Production and characterization of a monoclonal antibody to human saposin C. A murine IgG1 monoclonal antibody, termed 68-12, was produced against purified human saposin C. Immunoprecipitation and binding analysis indicated that the antibody reacted only with saposin C. Dot blotting and Western analysis demonstrated that antibody 68-12 also reacted with prosaposin and a higher molecular weight protein(s) in murine spleen and cerebral grey matter. Solid phase competitive radioimmunoassay against 125I labeled saposin C (0.25 micrograms/ml) showed no cross reactivity for saposin A, B and D up to 15 micrograms/ml. At a concentration of 50 micrograms/ml saposin A, B and D cross reacted 21, 1.5, and 49% respectively. Monoclonal antibody 68-12 appears to have potential utility in the purification, detection and quantitation of human saposin C and its precursor.


Production of monoclonal antibodies specific to major allergens of Cryptomeria japonica pollen. Monoclonal antibodies (MAbs) directed against Cryptomeria japonica pollen antigen (CPAg) were prepared. CPAg was precipitated with these MAbs and both MAbs CPA7 (IgG2a) and CPA9 (IgG1) recognized two major glycoproteins (m = 41 kDa and 46 kDa) of CPAg. MAb CPA7 or CPA9 was coupled to CNBr activated sepharose and affinity purification of the major allergen of CPAg from the crude extract was performed. The affinity purified CPAg bound IgE antibody present in patients with Cedar pollinosis.


Characterisation of eight monoclonal antibodies to involucrin. Involucrin is a precursor of the insoluble protein envelope that is assembled in the outermost layers of the epidermis. The coding sequence of the protein contains a number of short tandem repeats that have been greatly altered during mammalian evolution. We have characterised eight mouse monoclonal antibodies raised against human involucrin, all of which bind to the protein in immunoprecipitation, immunoblot and immunohistochemical preparations. Each antibody was screened for cross-reactivity with gorilla, owl monkey, dog and pig involucrin and with a fragment of the human protein, expressed in lambda gt 11, that includes the entire early region of the modern segment of repeats. Three antibodies recognised involucrin in all of these assays. Four antibodies recognised primate involucrins and the lambda gt 11 fragment. One antibody, which showed cross-reactivity with lower molecular weight proteins, only recognised primate involucrins and therefore bound outside the early region of the modern segment. Since the antibodies can be used to detect involucrin both biochemically and histologically, in a range of species, they will have applications in further studies of the expression, function and evolution of the protein.


Monoclonal antibodies to Escherichia coli beta-galactosidase and their use for detection and purification of recombinant expression products. A panel of seven mouse monoclonal antibodies (BG-01-BG-07) was prepared against beta-galactosidase derived from E. coli. The antibodies are beta-galactosidase specific, show no cross-reactivity with other E. coli proteins and can be used for identification and characterization of beta-galactosidase fusion proteins expressed in lambda expression vectors. One of the antibodies allows a simple, one-step isolation of the fusion proteins directly from the crude bacterial lysates using immunoaffinity chromatography.


[Isolation of H. pylori proteins and support for their suitability in serologic diagnoses]. H.pylori-proteins were separated using gel chromatographic methods. These antigens were tested for their suitability to detect H.pylori-specific antibodies. A complex of two proteins (62 kDa and 30 kDa) was a strong and specific antigen. A third protein (13 kDa) was a good but nonspecific antigen. Concerning these facts we compared two often used antigen preparations for serodiagnosing H.pylori-specific antibodies (acid-glycine preparation and sarcosyl-insoluble outer membrane proteins). The sarcosyl-insoluble material contains more specific antigens and lower levels of nonspecific proteins compared to the acid-glycine preparation. Based on these results we conclude that the outer membrane preparation seems to be more suitable for the serodiagnosing of H.pylori-specific antibodies.


[Alterations in lymphocyte subsets in variable immunodeficiency syndrome]. Besides functional defects, different phenotypical alterations of lymphocytes have been described in patients with CVID. A decrease in LAM-1- (= Leu8) and CD21 expression was observed on B cells. Some patients with CVID show alterations in their T-cell subpopulations: The most striking features of this subgroup are low CD4/CD8-ratio, an increase in CD57/CD8 double-positive lymphocytes, and a strong enhancement of HLA-DR and "memory" markers such as CD29 and LFA-3 on T cells.


[Pentoxifylline--an inhibitor of the synthesis of tumor necrosis factor alpha]. The growing knowledge on the pathophysiological role of cytokines in acute and chronic inflammatory processes stimulated efforts to control their synthesis and action pharmacologically in clinical situations. Recently, in our institute evidence was raised that pentoxifylline (POF) is able to suppress the synthesis of tumor necrosis factor-alpha (TNF) in cell cultures, in vivo, and to protect experimental animals against endotoxin shock. Studies in human experimental endotoxemia showed that pentoxifylline decreased circulating TNF without affecting endogenous formation of interleukins. The potency of this drug to interfere with TNF synthesis could also be demonstrated in cases of acute and chronic cytokine release syndromes such as OKT3 first-dose reaction and severe pulmonary tuberculosis, respectively. We suggest that POF may improve therapeutic strategies in various diseases in which TNF was identified as a causative pathophysiological factor.


[Impaired induction of chemiluminescence and function of polymorphonuclear neutrophilic granulocytes in diabetes mellitus]. The aim of this study was to investigate PMN-chemiluminescence response as a measure of respiratory burst activity and the phagocytic PMN function "ingestion" with regard to metabolic control parameters in diabetes mellitus (d.m.) in comparison to healthy controls. Our findings demonstrated a significant reduction of chemiluminescence response and ingestion in diabetic patients compared to controls (p less than 0.01 resp.); further an inverse relation of metabolic control parameters in d.m. and PMN impairment, suggesting inhibitory effects on PMN function, thus leading or contributing at least in part to altered host defense.


[Epidemiology and pathogenesis of streptococcal infection]. The appearance of the "streptococcal toxic shock-like syndrome" led to a growing interest in infections caused by Streptococcus pyogenes (group-A-streptococci). Since 1987 some 800 cases with a lethality of 20% or more were observed. Contrary to toxic scarlet fever the site of primary infection are the lower respiratory tract or soft tissue infections. Erythrogenic toxins and low molecular weight mitogens, inducing cytokines (IL-2, IL-3, IL-6, TNF-alpha, IFN-gamma) seem to be involved in the pathogenesis of these severe infections. Morphologically and culturally the strains isolated from cases of toxic shock-like syndrome cannot be differentiated from isolates of epidemic scarlet fever or sporadic cases. At the same time, when in Scandinavia an epidemic by S.pyogenes type 1 with many cases of toxic shock was observed, the same type caused a scarlet fever epidemic without complications in eastern Germany. Erythrogenic toxin type A or its toxoid, respectively, can be used for successful immunizations of rabbits. Another--antibacterial-immunization can be done with the M-protein of S.pyogenes, which is limited by its type-specificity. Streptococcal vaccination is required especially for developing countries with a high incidence of rheumatic fever. Infections due to Streptococcus agalactiae (group-B streptococci) are often underestimated though they have a first position in septicemia and meningitis of newborns. Taxonomy and nomenclature of streptococci are often changing; a list of the presently known species is presented in table I.


Intestinal intraepithelial lymphocytes and lymphoepithelial interactions in the human gastrointestinal mucosa. Although representing a major immunological apparatus, it is not known how the immune system of the intestinal mucosa differentiates between dietary antigens (resulting in systemic tolerance) and potential pathogens. It is thought that intraepithelial T lymphocytes (IEL) may play a central role in local intestinal immunity and are likely to be important in immunity to gastrointestinal neoplasms and rejection responses to gut allografts. However, the biology of IEL and their unusual immunological microenvironments in the gastrointestinal mucosa are little understood. IEL are predominantly CD8+ TcR alpha beta+ CD3+ T cells which differ from lamina propria and peripheral T cells in many respects. IEL show low expression of CD5, CD6, LFA-1 (CD11a/CD18) and VLA-4, and high expression of HML-1. TcR gamma delta + IEL, although a minority population, are also phenotypically distinct, insofar as they are 50% CD8+, mainly V delta 1+ V gamma 9- and CD4- CD5-. IEL show poor proliferative responses to PHA, anti-CD3 and phorbol ester/calcium ionophore in vitro and have no clear functional role: they neither provide helper nor suppressor functions for Ig synthesis by B cells and do not mediate spontaneous cytotoxicity. However, there is evidence that IEL show preferential activation in response to sheep erythrocytes, presumably signalling via CD2. As normal and inflamed intestinal epithelia do not express ICAM-1, it seems unlikely that the LFA-1/ICAM-1 interaction is of importance to IEL activation. Rather, the CD2 (LFA-2) interaction with LFA-3 expressed by enterocytes may serve both to anchor IEL and to provide an accessory stimulus for activation. Nevertheless, the questions of antigenic specificity and immunological role remain unanswered.


Monoclonal antibodies against metallothioneins from the human liver. Six hybridomas secreting monoclonal antibodies directed against human fetal liver metallothioneins (MTs) have been generated and characterized. One antibody, K5A6, was specific for MT-1, the others recognized all isoMTs present in the human fetal liver. Each of these antibodies showed a unique cross-reactivity pattern when tested with MTs from the livers of different mammals. A double-antibody sandwich enzyme-linked-immunosorbent-assay (ELISA) was developed with the antibodies L2E3 and L5G2. This assay allows the detection of 60 pg human fetal liver MT and exhibits a metal dependent response for Zn-, Cd- and Hg-MT.


