bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of C3H/HeJ cell activation by lipopolysaccharide endotoxin
Authors: Sultzer B M , Bandekar J R , Castagna R , Abu-Lawi K , Sadeghian M , Norin A J
Published: January 2020

Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of Salmonella typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the G1 stage of the cell cycle. Furthermore, [3H]uridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.
Humoral response to Porphyromonas (Bacteroides) gingivalis in rats: time course and T-cell dependence
Authors: Katz J , Leary R M , Ward D C , Harmon C C , Michalek S M
Published: January 2020

In this study, we describe the time course and T-cell dependence of the serum antibody response to the periodontopathogen Porphyromonas (Bacteroides) gingivalis in an experimental rat model. Normal Fischer rats were challenged by a local injection of P. gingivalis (2 x 10(8) bacteria) into gingival tissue or by the administration of a similar number of bacteria by the intravenous (i.v.) route on days 0, 2, and 4. Serum antibody activity was detected within 1 week and peaked at 8 weeks after gingival challenge. A similar but lower response was seen for rats challenged by the i.v. route. The response in both groups of rats was mainly of the immunoglobulin G (IgG) isotype; some IgM but no IgA antibody activity was detected. Analysis of the IgG subclass revealed mainly IgG2c in animals challenged locally in the gingiva with P. gingivalis, whereas IgG2b predominated in rats challenged by the i.v. route. The importance of T cells in the response was established by demonstrating the absence of serum IgG antibodies in nude rats after a local challenge of gingival tissue with P. gingivalis. Nude rats given purified splenic T cells from normal rats immunized systemically with P. gingivalis prior to a local gingival challenge showed a rapid appearance of serum antibody activity that peaked between 4 and 6 weeks. This initial peak occurred 2 to 4 weeks earlier than that seen in normal animals. Fluorescence-activated cell sorter analysis of splenic lymphoid cells from these nude rats revealed a helper T-cell population. The levels of serum IgG antibodies in nude rats given nonimmune T cells rose slowly, and the antibodies were mainly of the IgG2a and IgG2b subclasses. Nude rats given immune T cells showed a rapid increase primarily in IgG2b antibody levels following a local gingival challenge. These findings suggest that the immune helper T-cells contributed to the rapid development of the response to P. gingivalis. Furthermore, it is likely that the IgG subclass response to P. gingivalis in these nude rats was related to the splenic origin of the T cells used for adoptive transfer.
Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni
Authors: Hong Z , Kosman D J , Thakur A , Rekosh D , LoVerde P T
Published: January 2020

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.
Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli
Authors: Sommerfelt H , Grewal H M , Svennerholm A M , Gaastra W , Flood P R , Viboud G , Bhan M K
Published: January 2020

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.
Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli
Authors: Heinzen R A , Frazier M E , Mallavia L P
Published: January 2020

A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.
Induction of circulating group II phospholipase A2 expression in adults with malaria
Authors: Vadas P , Keystone J , Stefanski E , Scott K , Pruzanski W
Published: January 2020

High levels of interleukin 1 and tumor necrosis factor are found in both cases of malaria and cases of septic shock. Since both interleukin 1 and tumor necrosis factor induce expression of the proinflammatory enzyme phospholipase A2 (PLA2), we examined serum PLA2 levels in 14 adults with malaria. Mean serum PLA2 activity was elevated 40-fold above normal (P less than 0.001). Serum PLA2 activity correlated with PLA2 immunoreactivity (r = 0.987; P less than 0.001) by an enzyme-linked immunosorbent assay specific for human group II PLA2, showing that serum PLA2 in cases of malaria is host derived. This article describes the novel finding of elevated PLA2 levels in cases of malaria, further strengthening the notion that mediators of the host response in cases of malaria are similar to those in cases of septic shock.
Immunoglobulin M and G antibody responses to Plasmodium falciparum glutamate-rich protein: correlation with clinical immunity in Gambian children
Authors: Dziegiel M , Rowe P , Bennett S , Allen S J , Olerup O , Gottschau A , Borre M , Riley E M
Published: January 2020

