal models.

Rats and rabbits were immunized intraintestinally with different doses of virulent and nonvirulent live Shigella flexneri 2a and Shigella sonnei I strains. The nonvirulent strains had one or two attenuating markers. The period of excretion with the faeces of the bacteria and of their polysaccharide antigens, the proliferation of antigen-binding and antibody-producing cells in the spleens and gut-associated lymphoid tissues and the levels of antibodies in sera and faeces were studied. Attenuated strains S. flexneri 2a 77 and S. sonnei I 3359 induced the most potent and long lasting local immune response.

Role of veiled cells in lymphocyte activation.

Peripheral blood lymphocytes from normal rabbits or from rabbits hyperimmunized with human immunoglobulin were cultured in 20-microliters hanging droplets. The cultures were stimulated with concanavalin A or, in the case of cells from sensitized animals, with human immunoglobulin. The addition to the cultures of small numbers of autologous or allogeneic veiled cells separated from afferent lymph increased the responses to low doses of the stimulants, particularly when the cells were cultured at low cell densities or for short periods of time. At high cell densities or at longer periods of culture, stimulation occurred in the absence of added veiled cells and was associated with clumping of the lymphoid cells and development of cells which morphologically resembled veiled cells found in afferent lymph. The enhanced responses upon addition of small numbers of veiled cells were also correlated with the formation of lymphoid aggregates which were frequently held together by the elongated processes of a single veiled cell. The observations support the view that the veiled cells are lymph-borne precursors of dendritic cells in the lymph nodes. This may provide an in vitro model for the cellular relationships which occur in paracortical cords of lymph nodes following antigenic stimulation.

The effect of veiled cells on lymphocyte function.

The response of lymphocytes to stimulation with Con A has been studied in the presence of veiled cells collected from the afferent lymph. In these enriched cultures the response occurred earlier with smaller numbers of lymphocytes and at lower concentrations of Con A. Veiled cells also caused clumping of lymphocytes in unstimulated cultures. In stimulated cultures small cells with veil-like projections appeared after 48 hr, but were not seen in unstimulated cultures.

[Local Immune response in rabbits following enteral immunization with live attenuated bacterial Enterobacteriaceae vaccines]. / Lokale Immunantwort nach enteraler Impfung von Kaninchen mit attenuierten bakteriellen Enterobacteriaceae-Lebendimpfstoffen.

Streptomycin-dependent and inactivated Shigella flexneri 2a and Shigella sonnei strains were intra-intestinally applied to rabbits for immunisation. Rosette and plaque tests and well as indirect haemagglutination gave short-time secretion of low titres of specific copro-antibody, following monovaccines and bivaccines. High titres of secretory antibody were induced, depending on doses, by re-immunisation. No antigen competition was established. The localised immune response caused by Shigella live vaccines was found to be much stronger than that induced by inactivated vaccines

Contact sensitivity in the pig. II. Induction by intralymphatic infusion of DNP conjugated cells.

Pigs were skin painted with the contact sensitizing agent DNFB and afferent lymph was collected for the next 24 h. The lymph cells carried a small amount of DNP and were able to sensitize 40% of homologous recipients. Peripheral blood lymphocytes conjugated with DNFB in vitro to the same degree as afferent lymph cells sensitized 80% of autologous and 40% of homologous recipients: lymphocytes conjugated at higher levels sensitized 75% of animals in each group. Lightly conjugated cells were capable of survival and able to respond to stimulation by mitogens and their ability to sensitize autologous recipients was abolished by heat killing. Highly conjugated cells were not capable of survival, their sensitizing ability was not altered by heat killing and they were able to sensitize incompatible recipients. It is suggested that highly conjugated cells sensitize by a different mechanism which depends on the cooperation of non-lymphocytic cells, not easily mobilized from lymphoid tissue.

Contact sensitivity in the pig. III. Induction by intralymphatic infusion of DNP-conjugated cell membranes and soluble proteins, free DNFB and some small molecular weight derivatives.

DNP-conjugated lymph node cell plasma membranes, thymocyte plasma membranes and red cell ghosts were prepared and tested for their ability to induce contact sensitivity, using pigs as experimental animals. Lymph node cell membranes and red cell ghosts were able to sensitize, provided the dose of DNP was very large, but thymocyte membrane failed to sensitize most pigs. DNP-conjugated lymph proteins coming from the site of application of DNFB were also able to sensitize if large amounts were administered, but free DNFB itself, infused directly into an afferent lymphatic, was much more efficient. Since DNFB can often be detected in lymph folowing skin painting, it may conjugate cells within the node which later contact the recirculating population of lymphocytes and so sensitize the animal by a central mechanism. The best-equipped cells would be the macrophage-like lymph cells which are closely related to the epidermal Langerhans cells and are known to migrate to the paracortex of the node.