Isotypic and allotypic analysis with monoclonal antibodies and jacalin of 309 serum monoclonal IgA from French and Japanese myeloma patients. The subclass and allotype distribution of serum monoclonal IgA from myeloma patients was determined by ELISA with monoclonal antibodies in two French and one Japanese laboratory. In addition, the French sera were tested for their reactivity with the lectin jacalin. No significant difference in the isotypic distribution between French and Japanese series could be demonstrated: kappa/lambda ratios were 0.99 and 1.17, and the IgA1 subclass accounted for 93.9% and 91% of cases in the French and Japanese studies, respectively. Five out of 7 myeloma IgA2 from Japan and only one of the 12 IgA2 from France belonged to the A2m(2) allotype (P less than 0.01). All 219 IgA1 tested reacted with jacalin by immunoelectrophoresis (IEP), although with variable intensities. Among IgA2 proteins, only one (of the A2m(1) allotype) yielded a precipitating line with jacalin by IEP. Molecular analysis demonstrated that this protein was an IgA1-IgA2 hybrid bearing most of the A2m(1) epitopes.


The human lymphokine leukoregulin induces cell resistance to complement-mediated lysis. Leukoregulin (LR) is a lymphokine secreted by human natural killer (NK) cells. Its effect on the susceptibility of K562 human erythroleukemic cells to lysis by antibody and complement was examined. As reported here, treatment of K562 cells with LR for 60 min at 37 degrees C confers on them resistance to complement damage. The LR-induced state of complement resistance is transient and the cells recover within 4-6 h unless a second dose of LR is added. The protective action of LR was observed using both conventional 51Cr-release and trypan blue inclusion assays. The protein synthesis inhibitors puromycin and cycloheximide and the protein kinase inhibitors tamoxifen, polymyxin B and W-7, could each block this action of LR. Fewer membrane attack complexes were found, following complement activation, on LR-treated than control cells. These results suggest that LR increases the capacity of K562 cells to down-regulate complement activation or repair the complement damage, possibly by inducing synthesis of defense proteins and/or activation of protective protein kinases.


Purification of a novel B cell growth and differentiation factor associated with lupus syndrome. We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mice produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55-kDa protein that we termed nucleobindin (Nuc). Nuc showed not only induction of anti-ssDNA IgG antibody in cultures of B cells from MRL/l mice (greater than 16 weeks old), but also growth activity. Furthermore, antibodies against existing cytokines have so far not been shown to block Nuc activity on these B cells. In view of the fact that Nuc did not boost anti-ssDNA IgG antibody production in cultures of spleen cells of comparable age from MRL/n mice, which develop a mild form of lupus after the age of one year, Nuc may act on pre-activated B cells to help IgG anti-DNA antibody production. Taken together, Nuc is a new kind of growth and differentiation factor associated with lupus syndrome.


Synthetic peptides comprising defined sequences of CH-2 and CH-3 domains of human IgG1 induce prostaglandin E2 production from human peripheral blood mononuclear cells. Synthetic peptides Y48 and Y75 comprising sequences at exposed sites within the CH-2 and CH-3 domains of human IgG1 at a concentration of 10(-5) M, increase PGE2 production by human peripheral blood mononuclear cell (PBMC) cultures. An increase of leukocyte migration inhibitory factor (LMIF) production in PBMC cultures--as a result of synthetic peptide treatment--was also observed. This LMIF activity, to some extent, is attributed to the PGE2 production by the cells; the inhibition of leukocyte migration being abolished by the presence of indomethacine or antibody to PGE2.


Gut mucosal lymphocyte subpopulations in the host-defence of Shigella infected guinea-pigs. Lymphocyte subpopulations from the gut mucosa were studied in Shigella-susceptible guinea-pigs to evaluate their role in mucosal immune responses to such organisms. Four weeks after infection, isolated lymphocytes were characterised by an immunofluorescent technique. The increase in the T cell intra-epithelial population was less (48%) in comparison to that in normal uninfected animals (74%). On the other hand, Ig bearing B cells in the lamina propria showed an increased incidence and more than 15% of the total mucosal lymphocytes lacked any surface marker, which indicated that they were non-T and non-B cells. A significant rise in the intraepithelial T cell population (P less than 0.01) was observed 6 weeks after infection in comparison to that observed after 4 weeks.


IP-10, a gamma-interferon-inducible protein related to interleukin-8, lacks neutrophil activating properties. IP-10, a small, gamma-interferon-inducible protein with structural homology to interleukin-8 (IL-8), was prepared by automated chemical synthesis and compared with synthetic IL-8, GRO alpha and neutrophil-activating peptide 2 (NAP-2) for neutrophil stimulating activity. The following functions were tested: cytosolic free calcium changes, chemotaxis in vitro, respiratory burst, exocytosis of azurophil and specific granules from cytochalasin B-pretreated cells, and competition for IL-8 receptor binding. At 1, 10, 100 and 1000 nM, IP-10 was inactive in all assays, in contrast to the reference peptides which exhibited the expected neutrophil stimulating effects. In addition, IP-10 did not induce neutrophil accumulation after intradermal injection in rats, and did not act as IL-8 antagonist.


Enhancement of lymphocyte proliferation by mouse glandular kallikrein. Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.


G protein mediated signal transduction: membrane immunoglobulin associated phosphoproteins identified in octyl-beta-glucoside lysates of normal B cells. Several recent studies have shown that the antigen receptors (mIgM and mIgD) on normal B cell surfaces are part of larger multimolecular complexes, as each is noncovalently coupled to at least two other proteins whose phosphorylation is inducible upon G protein activation. However, B cell receptor complexes appear to be extremely labile, so that extraction of membrane immunoglobulin (mIg) from cell lysates in physical association with noncovalently coupled proteins has been shown to be highly dependent on the choice of detergent. Recent progress has to a large extent been dependent upon the introduction of digitonin, a detergent in which B cell receptor complexes are at least partially stable, as an agent to solubilize B cell membranes. Studies presented here demonstrate that if another well known detergent, octyl-beta-D-glucopyranoside (octyl glucoside), is utilized in place of digitonin, mIg receptors are immunoprecipitated from B cell lysates in association with several newly identified proteins, whose phosphorylation is also G protein dependent. In particular we find three to four mIg associated phosphoproteins in the molecular weight range of 47 to 57 kDa, and at least two others at 82 and 112 kDa. Some of these proteins may correspond to polypeptides that have been functionally linked to mIg dependent signal transduction, but have not as yet been shown to be physically coupled to the receptor.


Antigen-induced inhibition of the autoimmune response to rat male accessory glands: bone marrow dependence of the enhancement of IA+ but not IE+ antigen-presenting cells. IE+ peritoneal cells (PC), involved in the induction of suppression of autoimmune response to rat male accessory glands (RAG), are obtained from rats 2 h after i.p. injection of a purified fraction (FI) of RAG (FI-PC2h). In contrast, IA+ PC, involved in the induction of autoimmune response to RAG, are obtained from rats 24 h after FI of RAG injection (FI-PC24h). The present report analyzes the effect of irradiation or irradiation/bone marrow reconstitution on the induction of both populations of PC. Peritoneal cell donor rats were irradiated in a telegamma therapeutic Cs137. Twenty hours later half of them were i.v. reconstituted with 40 x 10(7) bone marrow cells. Six days later rats were i.p. injected with 200 micrograms of FI of RAG and 10(7) resident PC. The PC were harvested 2 h or 24 h later. The ability of resident PC to yield IE+ FI-PC2h involved in the induction of suppression is not impaired by irradiation, but the ability of resident PC to yield IA+ FI-PC24h involved in the induction of a positive response is impaired by irradiation and restored by bone marrow reconstitution of irradiated rats. Culture of normal PC with FI of RAG for 2 h or 24 h shows a selective increase in IE+ cells able to induce suppression to RAG.(ABSTRACT TRUNCATED AT 250 WORDS)


Defect in generation of LAK cell activity under oxygen-limited conditions. In general, the in vitro induction of lymphokine activated killer (LAK) cell activities by interleukin 2 (IL-2) in human peripheral blood mononuclear cells (PMNC) has been performed in an atmosphere of 5% CO2 in air (20% O2), whereas IL-2-induced LAK cell activities are considerably reduced under concentrations of 5% O2 equal to arterial blood oxygen tension (100 mmHg) and 2% O2 equal to venous blood oxygen tension (40 mmHg). Cultured cell viability, IL-2 receptor-beta expression on large granular lymphocytes (LGL), the percentage of IL-2 receptor-beta positive LGLs and cell proliferation were not affected by oxygen-limited conditions. LAK cells were induced by IL-2 over 5 days at 20% O2, at which time the LAK cells were further stimulated by IL-2 in 2% O2 and 20% O2. Under these conditions the activity of LAK cells in 2% O2 decreased day by day, while that of LAK cells induced in 20% O2 was maintained at least until day 10 of the original culture. LAK effector cell-mediated lysis was not influenced by oxygen-limited conditions. These results point to more successful applications of the combination of oxygen therapy and adoptive cellular immunotherapy in the clinic.


Haemolysis mediated by anti-D monoclonal antibodies in direct and cold target competition ADCC assays. Thirteen IgG anti-D human monoclonal antibodies (mAbs) were compared for their ability to mediate lysis of D-positive erythrocytes by PBMC in direct and cold target competition antibody-dependent cell-mediated cytotoxicity (ADCC) assays. In the latter assay, lysis of fluid-phase anti-D-sensitised O Rh D-positive papainised erythrocytes (E-IgG) was inhibited by A (or B) Rh D-negative papainised erythrocytes sensitised by fluid-phase anti-A (or anti-B) mAbs. The competitive and lytic activities of the anti-D mAbs were characterised by the equilibrium dilution (ED) values, which were the reciprocal of the dilution of anti-A (or anti-B) at which lysis of target E-IgG and competitor E-IgG were identical. There was a poor correlation between the number of erythrocyte-bound anti-D molecules and either the sensitivity of E-IgG anti-D to haemolysis in the direct ADCC assay, or to the ED values of the mAbs obtained in the cold target competition ADCC. The discriminatory power of the cold target competition ADCC was better than than of the direct ADCC to detect differences in the lytic potential of the anti-D mAbs.