The aims of the present study were to describe the age-related immunoglobulin M (IgM) and IgG response to part of a 220-kDa glutamate-rich protein (GLURP) from Plasmodium falciparum and to determine possible correlations of possession of these antibodies with malaria morbidity. IgM and IgG levels were measured with a recombinant fusion protein consisting of the carboxy-terminal 783 amino acids of the GLURP. Samples for the study were obtained during a longitudinal malaria morbidity survey performed in The Gambia; cross-sectional surveys were performed at the beginning of the transmission season in May and in October. Seropositivity rates increased with age to a maximum of 77% for IgM and 95% for IgG in adults. High prevalences of seropositivity were associated with certain human leukocyte antigen class II alleles (DRw8, DR9, DR7, DR4, DQw7, and DQw2) or haplotypes. The relationship between anti-GLURP489-1271 antibodies and clinical immunity is not clear; asymptomatically infected children aged 5 to 8 years had significantly higher levels of IgG than clinically ill children of the same age, suggesting that antibodies to the carboxy-terminal part of the GLURP may contribute to immunity to P. falciparum. However, this was not significant for younger children.
Major stable peptides of Yersinia pestis synthesized during the low-calcium response
Authors: Mehigh R J , Braubaker R R
Published: January 2020

It is established that the medically significant yersiniae require the presence of physiological levels of Ca2+ (ca. 2.5 mM) for sustained growth at 37 degrees C and that this nutritional requirement is mediated by a shared ca. 70-kb Lcr plasmid. The latter also encodes virulence factors (Yersinia outer membrane proteins [Yops] and V antigen) known to be selectively synthesized in vitro at 37 degrees C in Ca(2+)-deficient medium. In this study, cells of Yersinia pestis KIM were first starved for Ca2+ at 37 degrees C to prevent synthesis of bulk vegetative protein and then, after cell division had ceased, pulsed with [35S]methionine. After sufficient chase to ensure plasminogen activator-mediated degradation of Yops, the remaining major radioactive peptides were separated by conventional chromatographic methods and identified as Lcr plasmid-encoded V antigen and LcrH (and possibly LcrG), ca. 10-kb Pst plasmid-encoded pesticin and plasminogen activator, ca. 100-kb Tox plasmid-encoded fraction 1 (capsular) antigen and murine exotoxin, and chromosomally encoded antigen 4 (pH 6 antigen) and antigen 5 (a novel hemin-rich peptide possessing modest catalase activity but not superoxide dismutase activity). Also produced at high concentration was a chromosome-encoded GroEL-like chaperone protein. Accordingly, the transcriptional block preventing synthesis of bulk vegetative protein at 37 degrees C in Ca(2+)-deficient medium may not apply to genes encoding virulence factors or to highly conserved GroEL (known in other species to utilize a secondary stress-induced sigma factor).
T-lymphocyte response in a guinea pig model of tuberculous pleuritis
Authors: Phalen S W , McMurray D N
Published: January 2020

The ability to induce tuberculous pleuritis in Mycobacterium bovis BCG-vaccinated guinea pigs was investigated as a model of human disease. A pleural effusion of 5 to 10 ml was obtained 6 to 7 days after the bilateral pleural injection of a suspension of heat-killed M. tuberculosis cells. Histological lesions were indicative of granulomatous pleuritis. Comparative studies of T lymphocytes obtained from pleural fluid and peripheral blood revealed increased antigen-driven lymphoproliferation and E rosette formation in pleural effusion lymphocytes. The CD2+ T-lymphocyte population appeared to be expanded or concentrated in pleural fluid, suggesting a compartmentalization of antigen-reactive T lymphocytes. These data demonstrate that experimental tuberculous pleuritis with effusion, closely resembling the human disease, can be produced in BCG-vaccinated guinea pigs.
Genetic structure of populations of Porphyromonas gingivalis associated with periodontitis and other oral infections
Authors: Loos B G , Dyer D W , Whittam T S , Selander R K
Published: January 2020

One hundred isolates of the oral pathogenic bacterium Porphyromonas gingivalis were genetically characterized by determining the electrophoretic mobilities of 16 metabolic enzymes and the presence or absence of catalase activity. A total of 78 distinct electrophoretic types (ETs), representing multilocus genotypes, were identified, and cluster analysis placed them in three major phylogenetic divisions. Division I (71 ETs) included all 88 human isolates examined, most of which had been recovered from patients with periodontitis, together with 4 monkey isolates. The strains in division II (four ETs) and division III (three ETs) are strongly differentiated from those in division I and apparently represent two previously unclassified (cryptic) species. The mean genetic diversity per enzyme locus among the 92 isolates of division I (P. gingivalis, strict sense) was 0.321, and the strains were distributed among 14 phylogenetic clusters and single-ET lineages. The population structure is basically clonal, with some clonal genotypes being widespread, and even global, in distribution. There was no evidence of association between specific genetic lineages or clusters of ETs and the type of disease (periodontitis or root canal infections), invasive potential, serogroup, or fimbrial restriction fragment length polymorphism group. The finding that dental patients are infected by strains of a wide variety of chromosomal genotypes suggests that interstrain variation in pathogenicity is small. On the basis of the observed genetic structure of natural populations of P. gingivalis, we hypothesize that the role of this microorganism in the pathogenesis of periodontitis and other dental infections is largely opportunistic.
Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388
Authors: Kulich S M , Frank D W , Barbieri J T
Published: January 2020

