Lipid analogs reveal features critical for hemolysis and diminish granadaene mediated Group B Streptococcus infection.

Although certain microbial lipids are toxins, the structural features important for cytotoxicity remain unknown. Increased functional understanding is essential for developing therapeutics against toxic microbial lipids. Group B Streptococci (GBS) are bacteria associated with preterm births, stillbirths, and severe infections in neonates and adults. GBS produce a pigmented, cytotoxic lipid, known as granadaene. Despite its importance to all manifestations of GBS disease, studies towards understanding granadaene's toxic activity are hindered by its instability and insolubility in purified form. Here, we report the synthesis and screening of lipid derivatives inspired by granadaene, which reveal features central to toxin function, namely the polyene chain length. Furthermore, we show that vaccination with a non-toxic synthetic analog confers the production of antibodies that inhibit granadaene-mediated hemolysis ex vivo and diminish GBS infection in vivo. This work provides unique structural and functional insight into granadaene and a strategy to mitigate GBS infection, which will be relevant to other toxic lipids encoded by human pathogens.

Development of monoclonal antibodies for the fusarin mycotoxins.

The fusarins are a group of mycotoxins produced by fungi that commonly infest cereal crops, in particular by the fungus Fusarium verticillioides. This group of compounds is characterized by a substituted 2-pyrrolidone ring attached to a 12-carbon polyunsaturated backbone. Several of the fusarins contain an epoxide substitution on the pyrrolidone ring and are highly mutagenic. This paper describes the development of seven monoclonal antibodies and immunoassays for detecting fusarins C and A. Fusarin C was isolated and conjugated to ovalbumin to produce the immunogen. Competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) were developed based upon the isolated monoclonal antibodies. The concentrations of fusarin C able to inhibit colour development by 50% (IC(50)) in CI-ELISAs were 1.0, 2.0, 3.6, 23.4, 28.9, 31.4, and 66.7 ng ml(-1) for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Cross-reactivity with fusarin A was 44.8, 51.4, 41.1, 174.0, 62.6, 78.2, and 98.0% for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Given the sensitivity of these antibodies for fusarins it is expected that, with further development, they may be useful for detecting fusarins at relevant levels in foods.

Polyene substrates with unusual methylation patterns to probe the active sites of three catalytic antibodies.

The synthesis of two tetraenes that differ in their methylation pattern from the natural substrate in lanosterol biosynthesis, 2,3-oxidosqualene, and their examination with three catalytic antibodies is described. The design of these novel, linear terpenoid structures was governed by initial results obtained from the characterization of the three catalytic antibodies. These were generated by immunization with a steroidal hapten that mimics multicyclization without the necessity for anti-Markovnikov additions or ring expansions. Such a reaction cascade would represent a more 'primitive' version compared to the oxidosqualene cyclization observed in lanosterol, cycloartenol and beta-amyrin biosynthesis and would not require a tail-to-tail connection of the third and fourth isoprene unit as seen in squalene. The first tetraene design (A) only contains trisubstituted double bonds and hence its synthesis starts from farnesol and tris-norgeraniol. The second tetraene design (B) is considered the more precise match to the inducing hapten that generated the antibody collections by exhibiting one disubstituted double bond and its synthesis utilizes a tris-norgeraniol derivative and a symmetrical bis-allylic alcohol as key building blocks. Chromatographic comparison studies lead to the conclusion that the currently studied antibodies also produce monocyclic products from the two substrates as has been formerly observed with a squalene-derived substrate. In contrast, 2,3-oxidosqualene is not accepted by these catalysts supporting the notion that the current substrates are fully bound by recognition of both terminal functional groups.

Immunopharmacology of rapamycin.