Suppression of antibody responses in rats to murine anti-CD4 monoclonal antibodies by conjugates with monomethoxypolyethylene glycol. The effectiveness of therapeutically relevant xenogeneic monoclonal antibodies (MoAb) may be counteracted by their inherent immunogenicity. Since conjugates of diverse proteins with mono-methoxypolyethylene glycol (mPEG) were shown to induce Ag-specific tolerance in mice and rats, we used outbred rats in this study as an experimental model for establishing the tolerogenicity of mPEG conjugates of murine MoAb. The results demonstrate that: (i) murine anti-rat CD4 MoAb (W3/25) were more immunogenic than murine anti-human CD4 MoAb (MAX.16H5) in rats; (ii) W3/25 preferentially induced an anti-idiotypic (anti-id) antibody response; and (iii) antibodies to both common and idiotypic determinants could be suppressed in rats by treatment with W3/25(mPEG)28.


Identical point mutation leading to low levels of mannose binding protein and poor C3b mediated opsonisation in Chinese and Caucasian populations. A common opsonic defect occurring in 7% of the Caucasian population is associated with low serum levels of the lectin mannose binding protein (MBP). This study sought to determine whether the deficiency was also present in a Chinese population using sera obtained from 100 healthy Chinese children (age range 6 weeks-16 years). The distribution profiles of MBP levels and C3b/C3bi fragments binding to mannan coated plates were both bimodal and similar to the corresponding Caucasian profiles. Serum MBP levels were low in 9% of the Chinese children and all of these sera generated low levels of C3b/C3bi fragments. Overall there was a high significant correlation between MBP levels and C3b opsonin generation (r = 0.77; P less than 0.001). By analogy with similar findings in a Caucasian population we believe this correlation to be a reflection of antibody independent complement activation by MBP. In a pilot study of DNA obtained from three adult Chinese with low MBP levels the point mutation causing MBP deficiency in Caucasians was identified in all three cases.


Recombinant human interleukin 1 beta and tumor necrosis factor affect glycosylation of serum alpha 1-acid glycoprotein in rats. Serum concentration and glycosylation of rat alpha 1-acid glycoprotein (alpha 1-AGP) were evaluated after the in vivo administration of recombinant human interleukin-1 beta (rhIL-1 beta) and tumor necrosis factor alpha (rhTNF-alpha), alone or associated. The effect of LPS and turpentine was also studied. In all models, serum alpha 1-AGP concentrations were increased and glycosylation was altered. The alpha 1-AGP levels reached 1.8 g/liter with cytokines alone, 2.1 g/liter with cytokines associated or LPS, and 3.4 g/liter with turpentine. Analysis by concanavalin A (Con A) affinoimmunoelectrophoresis (CAIE) revealed that the relative proportion of Con A unreactive form always decreased whatever the inducing agent. On the other hand, the resulting effect on the concentrations of Con A unreactive alpha 1-AGP concentrations was an increase with cytokines alone or LPS and a decrease with cytokines associated or turpentine. These results suggest a dissociation between the alteration in the level of alpha 1-AGP synthesis and in the pattern of its glycosylation in the various inflammatory models.


Prekallikrein activation in human, bovine, and rabbit plasmas: presence of an inhibitor in bovine plasma. Contact of human plasma with a negatively charged surface such as dextran sulfate activates prekallikrein to kallikrein, which releases the vasoactive peptide bradykinin from high-molecular-weight kininogen. The dextran sulfate-induced activation of prekallikrein at 0 degree C (assayed by its amidolytic activity on the chromogenic substate S-2302) could not be observed in either bovine or rabbit plasmas when compared to human plasma. Neither bovine nor rabbit plasma inhibited the amidolytic activity of contact-activated human plasma at 0 degrees C. The activation of prekallikrein in human plasma was significantly inhibited by the addition of bovine plasma but not by rabbit plasma. Bovine plasma (0.025 units, 1 unit = 1 ml of plasma) caused 68.8% inhibition of prekallikrein activation. Eighty percent of the inhibitory property of bovine plasma was present in the greater than 30,000-molecular-weight fraction. These results indicate the presence of an inhibitor(s) of prekallikrein activation in bovine plasma.


Comparison of oxidant production by bovine neutrophils and monocyte-derived macrophages stimulated with Brucella abortus strain 2308. Oxidant production by bovine monocyte-derived macrophages and neutrophils was compared after stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ), and B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera. Neutrophils responded more rapidly to all stimuli and produced up to 100-fold greater oxidant than did equal numbers of bovine monocyte-derived macrophages. Macrophages and neutrophils stimulated with PMA, OZ, and reactor-opsonized B. abortus had higher mean oxidant production than phagocytes exposed to B. abortus opsonized with autologous sera, fetal bovine serum, or nonopsonized bacteria. Stimulation of macrophages by opsonized zymosan, buffer, and B. abortus opsonized with autologous sera, reactor serum, or fetal bovine serum resulted in low levels of oxidant production that were not significantly different. Only PMA caused a significantly higher level of oxidant production by macrophages.


Arachidonic acid metabolites: effects on inflammation of fetal rabbit excisional wounds. Uncovered fetal rabbit excisional wounds do not exhibit any classic signs of healing; wounds covered with an impermeable cover do contract, reepithelialize, and exhibit inflammation. Prostaglandin E2 (PGE2) is elevated in amniotic fluid, acting as an immunosuppressant at the maternal-fetal interface. Full-thickness excisional wounds were made on 25-day gestational age rabbit fetuses. Half the wounds were covered with an impermeable cover. Tissue from covered, uncovered, and nonwounded fetuses was examined 72 h after wounding for arachidonic acid metabolites. Uncovered wounds had significantly (P less than or equal to 0.05) elevated levels of PGE2, PGF2 alpha, and 12-HETE versus covered wounds and control tissue. Covered wounds had significantly elevated levels of 15-HETE compared to uncovered and control tissue. The elevated PGE2 in uncovered wounds may act as a fetal immunosuppressant; covered wounds (lower PGE2) developed cellular inflammation. Further investigations of these interactions may permit modulation of adult inflammation.


Hematocrit and whole blood viscosity in newborns: analysis of 100 cases. Hematocrit (Hct) and whole blood viscosity was studied at a mean age of ten hours in 100 neonates. Group A (n = 25), were term normal newborns, Group B (n = 25) were preterms, Group C (n = 20) were term small for gestation (SGA) and Group D (n = 30) had perinatal hypoxia. Blood viscosity was estimated in all cases at shear rates 94.5, 51.2, 20.4 and 8.1 and intergroup variability in viscosity compared at shear rate 51.2. The mean hematocrit (Hct) (59.4%) and viscosity (8.2 cps) was higher in Group A as compared to other groups, but the difference was not significant (p greater than 0.05). The upper limit of viscosity in Group C (11.9 cps) was higher than in all other groups but this difference was also not significant (p greater than 0.05). With decrease in shear rates a reciprocal increase in viscosity was noted in all four groups. Seventeen neonates (17%) had polycythemia of which eight (47.5%) were SGA. Twelve per cent preterms were polycythemic. Only 3% of neonates had hyperviscosity. The mean Hct and viscosity of the 17 cases with polycythemia was 70.9 and 9.21 cps, respectively, which was significantly higher than mean Hct and viscosity of Group A (p less than 0.05). Partial exchange transfusions were done in five neonates with Hct greater than 75%, of which only one had hyperviscosity. Post-exchange viscosity was not estimated. Whereas, three neonates with polycythemia were symptomatic, none of these had hyperviscosity. A linear correlation between Hct and viscosity was observed (r = 0.67).


Correlation of plasma color index with serum bilirubin in neonatal hyperbilirubinemia. In this study, a simple method was evaluated for serum bilirubin estimation in neonates. The color of plasma was compared with various dilutions of potassium dichromate. Readings were then compared with serum bilirubin levels obtained by diazo reaction. The coefficient of correlation (r = 0.7947) between the two was statistically significant. It is concluded that the color index provides reliable estimates of serum bilirubin levels.


Reliability of capillary blood gas estimation in neonates. In order to compare the reliability of capillary blood gases to the arterial blood gases, we studied fifty one neonates with moderate birth asphyxia. A significant difference (p less than 0.05) was found between the capillary and the arterial blood gas values with respect to blood pH, PCO2 PO2 and oxygen saturation. However, the levels of blood bicarbonate as assessed by the 2 samples were comparable. Capillary blood gas values are unsatisfactory indicators of the arterial blood gas values and may result in inappropriate management.


Thermoregulatory alterations as a marker for sepsis in normothermic premature neonates. To evaluate the core-peripheral temperature alterations as a marker for sepsis in normothermic premature newborns, 50 normal term neonates and 11 preterms with sepsis and 11 normal preterms (controls) were studied. Axillary, rectal and sole temperatures were recorded in all babies using a single mercury-in-glass thermometer by a single observer. There was significant widening of the rectal-sole and axillary-sole temperatures in the preterms with sepsis (p less than 0.001). There was no significant difference (p greater than 0.05) between the axillary and rectal temperatures in the term, normal preterms or those with sepsis. With an overall accuracy of 90.9%, a rectal-sole temperature difference of greater than or equal to 2.3 degrees C (100% sensitivity) or greater than or equal to 3.2 degrees C (100% specificity) is a useful marker to differentiate normothermic preterms with or without sepsis. Using the axillary-sole temperature difference, the respective values were greater than or equal to 2.2 degrees C and greater than or equal to 3.0 degrees C.


Effect of pregnancy associated hypertension on immunoglobulin levels in newborns. In a prospective study of 124 neonates born to mothers with normal pregnancy and pregnancy associated hypertension (PAH), serum IgG, IgA and IgM were estimated by single radial immunodiffusion technique. Significantly low levels of IgG were found in mothers having PAH, as compared to normal pregnancy (p less than 0.001), whereas IgA and IgM showed no difference in the two groups. There was no statistical difference in maternal and cord blood IgG in either the control or study group. IgG was significantly higher (p less than 0.001) in cord blood of babies born by vaginal route as compared to forceps (via vaginal route) or cesarean section. IgA and IgM levels did not vary with mode of delivery.


