bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement and antibody participation in opsonophagocytosis of type IV and V group B streptococci
Authors: Hall M A , Edwards M S , Baker C J
Published: January 2020

Requirements for complement and antibody in neutrophil-mediated killing of serotype IV and V group B streptococci were investigated. Neutrophils from adults were tested in an opsonophagocytic assay with sera from healthy adults, healthy newborns, and hypogammaglobulinemic, agammaglobulinemic, and C4-deficient patients. For all serum sources, the bactericidal index for both serotypes exceeded 84% after 40 min of incubation. Heat inactivation of sera ablated killing. Blockade of neutrophil receptor FcIII effected a maximum of 16% inhibition of opsonophagocytosis, and FcII receptor blockade demonstrated negligible inhibition. When neutrophil complement receptor 1 or 3 blockade was employed, the maximum inhibition detected was 26%. Simultaneous blockade of complement receptors 1 and 3 effected maximum inhibition levels of 25 and 65% for serotypes IV and V, respectively. Blockade of complement receptor 3 and neutrophil receptor FcIII inhibited opsonophagocytosis by 56% for both serotypes. When serum complement concentrations were restricted, neutrophil-mediated killing diminished but was restored by the addition of hyperimmune rabbit antiserum. These findings suggest that complement and antibody are major participants in the opsonophagocytosis of serotypes IV and V group B streptococci. A low prevalence of carriage or mediation of efficient phagocytosis by interactions of neutrophil complement and Fc receptors may contribute to the rarity of human infections caused by these two serotypes.
Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells
Authors: Santucci L A , Gutierrez P L , Silverman D J
Published: January 2020

Human endothelial cells infected with Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, undergo striking morphological changes to the endoplasmic reticulum-outer nuclear envelope complex. These changes are accompanied by concurrent accumulation of intracellular peroxides. Both of these findings are consistent with the notion that cells undergo some form of oxidative stress. Since oxidant injury is often initiated or mediated through oxygen radicals, we examined superoxide radical generation when endothelial cells were exposed to R. rickettsii. We also examined the levels of superoxide dismutase, an enzyme induced in response to increased superoxide formation. The levels of both superoxide and superoxide dismutase increased when endothelial cells were exposed to R. rickettsii. These results, together with our previous findings, support our hypothesis that cells infected by this intracellular bacterium experience oxidant-mediated injury that may eventually contribute to cell death.
Streptococcal C5a peptidase is a highly specific endopeptidase
Authors: Cleary P P , Prahbu U , Dale J B , Wexler D E , Handley J
Published: January 2020

Compositional analysis of streptococcal C5a peptidase (SCPA) cleavage products from a synthetic peptide corresponding to the 20 C-terminal residues of C5a demonstrated that the target cleavage site is His-Lys rather than Lys-Asp, as previously suggested. A C5a peptide analog with Lys replaced by Gln was also subject to cleavage by SCPA. This confirmed that His-Lys rather than Lys-Asp is the scissile bond. Cleavage at histidine is unusual but is the same as that suggested for a peptidase produced by group B streptococci. Native C5 protein was also resistant to SCPA, suggesting that the His-Lys bond is inaccessible prior to proteolytic cleavage by C5 convertase. These experiments showed that the streptococcal C5a peptidase is highly specific for C5a and suggest that its function is not merely to process protein for metabolic consumption but to act primarily to eliminate this chemotactic signal from inflammatory foci.
Yersinia pestis YopM: thrombin binding and overexpression
Authors: Reisner B S , Straley S C
Published: January 2020

In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIb alpha. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human alpha-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeIII subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindIII-F fragment. To search for a possible regulator of YopM expression, the HindIII-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation of yopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.
Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1 beta or tumor necrosis factor alpha
Authors: Takashiba S , Takigawa M , Takahashi K , Myokai F , Nishimura F , Chihara T , Kurihara H , Nomura Y , Murayama Y
Published: January 2020