Exoenzyme S was purified > 1,500-fold from the culture supernatant fluid of Pseudomonas aeruginosa 388 at high yield without utilization of solvents or detergents. Two proteins, with apparent molecular sizes of 53 and 49 kDa, cofractionated with exoenzyme S activity. Rabbit anti-49-kDa-protein immunoglobulin G was prepared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified 49-kDa protein as immunogen. Anti-49-kDa-protein IgG inhibited the ADP-ribosyltransferase activity of purified exoenzyme S in a dose-dependent manner, which indicated a role for the 49-kDa protein in the ADP-ribosylation reaction. Analysis by ultrafiltration showed that exoenzyme S activity and the 53- and 49-kDa proteins cofractionated and that exoenzyme S was apparently > 300 kDa in size. Urea (8 M) and 1.0% Triton X-100 reversibly decreased the apparent molecular sizes of exoenzyme S activity and the 53- and 49-kDa proteins to between 30 and 100 kDa.
Rat model of congenital toxoplasmosis: rate of transmission of three Toxoplasma gondii strains to fetuses and protective effect of a chronic infection
Authors: Zenner L , Darcy F , Cesbron-Delauw M F , Capron A
Published: January 2020

The incidence of congenital toxoplasmosis in Fischer rats infected between the 8th and 12th days of pregnancy with three different strains of Toxoplasma gondii (RH, 76K, and Prugniaud) were 58.2, 35.2, and 62.8%, respectively. No infected fetuses were collected from rats previously infected with RH or Prugniaud strain parasites, even if the rats were reinfected during pregnancy. Since pups from chronically infected mothers are protected from congenital toxoplasmosis, rat infection could thus constitute a relevant model for immunological studies and vaccine design.
Expression of the Haemophilus influenzae transferrin receptor is repressible by hemin but not elemental iron alone
Authors: Morton D J , Musser J M , Stull T L
Published: January 2020

The absolute requirement for elemental iron and the porphyrin nucleus for growth of Haemophilus influenzae led us to investigate the role of iron and hemin in regulation of expression of the H. influenzae transferrin receptor. H. influenzae type b strain H1689 was grown in brain heart infusion broth supplemented with beta-NAD and either 10 or 0.1 microgram of hemin ml-1. Transferrin-binding ability was determined with a dot blot assay using human transferrin-horseradish peroxidase conjugate. Cells grown in media with 0.1 microgram of hemin ml-1 bound transferrin, but organisms grown in media with 10 micrograms ml-1 did not. In hemin-restricted media, transferrin binding occurred despite addition of up to 10 mM ferric nitrate, ferric citrate, or ferric PPi, whereas addition of 10 micrograms of hemoglobin ml-1 repressed expression. The breadth of species distribution of this mode of regulation was determined with strains previously characterized by multilocus enzyme electrophoresis. When grown in hemin-restricted media, 24 of 28 type b strains and 52 of 57 serologically nontypeable strains exhibited transferrin binding, although none did so in hemin- and iron-sufficient media. Strain H1689 and serologically nontypeable strain HI1423 grown in heat-inactivated pooled normal human serum, human cerebrospinal fluid, or human breast milk exhibited transferrin binding. Growth in these fluids with 10 micrograms of added hemin ml-1 abolished transferrin binding, whereas addition of 10 mM ferric nitrate did not. These data suggest that the transferrin receptor of H. influenzae is regulated by levels of hemin but not elemental iron alone and that this property is widely distributed among several major cloned families in the species.
Coxiella burnetii penetration into the reproductive system of male mice, promoting sexual transmission of infection
Authors: Kruszewska D , Tylewska-Wierzbanowska S K
Published: January 2020

Coxiella burnetii bacteria penetrate different host organs via the bloodstream. In infected mice, bacteremia was observed during the first week of infection: later, bacteria were cultured from the spleens, livers, testes, epididymes, prostate, and semen; bacteriuria developed beginning from the second week of infection. On day 21 of infection, degenerative changes with aggregated macrophages containing bacteria were observed in capillary blood vessels and the surrounding tissues of the adipose envelope of the epididymis. C. burnetii was shed to semen from the urogenital tract, probably from an abscess in the epididymis. There they were bound to the surface of spermatozoal cells, mainly to their heads, suggesting specific adhesion. Bacteria shed to semen were transmitted to female mice by sexual contact; this was demonstrated by the detection of antibodies against C. burnetii antigens in the blood of females and later by the cultivation of bacteria from the spleens, livers, and amniotic fluids of female mice.
Chemical properties of lipopolysaccharides from spotted fever group rickettsiae and their common antigenicity with lipopolysaccharides from Proteus species
Authors: Amano K , Fujita M , Suto T
Published: January 2020