A new transport incubator for primary care of low birth weight babies. A new portable, cheap and indigenous incubator made of polystyrene has been devised for delivery of primary health care services to the newborn babies in the community. Twenty six babies with a mean weight of 1726 g (range 1388-1981g) and gestational age of 35.3 weeks (range 34-38 wks) were continuously evaluated for 2 hours observation period, in naked and clothed conditions. Rectal, abdominal skin, foot, ambient air and nursery temperatures were recorded. The baseline core temperature of the babies was 36.58 (+/- 0.21) degrees C; after incubator care it was recorded s 36.80 (+/- 0.10) degrees C in naked infants. The baseline core temperature of the clothed babies was 36.63 (+/- 0.21) while it was 37.01 (+/- 0.18) after 2 hours of incubator care. An ambient air temperature of 33-34 degrees C in the incubator (thermoneutral temperature range for these babies being 31.0-33.8 degrees C) was achieved within 30-60 minutes of incubator stay (nursery temperature being 28 +/- 0.6 degrees C). No evidence of carbon dioxide narcosis, hypoxia, acidosis, or adverse thermoregulatory behavior was observed. One baby had hypoglycemia (blood sugar less than 35 mg/dl) and another had sweating. There is a scope for providing additional facilities like administration of oxygen, phototherapy, X-rays through the incubator without disturbing the baby.


Growth and behavior development in rural infants in relation to malnutrition and environment. A group of 224 children from a rural cohort of 625 children registered from 1981 to 1983 in 10 villages of KV Block, Varanasi was assessed for morbidity, physical growth, and behavior development (Gesell's developmental schedule). By first birthday children of normal nutrition grade were reduced to one fourth and numbers in Grade II and III malnutrition doubled. This deterioration in nutritional status was probably due to high morbidity, i.e., gastrointestinal, respiratory infections, etc. The skull circumference was 43 cm at the age of one year, being lower by 3 cm than the average size. Children having Grades II and III malnutrition showed poor development in all the areas of behavior, i.e., motor, adaptive, language and personal social. Besides malnutrition, environmental factors like mother's involvement in teaching, encouraging the child, talking to him or being within the visual range; the parental education, their caste and the child's birth order contributed significantly to the development of the child during infancy.


Anti-nuclear antibody positivity in multi-transfused thalassemia major. The frequency of anti-nuclear antibodies (ANA) was evaluated in multi-transfused patients of thalassemia major. Twelve out of 83 patients (14.5%) had positive ANA at titres of 1:80 or above. The results were compared with age and sex matched healthy controls who showed positive results in only 1 of 52 cases (1.9%; p less than 0.05). Antibody against double stranded DNA was absent. ANA positivity was found to correlate with higher age (p less than 0.01), more amount of blood transfused (p less than 0.01), splenectomy status (p less than 0.01), higher levels of serum ferritin (p less than 0.01) and presence of hepatitis B surface antigen (p less than 0.01) and antihepatitis C antibody (p less than 0.01).


Clinical and etiological profile of acute viral hepatitis. There is a paucity of data on the incidence of sporadic viral hepatitis in Indian children. Clinical, biochemical and etiological profile of 54 patients with acute viral hepatitis was evaluated. Of these, 32 (59.25%) patients had Hepatitis A, 18 (33.33%) had NANB, 2 (3.7%) had Hepatitis B and 2 (3.7%) concurrent Hepatitis A and B infection. It was not possible to distinguish the etiological agents on the basis of the clinical and biochemical profile. Fulminant hepatitis was documented in 8 (14.8%) cases. Children with NANB infection were at a greater risk (p less than 0.05) of developing fulminant hepatitis as compared to Hepatitis A infection.


Health effects of consanguinity in Pondicherry. Of 1000 pregnant women studied, consanguinity was observed in 30.8%, with a higher frequency among women from rural areas, and among Hindus. In the consanguineous group, first cousin marriages were present in 47.4%, and uncle niece marriages in 23.4% of women. Coefficient of inbreeding was highest in Harijans (0.0258), followed by non-Brahmins (0.0220) and Brahmins (0.0204). Fertility was not influenced by consanguinity. There was a significantly higher rate of still births and infant mortality in consanguineous matings as compared with non-consanguineous. Total morbidity was higher in the consanguineous group as compared with the non-consanguineous (p less than 0.01), especially that due to neonatal infections and jaundice. There was no significant difference in the prevalence of congenital malformations, chromosomal and genetic disorders between the two groups, although the number of abnormal births in this group was small.


Neonatal gastrointestinal perforations.

Neonatal gastrointestinal perforations. Fourteen neonates presented with clinical and radiological evidence of gastrointestinal perforation, of which 13 were treated by operative intervention. No infant was formula fed, had rectal bleeding or radiological evidence of pneumatosis intestinalis. Operative findings revealed localized perforation in ten of which three were gastric, two distal ileal, three cecal and two in sigmoid colon. Gangrene with extensive perforation of jejunum, ascending colon and transverse colon were seen in one each. Blood cultures were positive in four, one grew E. coli and three Klebsiella while peritoneal cultures were positive in six, one grew E. coli and five Klebsiella. Ten neonates survived and are being followed up. Three of the four cases who died had white blood cell count greater than 25 x 109/L and grew Klebsiella on peritoneal culture. Factors predisposing to gastrointestinal perforations in neonates are discussed, emphasis is made on the cautious use of umbilical, gastric and rectal catheters, and the need for early surgical intervention.


Nosocomial rotavirus diarrhea in two medical wards of a pediatric hospital in Calcutta. One hundred eighty nine children suffering from different medical problems were admitted in two wards of a pediatric hospital in Calcutta during the period between November 18, 1985 and February 10, 1986. Amongst them, 36 children developed nosocomial diarrhea and rotavirus was detected from 80.5% of the cases. The nosocomial rotavirus diarrhea cases had lesser frequency of stools and only mild dehydration but the course of illness was longer in comparison to that of the hospitalized rotavirus diarrhea cases. There is a possibility of spread of infection via fomites, environmental surfaces and most likely mothers.


Causes of mortality in children with acute lymphocytic leukemia. Fifty five deaths between January, 1982 to September, 1989 in children with acute lymphoblastic leukemia (ALL) were evaluated to determine the cause of mortality. Fifty cases died during remission. Infection alone was responsible for death in 26 of 55 (47.3%) cases while hemorrhage was seen in 7 (12.7%) children. Infection and hemorrhage together were responsible in another 13 cases. Gastrointestinal tract and pulmonary system were the major sites of bleeding. Infections either alone or in combination with other factors were responsible for death in 42 of 55 (76.5%) of children. Septicemia (n = 11), gastrointestinal (n = 15) and pulmonary infections (n = 10) and meningitis in 2 cases were the major sites of infections. Pseudomonas and Klebsiella in 6 cases each accounted for 54.5% of isolates.


Blood pressure levels in south Indian adolescents. The present study was undertaken to develop normative data for the blood pressure (BP) levels in adolescent boys and girls aged 10-17 years residing the rural and urban areas of North Arcot Ambedkar District, Tamil Nadu State. Blood pressures were evaluated as part of an ongoing study on growth and development in adolescents. A total of 7028 BP measurements collected cross-sectionally were subjected to statistical analysis. The mean systolic (SBP) and diastolic (DBP) blood pressure levels were elevated in rural children as compared to that in urban children of respective sexes during 10 and 15 years of age. The mean BP levels were higher in both the rural and urban girls as compared to their male counterparts till the age of 15, after which the trend was reversed. While the mean SBP and DBP levels for rural children of either sex were stable over the age range considered, those for urban children exhibited consistent increase with age. In urban boys, the yearly mean increments in SBP and DBP were 2.5 mm Hg and 2.0 mm Hg and that in urban girls 2.4 mm Hg and 1.7 mm Hg, respectively. It is suggested that perhaps stress factors are responsible for the elevated mean BP levels among the rural adolescents as they are generally engaged in activities that exert considerable physical and psychological stress.


Age independent anthropometric index for identifying undernourished children in the age group 5-10 years. An age-independent anthropometric index to identify undernourished children in the age group of 5-10 years is described. Employing a mathematical approach (linear regression analysis) on data from 238 children, the index was derived to be wt/(ht)2. Employing this index, a classification rule for identifying the undernourished was evolved, using as the 'Gold standard' a WHO classification based on NCHS standards. The sensitivity of the classification rule was 95%, the specificity was 92% and the overall efficiency was 92%.


Errors in medication in a pediatric ward. Iatrogenic errors in medication were studied in a busy pediatric ward. The study was based on voluntary reporting of errors noticed by the doctors and nurses in the ward. The error rate was 6.4%. Prescription errors accounted for 37.7% of the errors and 2 of these were potentially fatal. Dispensing errors and missed dosages were other frequent errors. Overcrowding in pediatric wards is an obstacle to optimum patient care. Adequate number of nurses and support of pharmacists is essential for safe and optimum drug therapy.


Prevalence of SSPE: a serological study. The presence of measles antibodies in serum and cerebrospinal fluid (CSF) of 340 samples from children clinically suspected of subacute sclerosing panencephalitis (SSPE) were studied. One hundred and thirty eight (40%) of these children had SSPE based on the serological evidence. The mean age group of children affected was 8.2 years. The M:F ratio was 5:1. The titres of antibodies ranged from 1:2 to 1:32 in the CSF and from 1:16 to 1:512 in the serum.


Frequency and significance of electrolyte abnormalities in pneumonia. To determine the frequency of electrolyte disturbance in pneumonia, we studied 264 hospitalized children with pneumonia for serum sodium and potassium concentration, and plasma osmolality (Posm) on the day of admission. Urine osmalality (Uosm) and urine spot sodium concentration were measured in those who had a serum sodium less than or equal to 130 mEq/L. Hyponatremia was found in 27%, hypernatremia in 3.7%. Hypokalemia (serum potassium less than or equal to 3.5 mEq/L) in 19 and 2% had hyperkalemia (serum potassium greater than or equal to 6.5 mEq/L). Of all the hyponatremia, 68% were secondary to syndrome of inappropriate ADH secretion (SIADH) as suggested by a concomitant lowering of Posm less than or equal to 280 mosm/kg and increased urinary osmolality and sodium excretion. Hyponatremia was associated with 60% longer hospital stay, two fold increase in complications and the 3.5 times higher mortality compared to that of normonatremia. The above variables were affected further, if hypokalemia coexisted with hyponatremia.