Inflammatory mediators produced by cells in the gingiva have been implicated in the initiation and progression of periodontal disease, a common infectious disease. In this study, we examined the biological activity of neutrophil chemotactic factors and the kinetics of expression of interleukin-8 (IL-8) mRNA derived from normal gingival fibroblasts in response to inflammatory mediators in an in vitro model. Gingival fibroblasts stimulated by either recombinant human interleukin-1 beta or recombinant human tumor necrosis factor alpha produced neutrophil chemotactic factors after 4 h, whereas expression of cell-derived IL-8 mRNA was detected within 1 h after stimulation. Furthermore, in a neutralization assay, rabbit anti-recombinant human IL-8 antiserum inhibited neutrophil chemotactic activity to basal levels. These results provide evidence that gingival fibroblasts synthesize potent chemotactic factors such as IL-8 in the presence of the inflammatory mediators interleukin-1 beta and tumor necrosis factor alpha. The activity of these factors may contribute to neutrophil-mediated processes in the pathogenesis of periodontal disease.
Secretory immune responses to Mycoplasma pulmonis
Authors: Steffen M J , Ebersole J L
Published: January 2020

Formalinized Mycoplasma pulmonis, along with aluminum hydroxide as an adjuvant, was used to subcutaneously immunize rats in the vicinity of the salivary gland to examine the characteristics of the secretory immune response to this pathogen. The induction of specific antibody to this microorganism was detected in serum and the exocrine fluids, namely, saliva and lung lavage fluid. Both immunoglobulin G (IgG) and IgA isotype antibodies were detected in each of these fluids after primary and secondary local immunizations. Serum responses from immunized animals were significantly greater than in the control group, but a dose response was not observed in either IgG or IgA antibody at the dosages selected for immunization. Salivary IgG antibody responses peaked early after both the primary and secondary immunizations, exhibiting a clear dose response. Salivary IgA in immunized groups was significantly greater than that in the control group but displayed little dose-dependent kinetics, and, at the termination of the experiment, this response had not yet peaked. Lung lavage IgG and IgA were minimal after the primary immunization when the antibody was normalized to total protein but displayed dose-dependent kinetics after a secondary challenge. IgG peaked immediately after a secondary challenge, while IgA peak responses were observed only after 20 days. A positive correlation was noted between the serum, saliva, and lung lavage fluid IgGs after both primary and secondary immunizations and only after a secondary challenge for IgA. In this study we were able to elicit a secretory immune response, consisting of both IgG and IgA, which exhibited a dose-dependent characteristic in lung lavage fluid to this immunogen. Additionally, a positive correlation of antibody levels between saliva and lung lavage fluid suggests that saliva could be used as an indicator for monitoring specific antibody to M. pulmonis in lung lavage secretions without requiring invasive, deleterious procedures.
Antigenic diversity in Haemophilus ducreyi as shown by western blot (immunoblot) analysis
Authors: Roggen E L , De Breucker S , van Dyck E , Piot P
Published: January 2020

The antigenic diversity within a panel of 63 Haemophilus ducreyi isolates was examined by Western blot (immunoblot) analysis with a pool of 238 well-characterized human antisera. When a serum pool adsorbed on a mixture of Haemophilus influenzae, H. parainfluenzae, and H. parahaemolyticus was used, the immunoprofiles suggested that prominent antigenic proteins involved in the human immune response have apparent molecular masses of 63, 42, 34 to 30, and 28.5 to 28 kDa. Preliminary subcellular localization revealed that these antigens are associated with the cellular membrane. Two subsets of antigens were discriminated by detergent extraction. There was no evidence that the antigen composition is altered by changing the growth conditions. With a serum pool adsorbed on the Haemophilus spp. mixture supplemented with Actinobacillus actinomycetemcomitans, Pasteurella ureae, Neisseria gonorrhoeae, and Escherichia coli, antigenic determinants more specific for H. ducreyi were identified. An immunodominant 28.5- to 28-kDa protein was expressed by all H. ducreyi isolates. In the range from 34 to 30 kDa, 56 isolates revealed a dominant protein with variable molecular mass. By using both proteins (28.5 to 28 kDa and 34 to 30 kDa) as immunotypic markers, seven different immunopatterns were identified. Antigenic diversity among isolates from different geographical origins as well as from a single area was observed.
Induction of proliferative responses of T cells from Babesia bovis-immune cattle with a recombinant 77-kilodalton merozoite protein (Bb-1)
Authors: Tetzlaff C L , Rice-Ficht A C , Woods V M , Brown W C
Published: January 2020