The potent immunosuppressive drugs FK506 and rapamycin interfere with signal transduction pathways required for T cell activation and growth. The distinct inhibitory effects of these drugs on the T cell activation program are mediated through the formation of pharmacologically active complexes with members of a family of intracellular receptors termed the FK506 binding proteins (FKBPs). The FKBP12.FK506 complex specifically binds to and inhibits calcineurin, a signaling protein required for transcriptional activation of the interleukin (IL)-2 gene in response to T cell antigen receptor engagement. The FKBP12. rapamycin complex interacts with a recently defined target protein termed the mammalian target of rapamycin (mTOR). Accumulating data suggest that mTOR functions in a previously unrecognized signal transduction pathway required for the progression of IL-2-stimulated T cells from G1 into the S phase of the cell cycle. Here we review the immunopharmacology of rapamycin, with particular emphasis on the characterization of mTOR.

Brief treatment with rapamycin in vivo increases responsiveness to alloantigens measured by the mixed lymphocyte response.

Rapamycin (RPM) is a macrolide fermentation product that prolongs rodent allograft survival more potently and effectively than cyclosporin A (CsA) and FK506. Experiments in vitro have shown that RPM inhibits lymphoproliferation by mechanisms of action that are different from other immunosuppressants. Much less is known, however, about the effects of RPM on immune cells in vivo compared to other immunosuppressive drugs. Others have shown that in vivo treatment with CsA suppresses the responsiveness of cells in the mixed lymphocyte response (MLR). Therefore, to investigate the effects of RPM in vivo, rats were treated with RPM and their lymphoid cells used as responder cells in the MLR. We confirmed that the proliferation of cells in the MLR was decreased after treatment with CsA in vivo. In contrast, treatment with RPM in vivo greatly increased the proliferative response to alloantigen in the MLR. These findings show that the effects of RPM and CsA on immune cells in vivo differ. Perhaps the cells proliferating in the MLR after in vivo RPM treatment play a role in the regulation of the immune system that enables this immunosuppressant to prolong allograft survival so effectively in rodents.

Molecular mechanisms of immunosuppression.

The immunosuppressive drug cyclosporin A (CsA, Sandimmun, SIM) is currently being evaluated in a variety of autoimmune disorders with some remarkable successes. Despite the wide empiric application of CsA, the precise mechanism of action of this drug remains elusive. To identify the molecular mode of action of CsA in the process of T cell activation, we have compared the biological profile of cyclophilin-binding cyclosporin analogues (CBCA), which lack immunosuppressive properties, with CsA. We have found that CsA binding to its intracellular receptor (cyclophilin) is required but not sufficient for immunosuppression. Moreover, inhibition of the peptidyl-prolyl cis-trans isomerase activity of cyclophilin does not seem to be relevant for the inhibitory effects of CsA. In analogy to the immunosuppressants FK506 and rapamycin, a specific structure at the 'effector' domain of the CsA molecule different from the immunophilin 'binding' domain determines the biological activity. Overall, a significant understanding of the structure-activity relationship of CsA has emerged. This will have a major impact on the identification of the precise mechanism of action of CsA and its side effects in the process of immunosuppression.

Persistent diarrhea and malnutrition--the impact of treatment on small bowel structure and permeability.

Previous studies using dual sugar permeability tests suggested that damage to the small intestinal mucosa plays an important part in the development of persistent diarrhea in The Gambia. The present study has extended these findings by examining the effect of nutritional rehabilitation on intestinal permeability and mucosal morphology. Intestinal permeability, measured by lactulose:mannitol (L:M) absorption, and mucosal structure, measured by a quantitative, computerised morphologic technique, were evaluated in 20 children before and after such treatment. L:M ratios were high on admission, (0.66 +/- 0.36) and, despite some temporary improvement, did not significantly improve (0.49 +/- 0.30) following rehabilitation for one month. The changes in L:M ratio were largely due to an increase in lactulose absorption, showing that the small intestinal mucosa becomes more "leaky" as a result of nutritional rehabilitation. Although no correlation was found between measures of intestinal permeability and mucosal morphology, nutritional restitution was associated with a significant increase in size of the mucosal crypt cell compartment, but not in villous epithelial volumes during the same period. It is necessary to establish, by further prospective studies, the interval required for full restitution of small intestinal structure and function during treatment for persistent diarrhea.

Effects of selenium supplementation on immune parameters in gut failure patients on home parenteral nutrition.