Evaluation of pediatric training of interns in a teaching hospital. This study was undertaken in a teaching hospital with residency system to evaluate Pediatric teaching of interns admitted to MBBS course (using the questionnaire method) and to find out ways to improve the existing system. It was seen that the present system increased the Pediatric knowledge of interns significantly (p less than 0.001). The study highlights what the contents of the course should be from the learners' point of view, the hindrances to their learning and some suggestions to improve training in future.


Staphylococcal alpha toxin: a study with chronically instrumented awake sheep. The in vivo responses to staphylococcal alpha toxin are reported for 15 chronically instrumented awake yearling sheep. The data obtained from a total of 30 experiments are grouped into four categories of response: no response, noted in seven experiments done on 5 sheep; pressor response, obtained seven times in 4 sheep; fluid and solute exchange, noted on six occasions in 3 sheep; and acute heart failure and death, which occurred in 10 of the 15 sheep. "No response" denoted no change in any of the measured outcome variables. The group of sheep labeled as showing "pressor response" responded to alpha toxin infusion with an increase in pulmonary artery pressure, unaccompanied by changes either in lung lymph flow or in lung mechanics. "Changes in lung fluid and solute exchange" involve increases in lung lymph flow. The harbinger of the last category, acute left heart failure leading to death, was a marked elevation in left atrial pressure. The threshold response dose in sheep is approximately 21 micrograms/kg. A very steep dose-response curve is observed, with only a narrow window of doses, 15 to 25 micrograms/kg, between the group showing no response and the group showing death from acute heart failure. The data obtained in these studies indicate that the lethal effects of alpha toxin in sheep include acute heart failure, which may be due to direct toxicity to heart muscle and/or the coronary vasculature endothelium.


Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora. Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.


Molecular population genetic evidence of horizontal spread of two alleles of the pyrogenic exotoxin C gene (speC) among pathogenic clones of Streptococcus pyogenes. It has recently been demonstrated that the bacteriophage-borne gene (speC) encoding pyrogenic exotoxin C is harbored by phylogenetic lineages representing virtually the entire breadth of genomic differentiation present in the species Streptococcus pyogenes (J. M. Musser, A. R. Hauser, M. H. Kim, P. M. Schlievert, K. Nelson, and R. K. Selander, Proc. Natl. Acad. Sci. USA 88:2668-2672, 1991). To determine whether the speC genes occurring in association with divergent chromosomal genotypes (clones) are identical or represent a group of allelic variants, we sequenced speC from 23 S. pyogenes strains representing 15 clones identified by multilocus enzyme electrophoresis. Two alleles of speC are present in natural populations, and each allele occurs in clones that are well differentiated in overall chromosomal character; in one case, isolates of a single clone had different speC alleles. We interpret these patterns of toxin allele-clone distribution as evidence of occasional episodes of speC horizontal dissemination, presumably by bacteriophage-mediated gene transfer and recombination.


Elimination of the vitamin B12 uptake or synthesis pathway does not diminish the virulence of Escherichia coli K1 or Salmonella typhimurium in three model systems. The role of iron in infection is of great importance and is well understood. During infection, both the host and the pathogen go through many complicated changes to regulate iron levels. Iron and vitamin B12 share certain features. For example, Escherichia coli has similar transport systems for both nutrients, and binding proteins for both are located in gastric juice, liver, saliva, granulocytes, and milk. It is because of such parallels between iron and B12 that we have explored the role of B12 in virulence. A btuB::Tn10 insertion which disrupts the gene encoding the vitamin B12 receptor from E. coli K-12 was P1 transduced into a virulent E. coli K1 strain. In both an infant-rat model and a chicken embryo model, no difference in virulence between the wild-type and the mutant strains was found. Strains of Salmonella typhimurium with mutations in the cobalamin synthesis pathway (Cob) and in btuB were used in a mouse model of virulence. Mutation of the Cob locus or of btuB does not decrease virulence. Interestingly, the inability to synthesize vitamin B12 actually increases virulence compared with the wild type in the S. typhimurium model. This effect is independent of the B12 intake of the mice.


Granulocyte-macrophage colony-stimulating factor increases the infectivity of Leishmania amazonensis by protecting promastigotes from heat-induced death. We have studied the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the infectivity of promastigotes of Leishmania amazonensis, an obligate intramacrophage parasite. We measured the capacity of the promastigotes to infect macrophages after preincubation at different temperatures (28, 34, and 37 degrees C) with recombinant murine GM-CSF, as well as the effect of an anti-murine GM-CSF antibody on the in vitro and in vivo infectivity of the parasite. GM-CSF increases the capacity of the promastigotes to infect cells when preincubated at 34 and 37 degrees C, whereas the anti-GM-CSF antibody exerts the opposite effect: it decreases the internalization rate and the progression of infection in macrophage cultures and slows the growth of the lesion in infected BALB/c mice. Neither of the described effects were observed when the in vitro and in vivo infections were made with amastigotes. Promastigotes die in a time-dependent manner when incubated at temperatures higher than 28 degrees C in the absence of GM-CSF. They are protected from this heat-induced death by incubation with the recombinant hormone. Our interpretation of these data is that the increase in the infectivity of promastigotes when incubated with GM-CSF at the temperatures at which infection occurs (34 and 37 degrees C) is due to the larger number of surviving forms within the infecting population. The decrease in infectivity when they are incubated with the antibody is due to inhibition of the protection conferred by the GM-CSF produced by the macrophages during the in vitro and in vivo infections.


Synthesis of a conjugate vaccine composed of pneumococcus type 14 capsular polysaccharide bound to pertussis toxin. Type 14 is one of the common types isolated from patients of all ages with infections caused by Streptococcus pneumoniae. Its capsular polysaccharide (Pn14) is composed of a neutrally charged tetrasaccharide repeat unit. Pn14 does not elicit protective levels of antibodies in infants and children and is a less than optimal immunogen of the 23-valent vaccine for adults. Pertussis toxin (PT) is both a virulence factor and protective antigen of Bordetella pertussis: it is not soluble at neutral pH and forms insoluble complexes with acidic polysaccharides. Both Pn14 and PT are potential components of vaccines for infants and children. Accordingly, a synthetic scheme was devised to prepare a conjugate of Pn14 and PT. An adipic acid hydrazide derivative of Pn14 was bound to PT at pH 3.9 by carbodiimide-mediated condensation. The conjugation procedure inactivated the PT as assayed by CHO cell and histamine-sensitizing activity. The Pn14-PT conjugate elicited antibodies in mice to Pn14 at levels estimated to be protective in humans and elicited neutralizing antibodies to PT. We plan to evaluate Pn14-PT clinically.


Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin. Vibrio parahaemolyticus produces a thermostable direct hemolysin (TDH) that has been implicated in the pathogenesis of diarrheal disease caused by this organism. However, previous studies attempting to demonstrate the contribution of the hemolysin to virulence have been inconclusive. We investigated this putative virulence factor by using an isogenic TDH-negative (TDH-) strain constructed by specifically inactivating the two copies of the tdh gene encoding TDH. The enterotoxigenicities of the parent strain (AQ3815) and the mutant strain were tested by adding sterile culture supernatants to rabbit ileal tissue mounted in Ussing chambers. The culture filtrate of the parent strain produced a significant increase in short-circuit current (Isc), compared with the change induced by the TDH- mutant. The capacity of the culture filtrate of AQ3815 to increase the Isc was reduced by neutralization with anti-TDH serum, and the return of the cloned tdh gene to the TDH- mutant restored the ability to increase the Isc. These results were corroborated by rabbit ileal loop assays in which AQ3815 caused fluid accumulation but the TDH- mutant did not. No microscopic damage was seen in mucosal tissues exposed to the culture filtrate of either strain. These results indicate that TDH has an enterotoxigenic effect on rabbit small intestine and could be responsible for the watery diarrhea seen with V. parahaemolyticus.


Heterogeneity of phenol oxidases in Cryptococcus neoformans. Phenol oxidase enzymes, linked to virulence in Cryptococcus neoformans, were prepared from broken cells. More enzyme activity was found in the ultracentrifugation supernatant; less was found in the membrane fraction. Phenol oxidases were located in acrylamide gel electropherograms by activity staining with L-dihydroxyphenylalanine (DOPA). Mobility differences between soluble and solubilized membrane-bound phenol oxidases were not found. Comparison of enzymes produced at 25 and 37 degrees C revealed that the enzyme had lower activity and lower mobility at 37 degrees C. The mobility of 25 degrees C phenol oxidases from strains of C. neoformans var. gattii was lower than that of those from C. neoformans var. neoformans. Half of the phenol oxidase produced at 25 degrees C was bound by concanavalin A, while that produced at 37 degrees C was not bound. However, glucose starvation of cultures at 25 degrees C overnight resulted in increased amounts of enzyme which did not bind to concanavalin A. A given strain of C. neoformans produces different species of phenol oxidase under different culture conditions.


Cryptdins: antimicrobial defensins of the murine small intestine. Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria. Recent studies by Ouellette and associates (A. J. Ouellette, R. M. Greco, M. James, D. Frederick, J. Naftilan, and J. T. Fallon, J. Cell Biol. 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported. We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells. Both peptides contained 35 amino acid residues, including the characteristic defensin "signature" of six invariantly conserved cysteines. Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J. Cell Biol. 108:1687-1695, 1989). Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro. Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages. Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S. typhimurium 14028S. Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties. The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L. monocytogenes and Salmonella species.


Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice. The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.