A major portion of a Babesia bovis-specific gene encoding a 77-kDa merozoite protein (Bb-1) produced during natural infection in cattle and in microaerophilous culture was subcloned into the pGEX1N expression vector. Recombinant Bb-1 protein fused to glutathione S-transferase (Bb-1-GST) was used to examine cellular immune responses in B. bovis-immune cattle. Sera from rabbits immunized with Bb-1-GST reacted with fusion protein and with the native antigen present in crude B. bovis but not with B. bigemina merozoites. Bb-1-GST but not GST induced strong proliferation of T lymphocytes from these immune cattle, and Bb-1-reactive T-cell lines which consisted of a mixed population of either CD4+ and CD8+ cells or CD4+, CD8+, and "null" (gamma delta T) cells were established by in vitro stimulation of peripheral blood mononuclear cells with the recombinant fusion protein. Three CD4+ CD8- and three CD4- CD8+ Bb-1-specific T-cell clones were identified after limiting-dilution cloning of the cell lines. The studies described here demonstrate that the 77-kDa protein of B. bovis contains T-cell epitopes capable of eliciting proliferation of two types of T cells in immune cattle, an important consideration for the design of a recombinant subunit vaccine.
Geographically restricted heterogeneity of the Plasmodium falciparum circumsporozoite protein: relevance for vaccine development
Authors: Doolan D L , Saul A J , Good M F
Published: January 2020

The design of a subunit vaccine against the malaria parasite relies on the epitopes recognized by T cells being identified and polymorphisms therein being defined. Here we present the analysis of a 354-bp fragment of the circumsporozoite (CS) protein encompassing defined proliferative and cytotoxic T-cell recognition regions. We reveal that the polymorphism of CS protein T-cell sites appears to be very limited among Plasmodium falciparum isolates prevalent in certain geographical regions, in particular Papua New Guinea. Furthermore, the more extensive polymorphism noted in other areas appears to be restricted. Although the extent of variation observed for the T-cell recognition domains suggests that any vaccine designed to stimulate this form of immunity will need to be polyvalent, this variation appears to be finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways to increase immune responsiveness can be found, then a vaccine designed to stimulate CS protein-specific T-cell activity may prevent malaria.
Distinct T-cell receptor V beta gene usage by human T lymphocytes stimulated with the streptococcal pyrogenic exotoxins and pep M5 protein
Authors: Tomai M A , Schlievert P M , Kotb M
Published: January 2020

A number of streptococcal products, including the streptococcal pyrogenic exotoxin (SPE) types A, B, and C as well as a 22-kDa fragment of M type 5 protein (pep M5), are potent stimulants of human T-lymphocyte blastogenesis and belong to the newly designated family of superantigens. The V beta usage of human T cells stimulated with these toxins was investigated by using the polymerase chain reaction. We demonstrate that SPE A, B, and C as well as pep M5 stimulate the proliferation of T cells in a dose-dependent manner. pep M5 stimulates cells bearing V beta 2, 4, and 8 elements of the T-cell receptor (TCR), whereas SPE A stimulates TCR V beta 2-, 12-, 14-, and 15-bearing cells. SPE B stimulated only cells expressing TCR V beta 8 elements, while SPE C stimulated cells expressing V beta 1, 2, 5.1, and 10. These studies reveal that the preferential usage of particular V beta elements is distinct for these different superantigens, which may be important in the pathogenesis of various streptococcal diseases.
T lymphocytes mediate protection against Yersinia enterocolitica in mice: characterization of murine T-cell clones specific for Y. enterocolitica
Authors: Autenrieth I B , Tingle A , Reske-Kunz A , Heesemann J
Published: January 2020