The lipopolysaccharides (LPS) isolated from spotted fever group (SFG) rickettsia strains Thai tick typhus TT-118 and Katayama were characterized by chemical analyses, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. These LPS did not contain heptose, but they contained 3-deoxy-D-manno-octulosonic acid (KDO), glucosamine, quinovosamine, phosphate, ribose, an unknown neutral sugar, and palmitic acid. Resolution of the apparent molecular masses of these LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with silver showed ladder-like bands. In an ELISA, convalescent-phase sera from 10 patients with Japanese spotted fever reacted with LPS from the Katayama strain, and 90% (9 of 10) of these sera also reacted with LPS isolated from Proteus vulgaris OX2. Immunoblotting revealed that the sera reacted with the high-molecular-mass bands of LPS from SFG rickettsiae, in addition to those of OX2 LPS. In an ELISA, immunoglobulin M antibodies from these sera reacted with the O-polysaccharide and lipid A portions of LPS from P. vulgaris OX2. The epitopes common to LPS of SFG rickettsiae and P. vulgaris OX2 may be in the O-polysaccharide and lipid A portions.
Differential regulation of cytokine production in lipopolysaccharide tolerance in mice
Authors: Erroi A , Fantuzzi G , Mengozzi M , Sironi M , Orencole S F , Clark B D , Dinarello C A , Isetta A , Gnocchi P , Giovarelli M
Published: January 2020

We investigated the pattern of down-regulation of cytokine production in endotoxin (lipopolysaccharide [LPS]) tolerance. A 4-day treatment with LPS (35 micrograms per mouse) was followed by a challenge on day 6 with one more injection of LPS. Circulating tumor necrosis factor (TNF) and interleukin-6 (IL-6) could not be induced (> 99% inhibition) by LPS in LPS-tolerant mice; colony-stimulating factor (CSF) was also down-regulated by more than 95%, whereas interferon (IFN) and IL-1 syntheses were only partially inhibited. To study the mechanism of cytokine down-regulation in tolerance, we attempted to reverse the tolerant state by pretreatment with phorbol 12-myristate 13-acetate (PMA) (4 micrograms per mouse) 10 min before the LPS challenge. PMA completely restored IL-6 production and partially that of CSF. PMA had no effect on IFN production and inhibited the induction of IL-1. TNF production was also not restored by PMA. To investigate the role of endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-1 alpha, using the same treatment schedule as that for LPS. Whereas IL-6 had no effect, IL-1 alpha or TNF induced partial tolerance to LPS in terms of inhibition of LPS-stimulated TNF and IL-6 production. However, a full LPS-tolerant state could not be induced by administration of recombinant cytokines, suggesting the existence of additional mechanisms, such as a loss of LPS receptors or changes in release of soluble binding proteins.
Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes
Authors: Ollert M W , Söhnchen R , Korting H C , Ollert U , Bräutigam S , Bräutigam W
Published: January 2020

We established an in vitro adherence model with primarily cultured human keratinocytes as target cells which allows for the investigation of the molecular mechanisms that are responsible for Candida albicans host cell attachment in the initiation of cutaneous candidosis. The extent of C. albicans binding to cultured human keratinocytes was dependent on the yeast inoculum size and the incubation temperature. Heat and paraform-aldehyde treatment of yeasts completely abolished the binding activity of C. albicans. Of the different Candida species tested, C. albicans was by far the most adhesive species. C. albicans adherence was blocked by the acid protease inhibitor pepstatin A and the metabolic inhibitor sodium azide. The latter, however, was much less effective when yeasts were preincubated, suggesting that sodium azide was mainly acting on the keratinocytes. The extracellular matrix protein fibronectin was slightly inhibitory, whereas the fibronectin-derived peptides RGD and RGDS were not able to prevent attachment. PepTite-2000, another RGD-containing synthetic peptide, reduced C. albicans adherence by a margin of 25% (P < 0.005). CDPGYIGSR-NH2, which is a synthetic adhesive peptide derived from the laminin B chain, was much more efficient in its inhibitory activity than the RGD peptides and reduced C. albicans adherence to cultured human keratinocytes up to 76% (P < 0.001). Laminin itself and the synthetic pentapeptide YIGSR were less active. A dose-dependent reduction in adherence was also observed with collagen type III. Additionally, saccharides were tested for their potential to inhibit C. albicans attachment to keratinocytes. The most potent competitive saccharide inhibitors of C. albicans adherence to human keratinocytes were the amino sugars D-(+)-glucosamine and D-(+)-galactosamine with one isolate of C. albicans (4918) and D-(+)-glucosamine and alpha-D-(+)-fucose with another C. albicans isolate (Sp-1). Collectively, our data suggest the existence of multiple molecular mechanisms such as protein-protein, lectin-carbohydrate, and yeast-yeast coaggregational interactions that are responsible for optimal C. albicans attachment to cultured human keratinocytes.
Increased sensitivity of gonococcal pilA mutants to bactericidal activity of normal human serum
Authors: Taha M K
Published: January 2020