Protective effect of a T-cell-dependent immunosuppressive, B-cell-mitogenic protein (F3'EP-Si, or P90) produced by Streptococcus intermedius. The role of a previously described bacterial protein (F3'EP-Si), now designated P90, in the survival of Streptococcus intermedius in the host was investigated, and the immunosuppressive and B-cell-mitogenic effects of this protein were further characterized. C57BL6 mice treated with P90 were about 50 times more susceptible to infection with this bacterium than untreated mice. One of seven splenocytes of C57BL/6 mice were activated by P90. Marked splenomegaly was observed in mice treated with P90, with increased numbers of splenic mononuclear cells and polyclonal immunoglobulin-secreting plaque-forming cells. Peak responses were seen on day 3 for immunoglobulin M (IgM) and on day 5 for IgG, with an isotypic pattern consisting predominantly of IgG2a and IgG2b. When mice were treated with P90 before being primed with sheep erythrocytes, polyclonal immunoglobulin synthesis was accompanied by an ephemeral stimulation of the specific immune response against sheep erythrocytes that was quickly replaced by a dramatic immunosuppression. In contrast, when mice were treated with P90 after being primed, the polyclonal activation was comparatively much less evident and there was no suppression of the specific immune response. Immunosuppression was considerably reduced in mice thymectomized as adults or depleted of CD8+ cells. Adoptive transfer experiments showed that B cells obtained from P90-treated mice were less able to respond to an antigenic challenge, even in the presence of normal T cells, and that T cells obtained from P90-treated mice could actively suppress the specific immune response of normal B cells.


Lymphokine-activated killer cell regulation of T-cell-mediated immunity to Candida albicans. Monocytes are important accessory cells in the activation of T cells for specific antigen recognition yet little is known of their regulation. We demonstrated here that interleukin-2 (IL-2)-induced human lymphokine-activated killer (LAK) cells can inhibit monocyte antigen presentation, depending on the state of differentiation of the monocytes. Adherent monocytes cultured for 4 days in medium or granulocyte-macrophage colony-stimulating factor (GM-CSF) were found to equally process and present intact Candida albicans to autologous Percoll gradient-isolated T cells, as measured by [3H]thymidine uptake. However, only the GM-CSF-cultured monocytes were functionally inhibited by autologous 4-day IL-2-induced LAK cells. Even soluble candidal cell wall mannoprotein antigens could not be presented by these monocytes after exposure to LAK cells. Pretreatment of these monocytes with LAK cells for 1 h, followed by subsequent removal of the nonadherent LAK cells, was sufficient to cause significant inhibition, with maximal inhibition observed after 4 h. Northern (RNA) blot analysis indicated that mRNA expression for IL-1 alpha and IL-1 beta in response to C. albicans stimulation was also down-regulated in GM-CSF-cultured monocytes exposed to LAK cells. Interestingly, freshly isolated, Percoll gradient-purified large granular lymphocytes did not suppress antigen presentation in GM-CSF-treated monocytes. Another important finding was the inability of LAK cells to suppress the ability of freshly isolated or gamma interferon-cultured monocytes, which are resistant to LAK cell-mediated lysis, to present antigen to T cells. In contrast, IL-3 was similar to GM-CSF in inducing LAK cell susceptibility in monocytes. Taken together, these results indicated that IL-2 can induce LAK cells to down-regulate antigen presentation function in a select set of monocytes that have been activated by colony-stimulating factor (GM-CSF and IL-3) but not by gamma interferon. LAK cells may therefore play an important role in regulation of monocytes and their function, depending on their differentiation state.


Isolation of nonchemotactic mutants of Campylobacter jejuni and their colonization of the mouse intestinal tract. Three nonchemotactic mutants (D54, Y14, and N74) of Campylobacter jejuni were isolated from wild-type strain FUM158432 by either the negative swarming or liquid gradient method with brucella broth as the attractive substance. Strains D54 and Y14 were isolated after mutagenesis with methyl methanesulfonate, and N74 was isolated from a nonmutagenized culture. These mutants all failed to swarm on a semisolid medium and did not show any chemotactic behavior in the hard-agar plus assay method for any of the chemicals which act as attractants for the wild-type strain. They had intact flagella and were actively motile. Swimming behavior examined by a video tracking technique showed that the mutants swim only straight, without any tumbling. When suckling mice were challenged orally with approximately 10(5) CFU of these mutant strains, all of the mutants were cleared from the intestinal tract by 48 h. In contrast, the wild-type strain colonized the intestinal tracts of all mice challenged with 10(2) CFU. We concluded that chemotactic movement is important for colonization of the intestinal tract of suckling mice by C. jejuni.


Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci. Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.


Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes. Listeria monocytogenes is able to escape from the phagolysosome and grow within the host cell cytoplasm. By 3 h after initiation of infection, actin filaments begin to concentrate at one end of the bacterium. Polarization of F-actin is associated with intracellular bacterial movement, long projections of actin filaments forming directly behind the moving bacteria. New actin monomers are added to the region of the projection in proximity to the bacterium. The rate of new actin filament growth correlates closely with the speed of bacterial migration. This actin structure is anchored within the cytoplasm, serving as a fixed platform for directional expansion of the actin filament network. The actin projection progressively lengthens as the bacterium migrates. Cytochalasin blocks both elongation of the projection and bacterial movement but does not result in complete depolymerization of the bacterially induced actin structure, residual actin and alpha-actinin persisting in proximity to one end of the bacterium. Bacteria initially migrate within the cortical cytoplasm but later move to the peripheral membrane, where they form filopodiumlike structures which pivot and undulate in the extracellular medium. In the filopodia, bacteria are occasionally seen to abruptly change direction, turn 180 degrees, and move back into the medullary region of the host cell. All filopodium movement ceases once the bacterium containing the F-actin projection returns to the cortical cytoplasm. These results indicate that host cell actin polymerization is necessary for intracellular migration of listeriae and suggest that directional actin assembly may in fact generate the propulsive force for bacterial and filopodial movement.


Molecular analysis of the serotyping antigens of Neisseria meningitidis. Molecular approaches to the rapid analysis of the serotyping antigens of Neisseria meningitidis, the class 2 and 3 outer membrane proteins (OMPs), were developed, evaluated, and used to study 12 antigenic variants of these proteins. A primer set for the polymerase chain reaction (PCR) amplification of the genes encoding these antigens was devised. Low-stringency amplification of meningococcal chromosomal DNA with this primer set resulted in the amplification of two products from each strain, whereas at higher stringencies only one product was amplified in most strains. Southern hybridization techniques and restriction analyses were used to differentiate the PCR products amplified at high stringencies from strains expressing class 2 or class 3 OMPs; these PCR products were further characterized by the determination of their nucleotide sequences, confirming that they represented the amplified class 2 and class 3 OMP genes. Analyses of these and other nucleotide sequences enabled the construction of a phenogram illustrating the interrelationships between Neisseria OMP genes. The comparative analysis of deduced amino acid sequences revealed conserved and variable regions of the proteins; the latter probably correspond to surface loops on the protein and hence are potentially exposed to the immune system. Further analyses of the primary structures of these related porins from Neisseria species enabled construction of models of the secondary structure of these antigens and comparison of these models with those previously published. The methods reported in the present work are rapid reproducible procedures for the analysis of antigenic variants of these proteins.


Borrelia burgdorferi stimulates release of interleukin-1 activity from bovine peripheral blood monocytes. Infection with Borrelia burgdorferi is suspected to be a cause of lameness and arthritis in cattle. Interleukin-1 (IL-1) activity has been detected in joint fluids from human patients affected by various arthritides, including Lyme arthritis. In addition, human monocytes and murine macrophages have been reported to release IL-1 activity when incubated with B. burgdorferi in vitro. To address a possible mechanism by which B. burgdorferi might cause a bovine arthritic syndrome, we determined whether bovine peripheral blood monocytes released IL-1 activity when coincubated with B. burgdorferi in vitro. High-passage and low-passage isolates of B. burgdorferi stimulated release of IL-1 activity from bovine monocytes. The amount of IL-1 activity released was dependent on the number of borreliae added to the monocyte cultures. In addition, live and heat-killed B. burgdorferi cells stimulated release of similar amounts of IL-1. We also obtained no evidence that soluble components released from in vitro-cultured B. burgdorferi stimulated IL-1 release from bovine monocytes. A recombinant IL-1 receptor antagonist blocked the proliferative activity of monocyte-conditioned medium in a thymocyte costimulation assay, thus demonstrating that the costimulatory activity detected was due to IL-1.


Contributions of complement and immunoglobulin to neutrophil-mediated killing of enterococci. Enterococci have become a frequent causative agent in neonatal sepsis. The relative contributions of antibody and complement and their interactions in the neutrophil-mediated bacterial killing of 11 Enterococcus strains from neonates were investigated. Polymorphonuclear leukocytes (PMNL) from adult and term newborn infants were tested with normal human serum, adult hypogammaglobulinemic serum, and normal newborn serum in a neutrophil bactericidal assay. Neutrophil bactericidal activity for enterococci was not influenced by the serum source but was essentially ablated after heat inactivation of complement in all sera. No differences were observed in the killing capacity of healthy newborn versus adult PMNL regardless of serum source. Representative Enterococcus strains were then tested with agammaglobulinemic serum or C4-deficient serum, resulting in neutrophil bactericidal activities consistently exceeding 90%. A neutrophil bactericidal assay performed with normal rabbit serum and hyperimmune rabbit serum against enterococci showed that antibodies to enterococci enhanced neutrophil-mediated killing of this organism. Thus, neutrophil killing of enterococci appears to be mediated primarily by complement, with antibody playing a less essential but potentially important role. PMNL from adult and healthy term infants functioned with equal efficiency in the neutrophil killing of enterococci.


Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni. Our laboratories previously isolated a putative extracellular or membrane-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene, designated a signal peptide-containing (SP) Cu/Zn-SOD, from Schistosoma mansoni. SOD activity was thus investigated throughout the life cycle of S. mansoni and found in all stages: eggs, miracidia, cercariae, schistosomula, lung-stage worms, and adult worms. The adult worms had the highest SOD activity (53 +/- 9 nitrite units), which was five times higher than that of eggs or miracidia and twice as high as that of 3-h-old mechanically transformed schistosomula. Cu/Zn-SOD constituted over 95% of the total SOD activity found in S. mansoni, compared with that of Mn-SOD. Most of Cu/Zn-SOD specific activity was associated with a detergent-extractable fraction of the parasite. Isoelectric focusing gel electrophoresis analysis revealed that there were four major pI variants of Cu/Zn-SOD present in the adult worms. Only two of these Cu/Zn-SOD pI variants were present in the 3-h-old mechanically transformed schistosomula. Fast protein liquid chromatography gel filtration fractionation of adult parasite extract was carried out to correlate the SP Cu/Zn-SOD with the SOD activity by using anti-SP Cu/Zn-SOD monoclonal antibodies, which separated the immunoreactive gene product and the SOD activity into different fractions. Quantitative tissue fractionation also revealed a discordant distribution of the gene product compared with that of Cu/Zn-SOD activity. These results indicated the existence of another Cu/Zn-SOD(s) in the parasite. Purification of the Cu/Zn-SOD activity from the adult worms showed that it represented the two lower-pI variants found in both adult worms and 3-h-old schistosomula. Peptide sequence analysis of the purified Cu/Zn-SOD confirmed that there is a second form of Cu/Zn-SOD in the parasite.


Characteristics of Helicobacter pylori variants selected for urease deficiency. The urease of Helicobacter pylori is suspected to play a role in the pathogenesis of gastritis. Although all clinical isolates of H. pylori are urease positive (U+), we have selected and characterized several spontaneously arising urease-negative (U-) variants from wild-type strain 60190. Urease-negative variants were identified by growth in medium containing 60 mM urea and arose at a frequency of 10(-5) to 10(-6). The urease activity of the wild-type strain inhibited growth of this strain in the presence of 60 mM urea. U- variants retained the U- phenotype for more than 100 passages on medium with or without urea. The urease activities of the original U+ and derived U- cells were 9.55 to 16.7 and 0.01 to 0.17 U/mg of protein, respectively. Colonial growth and other biochemical characteristics were identical for the strains. U- variants showed three classes of whole-cell sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles: (i) identical to U+; (ii) change in the migration of the 61-kDa urease subunit; and (iii) lack of 61- and 30-kDa subunits. These differences were confirmed by immunoblotting and by protein separation using fast protein liquid chromatography. The U+ strain but not U- variants tolerated exposure to pH 4.0 for 60 min in the presence of urea. Supernatants of the U+ strain and U- variants contained vacuolating cytotoxin activity for HeLa cells in similar titers. By enzyme-linked immunosorbent assay, human serum samples recognized water extract from the U+ strain significantly better than extract from a U- variant lacking urease subunits. In conclusion, this study demonstrates that U- H. pylori variants may arise spontaneously, that urease activity enhances survival at acid pH, and that urease and cytotoxin activities are disparate phenotypes.


Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids. Lipoteichoic acids (LTAs) from various bacterial species, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Listeria monocytogenes, were examined for the ability to induce secretory and cellular responses in a pure population of bone marrow-derived mononuclear phagocytes. Some of the highly purified LTAs, in particular LTAs from Bacillus subtilis, S. pyogenes, E. faecalis, and Enterococcus hirae, were able to affect each of the macrophage parameters measured, i.e., reductive capacity, secretion of tumor necrosis factor and nitrite, and tumoricidal activity. As after stimulation with whole organisms or other bacterial products, secretion of tumor necrosis factor induced by these LTAs reached its maximum within the first few hours of the interaction, while secretion of nitrite and tumoricidal activity required 24 to 36 h for full expression. Other purified LTAs, i.e., LTAs from Streptococcus sanguis, S. pneumoniae, and L. monocytogenes, as well as lipomannan from Micrococcus luteus affected only some of these parameters, while native LTA from S. aureus was inactive. There was no obvious correlation between biological activity and chain length, kind of glycosyl substituents, glycolipid structures, or fatty acid composition of LTAs. Deacylation of LTAs resulted in a complete loss of activity, and deacylated LTAs did not impair the activity of their acylated counterparts, suggesting that acyl chains may be essential for binding of LTA to the cell surface. The results demonstrate that some LTA species are potent inducers of macrophage secretory and cellular activities.


Characteristics and cariogenicity of a fructanase-defective Streptococcus mutants strain. Polymers of D-fructose produced by a variety of oral bacteria are believed to function as extracellular carbohydrate reserves. Degradation of these polysaccharides in plaque following exhaustion of dietary carbohydrates is thought to contribute to the extent and duration of the acid challenge to the tooth surface and thus to the initiation and progression of dental caries. Streptococcus mutans produces a fructanase, the product of the fruA gene, which is capable of degrading beta(2,6)- and beta(2,1)-linked fructans that are commonly synthesized by dental plaque microorganisms. To evaluate the role of the FruA protein in exopolysaccharide metabolism and to assess the contribution of this enzyme to the pathogenic potential of S. mutans, a fructanase-deficient strain of S. mutans was constructed. Inactivation of a cloned fruA gene was accomplished in Escherichia coli by using a mini-Mu dE transposon, and then an isogenic mutant of S. mutans UA159 was constructed by allelic exchange. Successful inactivation of fruA was confirmed through the use of biochemical assays, Western blotting (immunoblotting) with anti-recombinant FruA antisera, and Southern hybridization. The data indicated that FruA was the only fructan hydrolase produced by S. mutans UA159. Inactivation of fruA had no significant effects on glucosyltransferase or fructosyltransferase activity. In the rat caries model using animals fed a high-sucrose diet and ad libitum, there were no significant differences in the number or severity of smooth surface, sulcal, or root caries elicited by the fruA mutant and the wild-type organism.


Experimental model of intracerebral infection with Cryptococcus neoformans: roles of phagocytes and opsonization. A murine model of intracerebral (i.c.) infection with Cryptococcus neoformans in which naive mice receiving an i.c. fungal inoculation developed a severe disease has been established. The effect was strictly dependent on the number of microorganisms injected and evolved as lethal meningoencephalitis. Murine susceptibility to i.c. infection with C. neoformans was enhanced by treatment with chloroquine and colchicine, agents known to greatly affect the host phagocytic compartment. Furthermore, the life spans of both naive and drug-treated mice were significantly augmented when opsonized fungi were injected. Therefore, phagocyte-mediated mechanisms are likely involved in local resistance to i.c. infection with C. neoformans. Further support for this conclusion was supplied by in vitro data showing that microglial cells were proficient anticryptococcal effectors, provided opsonized microorganisms were used.


Group G streptococcal M protein exhibits structural features analogous to those of class I M protein of group A streptococci. We have previously studied a collection of group G streptococcal strains isolated from bacteremic human infections and demonstrated that such strains resist phagocytosis by human polymorphonuclear leukocytes but are type specifically opsonized by homologous antiserum. We have now performed Southern hybridization analysis on genomic DNA from eight blood isolates. All eight isolates showed DNA homology to a group A emm24 gene probe. The M-protein gene of one of the isolates, strain 1750, has now been isolated. This gene (emmG1) encodes a polypeptide of 67 kDa (MG1) which is reactive with antibodies to the partially purified M protein of the parent strain. The predicted amino acid structure of MG1 demonstrates significant identity with the carboxy terminus (C, D, and anchor domains) of M6 and M24 but only limited identity with the amino terminus (variable portion) of these group A M proteins. Southern hybridization of genomic DNA of the eight group G blood isolates with an emmG1 gene probe indicated there were at least four emm alleles associated with these strains. These studies indicate that M proteins of group G streptococci, like those of group A, are genetically heterogeneous. Moreover, MG1 appears to conform to the recently proposed class I structure of M-protein molecules and thus shares certain distinct structural features with the M proteins of well-established rheumatogenic group A streptococcal serotypes. Further comparison of the structures of group G and group A M proteins of throat and skin isolates may cast light on those configurations of the M protein molecules which are and are not critical for the expression of rheumatogenicity.


Comparison of the abilities of Mycobacterium avium and Mycobacterium intracellulare to infect and multiply in cultured human macrophages from normal and human immunodeficiency virus-infected subjects. Patients with AIDS commonly develop disseminated infections with Mycobacterium avium (MA) but not its close relative, M. intracellulare (MI). In non-AIDS patients who have these infections, the two species are about equally distributed. The higher incidence of infection with MA than with MI in AIDS patients might be due to the selective susceptibility of these patients to MA. This possibility was tested by comparing the abilities of MA and MI to infect and replicate in cultured macrophages from normal subjects and from patients with AIDS-related complex or AIDS. The macrophages were cultured in medium supplemented with 1 or 5% normal or patient sera or with 1% defined serum substitute. Replication of MA (serovar 4) or MI (serovars 16 and 17) in the macrophages was measured by CFU counts made from lysed samples of the macrophages taken at 0,4, and 7 days after macrophage infection. MA and MI in infected normal macrophages which were cultured in normal serum replicated in these macrophages at similar rates. MA but not MI multiplied abnormally rapidly in patient macrophages cultured in either normal serum or patient serum. The accelerated growth of MA in patient macrophages was macrophage dependent, because patient sera did not change the rate of MA replication in culture medium lacking macrophages. However, patient sera did increase the permissiveness of normal macrophages to MA but not MI. These results suggest that a selective increased susceptibility to MA compared with a retained normal resistance to MI in human immunodeficiency virus-infected patients as they progress from AIDS-related complex to AIDS accounts for the higher prevalence of MA than MI infection in AIDS patients. The results also indicate that the mechanisms of native resistance in human macrophages to MA and MI are different.


Immune specificity of murine T-cell lines to the major outer membrane protein of Chlamydia trachomatis. The antigenically variant Chlamydia trachomatis major outer membrane protein (MOMP) is a target of antibody-mediated neutralization in vitro, and it is an important protein for designing a subunit vaccine. Knowledge of MOMP T-cell determinants will be essential to elicit rapid and strong immune responses following an encounter with infectious organisms. C. trachomatis-specific T-cell lines were derived from MOMP-immunized BALB/c mice and selected with intact organisms. We used these short-term T-cell lines to identify determinants of MOMP that could be recognized by T cells following processing of the intact organism. T-cell line proliferation in response to overlapping MOMP peptides showed that only a peptide encompassing the third variable segment (VS3) elicited a strong proliferative response. We further mapped determinants within the VS3 peptide and found that a sequence-conserved portion of the VS3 peptide elicited T-cell proliferation of T-cell lines from BALB/c mice. Thus, unlike the response to several MOMP peptides with unselected T cells, development of short-term T-cell lines with intact organisms restricted the repertoire of antigens capable of being recognized by MOMP-specific T cells.