The relationships between some parameters of the immune response and selenium were investigated in five patients receiving home parenteral nutrition for short-bowel syndrome. They were first submitted to a relative depletion by providing 20 micrograms selenium/day as L-selenomethionine for 1 mo. Then, daily selenium intake was raised to 200 micrograms for 2-4 mo. On entering the study, the patients presented a relatively good health status, and immunological parameters were at the lowest limit of the normal range. Four patients rapidly responded to the 200-micrograms supplementation by a continuous increase in their plasma selenium levels, whereas the fifth patient showed a moderate and late increase. At the end of the trial, there was an improvement in the lymphocyte response to pokeweed and phytohemagglutinin mitogens in four patients and to CD3 in three patients. The response to two of three antigens (Candidin, Varidase) tested was also enhanced in the same patients, but the response to the third antigen (tetanus toxoid) was uniformly low in all patients. The only patient showing essentially no immune improvement after selenium supplementation was the one with a low and delayed increase in plasma selenium. This study supports a role for selenium in the maintenance of an optimal immune response in humans.

Antibodies to nystatin demonstrate polyene sterol specificity and allow immunolabeling of sterols in Saccharomyces cerevisiae.

Polyclonal antibodies elicited by injection into rabbits of a nystatin-bovine serum albumin conjugate were reactive with both nystatin and amphotericin B. Upon labeling of polyene-treated Saccharomyces cerevisiae sterol auxotrophs grown on various sterols, nystatin reacted specifically with ergosterol, while amphotericin B did not react preferentially with ergosterol, cholesterol, or cholestanol. Time course labeling experiments demonstrated the rate of ergosterol transport into cholesterol-grown cells.

[Effect of immunoreactivity to candidin on phagocytic performance and intracellular killing of live fungi by polymorphonuclear granulocytes]. / Zum Einfluss der Immunreaktivität gegenüber Candidin auf die Phagozytoseleistung und intrazelluläre Abtötung lebender Sprosspilzzellen durch polymorphkernige Granulozyten.

Positive results of the intracutaneous test with Candida were associated with an increased phagocytosis (p-trend) and an increase of the intracellular killing (p less than or equal to 0.01) of Candida albicans by polymorphonuclear granulocytes.

[Comparison of the drop and capillary technics of the leukocyte migration inhibition test in the detection of sensitization to candidin]. / Vergleich von Tropfen- und Kapillartechnik des Leukozytenmigrationshemmtestes beim Nachweis einer Sensibilisierung gegenüber Candidin.

15 test persons with a positive and 3 with a negative intradermal test of candidin were examined with two modifications of the migration inhibition test. In these cases the agarose droplet test proved to be somewhat more sensitive in comparison to the capillary method. There were no correlations between the size of the cutaneous test reaction and the adequate migration index. The droplet test using culture residues (lymphokin assay) was less evident in comparison to the direct method. The two techniques (droplet test, capillary method) are well suited for the proof of cell-mediated immune reactions to candidin and other comparable antigens, respectively.

Uveitis with allergy to candidin.

By skin testing, 21 patients with uveitis were more reactive to Candida extracts than a control group of patients during the same 15-year follow-up period. Skin testing with candidin induced an obvious and transient change of the course of the ocular disease in 19 patients and led to systemic symptoms in 15 patients; 11 were atopic, 17 had intermediate uveitis. Antifungal treatment and/or desensitization improved the ocular condition in 16 patients. The real pathogeny of these uveitides remains hypothetical.

[Sensitization by repeated skin tests with candidin and PHA]. / Sensibilisation par tests cutanés itératifs à la candidine et à la PHA.