PilA is a pleiotropic transcriptional regulator in Neisseria gonorrhoeae, encoded by an essential gene, pilA. It regulates pilin gene expression and stress response and it is implicated in gonococcal adaptation to external signals. All these phenomena may participate in gonococcal virulence. In this report, I tested the role of PilA in another aspect of gonococcal virulence, resistance to the bactericidal effect of normal human serum. Gonococcal mutants with impaired PilA function were more susceptible to the bactericidal effect of normal human serum than the isogenic wild-type strain. However, the major outer membrane protein and the lipooligosaccharide, targets for complement-mediated killing by the serum, were unchanged in the mutants. I discuss the role of PilA in modulating gonococcal sensitivity and resistance to normal human serum.
Proliferative and cytokine responses to a major surface glycoprotein of Pneumocystis carinii
Authors: Theus S A , Linke M J , Andrews R P , Walzer P D
Published: January 2020

Naturally derived T-cell responses by rats to a 120-kDa major surface glycoprotein (MSG) of rat-derived Pneumocystis carinii were analyzed in vitro. Specific cytokines elicited by the T-cell response to the MSG were also identified. MSG was purified from rat-derived P. carinii by three different techniques: lectin affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution, and size-exclusion high-performance liquid chromatography. The cell-mediated immunity of spleen cells isolated from Lewis rats with and without natural exposure to P. carinii to the purified MSG was studied. Exposure to P. carinii was monitored by the presence or absence of serum antibodies to P. carinii antigens by Western blotting (immunoblotting). A T-cell proliferative response to the MSG was identified only with spleen cells isolated from rats exposed to P. carinii and peaked at 4 days. Flow cytometric analysis revealed that the percentage of CD4 cells was significantly increased during the proliferative response to MSG. MSG also elicited secretion of tumor necrosis factor alpha, interleukin-1, and interleukin-2, with peak activity of these cytokines occurring after 12, 24, and 48 h, respectively, of culture. These findings suggest that MSG is important in host T-cell recognition of and immune response to P. carinii by recruitment of inflammatory cells and cytokine production.
Pathology of congenital syphilis in rabbits
Authors: Froberg M K , Fitzgerald T J , Hamilton T R , Hamilton B , Zarabi M
Published: January 2020

We have developed a model for congenital syphilis in the rabbit. This report provides additional information on newborn tissue pathology in animals that were infected in utero. A total of 35 pregnancies were evaluated, each containing 6 to 12 newborns. In the infected group, the mortality was approximately 50%; of the live newborns, half appeared normal and half were hyperreflexic, weak, and runty. Gross pathology in the sickly newborns was quite prevalent and involved enlarged spleens with isolated spots of necrosis; enlarged livers that were overtly congested and hemorrhagic and had numerous granular, white spots; and brains with hemorrhage in the occipital area. Histopathology was apparent in different tissues. Lymphocytes, plasma cells, and vacuolated macrophages were prominent in livers, spleens, brains, and bones. A few actively motile treponemes were visualized by dark-field microscopy within extracts of spleen and within cerebrospinal fluid. Low numbers of treponemes were also demonstrated in sections of brain and liver by using the Warthin-Starry silver stain technique. Blood hematocrits were decreased, and extramedullary hematopoiesis was prominent within spleens and livers; this is consistent with anemia. This rabbit model exhibits many of the same pathologic features commonly found in human congenital syphilis.
Effect of type III group B streptococcal capsular polysaccharide on invasion of respiratory epithelial cells
Authors: Hulse M L , Smith S , Chi E Y , Pham A , Rubens C E
Published: January 2020