SCID mouse models of acute and relapsing chronic Toxoplasma gondii infections. Lymphodeficient scid/scid (SCID) mice died from acute infection with a strain of Toxoplasma gondii that causes chronic infection with mild symptoms in immunocompetent non-SCID mice. However, most SCID mice reconstituted with spleen cells from immunocompetent mice 1 month prior to T. gondii infection survived in good health after a transient period during which they appeared ill. Unreconstituted SCID mice given sulfadiazine in their drinking water from day 10 of Toxoplasma infection onward survived the acute phase of infection and lived for many weeks without overt symptoms. Histological examination revealed Toxoplasma cysts in their brains. However, if sulfadiazine was withdrawn from the drinking water of these chronically infected SCID mice, the mice died within 1 week with large numbers of trophozoites throughout their brains. These findings establish SCID mice as a potentially useful resource with which to study various aspects of immunological control of T. gondii infection during either its acute or chronic phase. Furthermore, the ability to produce chronic infections with avirulent T. gondii in SCID mice and to cause acute relapsing infections at will suggests that SCID mice may be helpful in evaluating potential therapies for acute and chronic T. gondii infections in immunocompromised patients.


In vivo induction of nitrite and nitrate by tumor necrosis factor, lymphotoxin, and interleukin-1: possible roles in malaria. Tumor necrosis factor and related cytokines are thought to be implicated in cell-mediated immunity and pathophysiology in malaria, but their mechanism of action has not been ascertained. Tumor necrosis factor has been reported to generate nitric oxide in vitro, so we have measured levels of this molecule and its products in the plasma of mice after they have received an injection of tumor necrosis factor, lymphotoxin, interleukin-1, gamma interferon, or interleukin-6, all of which have been reported to be increased in malaria. Total reactive nitrogen intermediate levels in plasma were assayed spectrophotometrically after exposing plasma to a copper-cadmium-zinc catalyst to convert nitrate to nitrite and then to Griess reagent. Tumor necrosis factor, lymphotoxin, and interleukin-1 all induced reactive nitrogen intermediates in vivo, with interleukin-1 showing the most activity. Tumor necrosis factor was then examined more closely. It induced more reactive nitrogen intermediates in malaria-infected mice than in normal mice, and appreciably more was in the form of nitrate than was in the form of nitrite. NG-methyl-L-arginine inhibited the in vivo generation of reactive nitrogen intermediates by tumor necrosis factor in a dose-dependent manner, implying that these molecules were arginine derived. These results are consistent with the possibility that tumor necrosis factor, lymphotoxin, and interleukin-1 may contribute to host pathology and parasite suppression through generation of nitric oxide.


Effects of cytokines, complement, and antibody on the neutrophil respiratory burst and phagocytic response to Plasmodium falciparum merozoites. The interaction between Plasmodium falciparum merozoites and human neutrophils, as well as the role of cytokines, complement, and antimalarial antibody on this interaction, was examined in vitro by measuring luminol-dependent chemiluminescence and phagocytosis. Merozoites, in the presence of heat-inactivated (56 degrees C/30 min) normal serum, had very little effect on the neutrophil chemiluminescence. This response was significantly enhanced by the addition of normal serum (containing normal complement activity). In the presence of serum or plasma containing anti-P. falciparum antibodies (IS) with no detectable complement activity, the merozoites induced a marked response characterized by an increase in initial peak rate of chemiluminescence and a sustained increased rate of chemiluminescence. However, this response was not further increased if IS containing complement activity was used. Pretreatment of neutrophils with either tumor necrosis factor alpha, lymphotoxin, or gamma interferon significantly increased the neutrophil response to IS-treated merozoites, reflected in an increased initial peak rate and sustained increased rate of chemiluminescence. The effects of cytokine treatment of neutrophils and IS opsonization of merozoites were synergistic. In association with the changes in the chemiluminescence responses, IS was shown to promote phagocytosis of merozoites by neutrophils, and this event was further increased by treating neutrophils with the cytokines. The results emphasize the importance of antibody and cytokines in neutrophil-mediated damage of P. falciparum merozoites.


Genetic analysis of scrA and scrB from Streptococcus sobrinus 6715. A DNA fragment containing scrA and scrB, which encode enzyme II of the phosphoenolpyruvate-dependent sucrose phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, was isolated from a lambda gt10 genomic DNA library of Streptococcus sobrinus 6715. Both genes were located on a 4.2-kb DNA fragment which was maintained stably in Escherchia coli on low-copy-number vector pGB2. The recombinant E. coli clone expressed sucrose-hydrolytic activity on MacConkey agar base supplemented with raffinose or sucrose. Results from deletion analysis showed that the sucrose-metabolic activity was contained within a 3.5-kb region. The lactic acid bacterium Lactococcus lactis subsp. lactis LM0230, which is devoid of sucrose-metabolic activity, was used to study the enzyme activities encoded by scrA and scrB from S. sobrinus 6715. L. lactis transformants carrying the 4.2-kb S. sobrinus-derived DNA fragment on E. coli-Streptococcus shuttle vector pDL278 were able to grow at the expense of sucrose and exhibited enzyme II and sucrose-6-phosphate hydrolase activities. Results from hybridization studies and a comparison of the restriction endonuclease maps of the scrA- and scrB-containing chromosomal regions from S. mutans GS5 and S. sobrinus 6715 suggested considerable divergence.


Immune cell lethality induced by streptococcal pyrogenic exotoxin A and endotoxin. Streptococcal pyrogenic exotoxin (SPE) A has many effects on the immune system, including immunolethality, which is characterized by a significant decrease in circulating immune cells as well as depletion of the spleen and lymph nodes prior to death of experimental animals. In this report, characterization of the mechanism of immunolethality has been undertaken. Synergistic induction of immunolethality was observed in vitro when human lymphocytes were treated with both SPE A and lipopolysaccharide (LPS). The same effect was demonstrated in the absence of a mitogenic response with the murine T-cell receptor, as well as in the absence of antigen-presenting cells and their secreted cytokines. The addition of antigen-presenting cells did not significantly affect lethality. SPE A directly interacted with LPS through interaction with ketodeoxyoctonate as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and iodinated exotoxin overlays. This interaction was demonstrated to be important for immunolethality, since simultaneous addition of SPE A and LPS was required, whereas sequential addition of SPE A and LPS did not result in lethality. LPS appeared to be acting, in part, to enhance the cell-binding ability of SPE A, since SPE A could only be detected in A.E7 cell membrane preparations after simultaneous incubation with SPE A and LPS.


Effects of gamma interferon on release of tumor necrosis factor alpha from lipopolysaccharide-tolerant human monocyte-derived macrophages. After an initial stimulation of human monocyte-derived macrophages with bacterial lipopolysaccharide (LPS), which produces substantial release of tumor necrosis factor-alpha (TNF-alpha), a subsequent exposure to LPS results in about an order-of-magnitude reduction in the levels of TNF-alpha released. We have shown that macrophages which have been stimulated with LPS and then maintained in culture without LPS for as long as 2 weeks do not regain their original capacity to secrete TNF-alpha upon a second LPS challenge. After 2 to 4 days in adherent culture, monocyte-derived macrophages which were not pretreated with LPS also experience a measurable decline in their capacity to release TNF-alpha in response to an initial LPS stimulation. When compared with these previously nonstimulated cells, however, the levels of TNF-alpha released by LPS-pretreated cells in response to a second LPS challenge decline by over 90% after 8 to 9 days in culture. Unstimulated cells spontaneously release barely detectable levels of TNF-alpha. In contrast to the release of TNF-alpha, unstimulated cells release significant levels of prostaglandin E2 continuously over time, and these levels are variably increased by no more than a factor of two in response to a single LPS stimulation. Prostaglandin E2 levels released by LPS-pretreated cells in response to a second LPS stimulation are much closer to the levels released by unstimulated cells. We have also demonstrated that gamma interferon (IFN-gamma) enhances TNF-alpha release from LPS-stimulated macrophages but not from phorbol myristate acetate-stimulated cells. Addition of IFN-gamma to macrophages either during the initial stimulation or during a second stimulation with LPS enhances levels of TNF-alpha released after the second LPS challenge. The greatest enhancement is observed when IFN-gamma is added during both exposures to LPS, but addition of IFN-gamma during only the initial LPS stimulation still results in marked enhancement of TNF-alpha release in response to a second stimulation with LPS 24 h later. If an interval of 2 days of culture in medium alone separates the first and second 24-h LPS stimulations, IFN-gamma enhances TNF-alpha release only when it is included during the second LPS exposure, indicating that, unlike the persistence of endotoxin tolerance, enhancement of TNF-alpha release by IFN-gamma is transient.


Recombinant Salmonella typhimurium strains that invade nonphagocytic cells are resistant to recognition by antigen-specific cytotoxic T lymphocytes. To address the question of whether Salmonella-infected nonphagocytic cells could serve as target cells for recognition by antigen-specific, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL), four recombinant Salmonella typhimurium constructs that expressed full-length, or fragments of, influenza A virus nucleoprotein (NP) were made. The bacteria were shown to infect Chinese hamster ovary (CHO) cells. Appropriate major histocompatibility complex restriction molecules, HLA-B27 and H-2 Db, were transfected into CHO cells, which were then infected with recombinant S. typhimurium and used as targets for NP-specific CTL. The cells in which NP was expressed by intracellularly replicating bacteria were not lysed by NP-specific CTL, although they were killed when appropriate influenza A virus or peptides were used. Thus, S.typhimurium bacteria within nonphagocytic cells were resistant to CTL recognition. In contrast to these results, mice infected with recombinant S.typhimurium that expressed fragments of NP in the periplasm were primed for NP-specific CTL responses. The results indicate that CTL responses specific to Salmonella antigens can be generated, but the bacteria may be safe from the CTL attack once they have entered the nonphagocytic cells.