Intra-dermal skin tests (IST) were applied with candidin (1:100) in 37 subjects and repeated 10 days later. The induration diameter of the second injection (read at 48 h) was greater (p less than 10(-8]. A correlation (r = 0.76, p less than 0.001) appears between the first and second induration diameters. In 17 of these patients (randomly distributed) we measured the absolute number of mononuclear cells bearing receptors of Fc IgG (RFc) before the first and before the second injection. The number of RFc was greater (p less than 0.02) after the second injection. It may be possible that candidin testing increase T suppressor cells. In 20 other subjects, we observed an increase of induration diameter (p less than 0.05) after a second IST of PHA (2 micrograms) applied 15 days after the first injection. Repeated IST with candidin and PHA induce a very high level of booster phenomenon. This fact may be kept in mind when interpreting serial IST in immunodeficiencies.

[Skin tests as immunologic parameters in cervical cancer]. / Hautteste als Immunparameter beim Zervixkarzinom.

Immunological skin-tests in 158 patients admitted at the 1st Department of Gynaecology and Obstetrics, University of Vienna, and in 25 healthy controls were done in a long-term prospective study. 123 examined patients were suffering from invasive cervical cancer stage I or Ii (group 1), 17 patients were suffering from preinvasive lesions of the cervix (group 2) and 18 patients were admitted for treatment of benign disorders (group 3). Recall-antigens--streptokinase-streptodornase (SKSD), purified protein derivate (PPD) and candida (CAND)--and a primary antigen (DNCB) were used testing cutaneous delayed hypersensitivity reactions. Preoperative total reactivity to recall-antigens calculated by a score was significantly reduced in cancer patients in comparison to other groups. Subdividing the cancer patients according to histological criteria of the malignancy we could find significantly differences in particular tests (SKSD and CAND) but not in total reactivity score. Comparing the preoperative reactivity of cancer patients with or without recurrence of malignancy within two years less pronounced differences were found. Reactivity to recall-antigens in all tests and in all patient groups were better three weeks after surgical treatment than preoperatively. The prior found differences between the groups remained significant (p less than 0,05). In contrast, cancer patients demonstrated severe anergy in postoperative DNCB sensitization test as only 1 of 33 patients exhibited normal hypersensitivity reaction. 113 out of 24 patients of group 2 and 3 (54%) demonstrated delayed hypersensitivity to DNCB in operative sensitization test.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired cell-mediated immunity in Plasmodium falciparum-infected patients with high-parasitemia and cerebral malaria.

Several cell-mediated functions were studied in vivo and in vitro in 63 Thai patients with acute falciparum malaria, including 21 cases with cerebral manifestations and 10 cases with initial parasitemia over 10%. Initial delayed cutaneous reactions to phytohemagglutinin and soluble protein antigens were negative in most cerebral malaria cases. In other patients, skin reactions were impaired or abolished as a direct function of parasitemia. No major alteration in the numbers of blood T and B lymphocytes was found. In lymphocyte cultures, proliferative responses to lectins were generally found within normal ranges; in contrast, proliferative responses to candidin were suppressed in parallel with delayed cutaneous responses to the same antigen. From these data, it can be concluded that the alteration of specific cell-mediated responses are predominantly detectable in acute cases with major parasite invasion, i.e., high parasitemia and/or cerebral manifestations. A direct role of Plasmodium falciparum was further suggested by the rapid restoration of cell-mediated functions observed in several cases under successful antimalarial therapy. These results do not support any evidence in favor of a preexisting cellular immune deficiency in relation with the occurrence of cerebral or high-parasitemia acute malaria in these patients.

Atopic dermatitis and delayed hypersensitivity.

Studies of patients with atopic dermatitis show both clinical and laboratory evidence of a depression of cellular immunity. In order to study one aspect of this disease in vivo we studied 40 patients with atopic dermatitis of both sexes, aged 2-30 years and 40 supposedly healthy subjects with similar characteristics in regard to age and sex who served as the control group. Both groups were subjected to tests of delayed hypersensitivity with Candidin, Trycophytin and Tuberculin. Candidin response was negative in 72.5% and 85% at 48 and 72 hours respectively. Trycophytin response was negative in 77.5% and 87.5% of the readings at the same time intervals, and Tuberculin response was negative in 85.5% of the patients with atopic dermatitis studied. The results are shown to be statistically significant. It is concluded that, cellular immunity should be evaluated in patients with atopic dermatitis.