Group B streptococcal (GBS) capsular polysaccharide is an important virulence factor, and its role in invasion of cultured respiratory epithelial cells was investigated. A type III GBS clinical isolate, COH1, and asialo and unencapsulated isogenic transposon capsule mutants of it were compared in an in vitro invasion assay. The results demonstrated that capsule attenuated the invasion process. Invasion was not affected when the A549 epithelial cells were preincubated with purified type III GBS capsular polysaccharide. Polyclonal type III GBS capsule antibody inhibited invasion by COH1 but did not affect invasion by the capsule mutants. Serotypes Ia, Ib, Ia/c, II, and III all invaded respiratory epithelial cells but demonstrated some strain variation in magnitude of invasion. These observations led us to conclude that type III capsular polysaccharide was not essential for invasion of respiratory epithelial cells by GBS and that bacterial factors other than capsule were responsible for respiratory epithelial cell invasion.
Virulence determinants in nontoxinogenic Escherichia coli O157 strains that cause infantile diarrhea
Authors: Schmidt H , Rüssmann H , Karch H
Published: January 2020

Ten sorbitol-fermenting Escherichia coli O157 strains that cause infantile diarrhea and are positive in the fluorescence actin staining test were determined to be negative for Shiga-like toxin (SLT) genes. We amplified their complete eae genes, contrasting them with those of SLT-producing E. coli O157 by restriction fragment length polymorphism analysis and nucleotide sequence analysis of a 400-bp stretch of the 3' end of eae. The data substantiated the presence of two eae genotypes within serogroup O157, one resembling eae of enteropathogenic E. coli (EPEC) strain E2348/69, found in nontoxinogenic E. coli O157 strains, and the other resembling eae of EHEC strain EDL 933, found in toxinogenic E. coli O157 strains. Another EPEC-specific virulence determinant was also shown to be large plasmids harboring EPEC adherence factor sequences. The SLT-negative E. coli O157 strains described here fall under the heading of EPEC, which serves as an explanation for their virulence in infants, and represent a third pathogroup within serogroup O157.
P1-antigen-containing avian egg whites as inhibitors of P adhesins among wild-type Escherichia coli strains from patients with urosepsis
Authors: Johnson J R , Ross A E
Published: January 2020

Among 58 Escherichia coli urosepsis isolates, P1-antigen-containing dove and pigeon egg whites were significantly more effective inhibitors of P-adhesin-specific agglutination than were chicken egg whites or globoside. Globoside's inefficacy may have resulted from a proadherence effect of globoside's lipid tail. Adhesin phenotypes determined with dove and pigeon egg whites as as agglutination inhibitors corresponded closely with phenotypes defined by comparative hemagglutination of human P1 and p erythrocytes. These data suggest that avian P1-antigen-containing substances may provide a useful alternative method for P adhesin inhibition among uropathogenic E. coli strains.
Protective immunity is induced by a Borrelia burgdorferi mutant that lacks OspA and OspB
Authors: Hughes C A , Engstrom S M , Coleman L A , Kodner C B , Johnson R C
Published: January 2020

A mutant of virulent Borrelia burgdorferi 297 was apparently selected for by long-term storage at 5 degrees C. This mutant was found to lack the plasmid which encodes outer surface protein A (OspA) and OspB. In addition to the loss of the OspA and OspB proteins, the mutant lacked two lipoproteins, of 20 and 7.5 kDa, that were observed in the wild type. Since the mutant was not recovered from the tissues or blood of hamsters injected with the mutant, the mutant was determined to be noninfectious. Hamsters vaccinated with noninfectious mutant 297 were protected completely from challenge with virulent wild-type 297 spirochetes. Prechallenge sera from hamsters vaccinated with mutant 297 lacked antibodies to OspA and OspB, while those from hamsters vaccinated with virulent wild-type 297 or avirulent 297 exhibited antibodies to these proteins. Hamsters vaccinated with virulent wild-type 297 or mutant 297 elicited antibodies to OspC and a 39-kDa protein (P39), whereas hamsters vaccinated with avirulent 297 lacked these antibodies. These results suggest that OspC and/or P39 are important for the development of a protective immune response. Study of this mutant may elucidate factors important to the development of a Lyme disease vaccine.
Yersinia lipopolysaccharide is modified by human monocytes
Authors: Wuorela M , Jalkanen S , Toivanen P , Granfors K
Published: January 2020

Reactive arthritis is usually self-limiting polyarthritis, which develops after certain gastrointestinal or urogenital tract infections, mostly in susceptible HLA B27-positive individuals. In the pathogenesis of this arthritis, it is probably important that structures of the causative bacteria are found in the affected joints. The structure found in the synovial fluid phagocytes of the patients with reactive arthritis after Yersinia, Salmonella, and Shigella infections has always been lipopolysaccharide (LPS) of the causative bacteria. It has been in a highly processed form but still immunoreactive. To follow the degradation process of LPS, we fed peripheral blood monocytes of healthy blood donors with heat-killed Yersinia enterocolitica O:3 bacteria in vitro and monitored the fate of LPS by immunofluorescence and immunoblotting methods. Heat-killed bacteria were used since Y. enterocolitica O:3 bacteria are able to live inside monocytes in vitro and dividing intracellular bacteria would have made it impossible to monitor the degradation process of LPS with these methods. Both the core region and the O-polysaccharide chain of LPS persisted in cytoplasmic vacuoles and on plasma membrane of monocytes through the 7-day follow-up time. Migration properties of processed LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested structural modifications of LPS. We also demonstrated that core epitopes appearing on the surface of Yersinia-fed monocytes on day 4 of incubation were processed intracellularly, suggesting that LPS-containing phagocytes are a constant source of membrane-active LPS in their microenvironment as well as in the joints of arthritic patients.
Presentation of mycobacterial antigens by human dendritic cells: lack of transfer from infected macrophages
Authors: Pancholi P , Mirza A , Schauf V , Steinman R M , Bhardwaj N
Published: January 2020

When exposed to a challenge of 10 Mycobacterium bovis BCG cells per antigen-presenting cell, most human monocytes engulf several organisms. In contrast, blood dendritic cells which are potent antigen-presenting cells for several antigens are not detectably phagocytic for mycobacteria. We investigated the possibility that infected macrophages might regurgitate antigens for presentation by populations of human blood dendritic cells. Macrophages were infected with M. bovis BCG, mixed with uninfected dendritic cells, and added to immune T cells, either bulk T cells or cloned populations from BCG vaccinees or patients recovering from tuberculosis. The macrophages were from donors who were mismatched to the T cells so that transfer of antigen to major histocompatibility complex-matched dendritic cells could be evaluated. As we describe, there was no evidence for the transfer of mycobacterial antigens from macrophages to dendritic cells in a form that was stimulatory for the T cells.
Antigenic switching of TSA 417, a trophozoite variable surface protein, following completion of the life cycle of Giardia lamblia
Authors: Meng T C , Hetsko M L , Gillin F D
Published: January 2020

Expression of TSA 417, the predominant cysteine-rich variable surface protein of Giardia lamblia WB clone C6 trophozoites, did not change during encystation in vitro. However, in vitro excystation of cysts derived in vitro or in vivo consistently produced TSA 417 nonexpressing trophozoite populations, suggesting that completion of the life cycle leads to antigenic switching.
Effects of antibody isotype and host cell type on in vitro neutralization of Chlamydia trachomatis
Authors: Peterson E M , Cheng X , Pal S , de la Maza L M
Published: January 2020

Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to IgG1, neutralization of C. trachomatis by MAb E-21 in HeLa cells is also corroborative evidence for the role of Fc gamma RIII receptors in this interaction. A fivefold enhancement of infectivity was seen when 10 and 1 micrograms of MAb E-4 per ml were tested in a CI assay with HeLa cells. In performing CI neutralization synergy studies in HeLa cells with MAbs E-4 and E-21, antagonism between MAbs E-4 and E-21 was observed at MAb E-4 concentrations of 10 and 1 micrograms/ml for all concentrations of MAb E-21 tested (10 to 0.1 micrograms/ml). When HaK cells were used in the same studies, no antagonism between the MAbs was found. In addition, when HeLa cells were used in a CI assay, polyclonal serum raised to a peptide representing variable domain IV of the major outer membrane protein inhibited the neutralizing ability of MAb E-21. The blocking of neutralization and the enhancement of infectivity by chlamydia-specific antibodies seen in this investigation with HeLa cells may have important clinical implications for developing preventive strategies for chlamydial infections.
The Salmonella typhimurium virulence plasmid increases the growth rate of salmonellae in mice
Authors: Gulig P A , Doyle T J
Published: January 2020

The virulence plasmids of Salmonella typhimurium and other invasive Salmonella serovars have long been associated with the ability of these bacteria to cause systemic infection beyond the intestines in orally inoculated animals. Genetic analysis of virulence genes on the high-molecular-weight plasmids has revealed that no more than five genes spanning a 6.2-kb region are sufficient to replace the entire plasmid for conferring virulence. However, the exact virulence function(s) encoded by these genes has not been elucidated. In this report, we measured the possible effect of the virulence plasmid on the growth rate of S. typhimurium in mice by two complementary procedures. The first procedure used segregation of a temperature-sensitive plasmid in vivo to provide a measure of bacterial divisions and the number of recovered marker plasmid-containing salmonellae as a measure of killing. In the second procedure, aroA deletions were transduced into virulence plasmid-containing and plasmid-cured S. typhimurium. Since AroA- salmonellae are inhibited for growth in vivo, if the virulence plasmid affected only growth rate, no difference in the recoveries of the paired AroA- strains would be seen. Virulence plasmid-containing S. typhimurium segregated the marker plasmid more rapidly than did the virulence plasmid-cured strain, and AroA- derivatives of both strains were recovered equally from mice. Therefore, the S. typhimurium virulence plasmid increased growth rate but had no detectable effect on killing or bacterial movement into deep tissues. To examine whether the plasmid accomplished this function by affecting the intracellular/extracellular location of bacteria, orally infected mice were injected with gentamicin to kill the extracellular bacteria. Wild-type and plasmid-cured S. typhimurium strains were equally resistant to gentamicin in vivo and hence most likely located intracellularly to equal degrees. When wild-type and plasmid-cured S. typhimurium strains were sequestered within peritoneal chambers in mice, the resulting extracellular growth was equal. Therefore, the virulence plasmid increases the growth rate of S. typhimurium in mice, probably within mouse cells.
Antibody responses in the serum and respiratory tract of mice following oral vaccination with liposomes coated with filamentous hemagglutinin and pertussis toxoid
Authors: Guzman C A , Molinari G , Fountain M W , Rohde M , Timmis K N , Walker M J
Published: January 2020

Mice were orally vaccinated with liposomes coated with filamentous hemagglutinin (FHA) and detoxified pertussis toxin (PT) of Bordetella pertussis. FHA- and PT-specific immunoglobulin G (IgG) was detected in serum, and both IgG and IgA were detected in lung washes following the immunization. Antibody responses in mice immunized with liposomes coated with FHA and PT were significantly higher than those in mice immunized with free FHA and PT, which demonstrated the adjuvanticity of the liposome carrier. The results indicate the potential usefulness of this approach for eliciting immune responses against FHA and PT (and perhaps other pertussis antigens) in humans and its possible utility in large-scale vaccination to protect against both B. pertussis infection and disease.
Modulation of growth and function of human gingival fibroblasts by fibroblast-activating factor derived from Porphyromonas gingivalis W50
Authors: Mihara J , Miyazawa Y , Holt S C
Published: January 2020

The effect of a 24-kDa fibroblast-activating factor (FAF) isolated from outer membrane vesicles of Porphyromonas gingivalis W50 on the modulation of [3H]thymidine uptake and cell proliferation was examined in selected fibroblast and transformed cell lines. FAF enhanced the proliferation of human gingival fibroblasts, human skin fibroblasts, and human umbilical vein endothelial cells in subconfluent and confluent cells, suggesting that FAF might be functioning as a competence factor. The transformed cell lines, U-937 and HEp-2, were not responsive. FAF and several human-derived growth factors showed a synergistic effect on proliferation. [3H]proline and [3H]leucine were rapidly incorporated into fibroblasts in the presence of FAF; however, there was no selective induction of collagen synthesis. FAF was not active in the induction of interleukin-1 and interleukin-6. It is hypothesized that FAF from P. gingivalis functions as a growth factor for human fibroblasts but is without activity for transformed cells.
Mycobacterium avium-M. intracellulare binds to the integrin receptor alpha v beta 3 on human monocytes and monocyte-derived macrophages
Authors: Rao S P , Ogata K , Catanzaro A
Published: January 2020

Mycobacterium avium-M. intracellulare is an intracellular pathogen responsible for the highest incidence of disseminated bacterial infection in patients with AIDS. Treatment of the infection is difficult and has been of limited efficacy. Attachment of the organism to macrophages is a critical early step in the establishment of the disease. In the present study, we isolated and identified a receptor that mediates attachment of M. avium-M. intracellulare to human peripheral blood monocytes and monocyte-derived macrophages. On Western blotting, (immunoblotting), the receptor was found to cross-react with antibodies against a human vitronectin receptor (alpha v beta 3). The receptor could be purified from monocyte extracts by using monoclonal antibodies (MAbs) against the alpha v subunit of vitronectin receptor coupled to CNBr-Sepharose 4B, as well as with the adhesive tripeptide sequence arginine-glycine-aspartic acid (RGD) coupled to CNBr-Sepharose 4B. Surface-bound MAbs directed against alpha v beta 3 were found to inhibit the attachment of M. avium-M. intracellulare to monocyte-derived macrophages in an in vitro inhibition assay, while MAbs directed against CD14, CD18, alpha 2 beta 1 and platelet glycoprotein gpIIb/IIIa receptors did not inhibit this attachment. These observations suggest that alpha v beta 3 on the surface of human monocytes and monocyte-derived macrophages may function as a receptor for M. avium-M. intracellulare. Identification of a receptor for M. avium-M. intracellulare on macrophages may offer new approaches to the prevention and control of M. avium-M. intracellulare infection at the cellular level.