Yersinia enterocolitica is enteropathogenic for humans and rodents, causing intestinal and extraintestinal diseases. The cellular immune response of the infected host has not yet been analyzed in detail. Therefore, we used a parenteral mouse infection model to determine the role of T lymphocytes in immunity against Y. enterocolitica. We report the generation and characterization of Y. enterocolitica-specific T-cell clones isolated from spleens of intravenously infected C57BL/6 mice. The T-cell clones obtained showed the phenotype of helper T cells (L3T4) or cytotoxic T cells (Lyt2). All T-cell clones were positive for the interleukin-2 (IL-2) receptor (Tac antigen, p55 subunit) and were negative for the gamma delta T-cell receptor. L3T4+ clones produced small quantities of IL-2 (less than 1 U/ml) when stimulated with heat-killed Y. enterocolitica, whereas Lyt2+ clones produced no or extremely low levels of IL-2. In contrast to IL-2 production, both L3T4+ and Lyt2+ T-cell clones produced considerable quantities of gamma interferon (500 U/ml). When transferred into nonimmune mice, some of the L3T4+, as well as the Lyt2+, T-cell clones could mediate at least partial protection against a challenge of a lethal dose of Y. enterocolitica. These data demonstrate for the first time the generation and characterization of Y. enterocolitica-specific T-cell clones and provide evidence that T cells may be involved in protection against enteropathogenic Y. enterocolitica.
Macrophage activation during Plasmodium chabaudi AS infection in resistant C57BL/6 and susceptible A/J mice
Authors: Stevenson M M , Huang D Y , Podoba J E , Nowotarski M E
Published: January 2020

Macrophage activation was examined in resistant C57BL/6 and susceptible A/J mice during the course of blood-stage infection with Plasmodium chabaudi AS. Three parameters of macrophage activation (lipopolysaccharide [LPS]- and malaria antigen-induced tumor necrosis factor [TNF] production in vitro, phorbol myristate acetate [PMA]-induced production of oxygen metabolites in vitro, and Ia antigen expression) were assessed during infection in populations of peritoneal and splenic macrophages recovered from infected mice of the two strains. The peak level of LPS-induced TNF production in vitro by splenic macrophages from both infected C57BL/6 and infected A/J mice occurred on day 7, which was 3 days before the peak of parasitemia. Although the kinetics of TNF production in vitro in response to either LPS, soluble malaria antigen, or intact parasitized erythrocytes varied in some of the other macrophage populations during infection, there was no significant difference in the peak level of production. Peritoneal and splenic macrophages from infected C57BL/6 mice exhibited significantly increased PMA-induced production of H2O2 in vitro on day 7. Peritoneal macrophages from infected A/J mice also exhibited significant PMA-induced H2O2 production on day 7, while production by splenic macrophages from these hosts was not increased in comparison with production by cells from normal animals. Only peritoneal macrophages from infected C57BL/6 mice produced significantly increased levels of O2-, and this occurred on day 7 postinfection. Ia antigen expression by both peritoneal and splenic macrophages from resistant C57BL/6 and susceptible A/J mice was significantly increased during P. chabaudi AS infection. However, the percentage of Ia+ peritoneal macrophages on days 8 and 10 postinfection and Ia+ splenic macrophages on day 3 postinfection was significantly higher in C57BL/6 than in A/J mice. Thus, these results demonstrate that macrophages from P. chabaudi AS-infected A/J mice exhibit defects in oxygen metabolism and Ia antigen expression which may contribute to the susceptibility of these hosts to this intraerythrocytic parasite. The cause-and-effect relationship between these defects and the susceptibility of A/J mice to P. chabaudi AS is unknown.
Structural basis of capacity of lipoarabinomannan to induce secretion of tumor necrosis factor
Authors: Chatterjee D , Roberts A D , Lowell K , Brennan P J , Orme I M
Published: January 2020

The results of this study show that lipoarabinomannans (LAM) isolated from a virulent strain and from an avirulent strain of Mycobacterium tuberculosis, which have recently been shown to differ markedly in terms of the structures of their nonreducing termini, also differ markedly in the capacity to induce the secretion of tumor necrosis factor from murine macrophages. It was found that LAM from the avirulent H37Ra strain was 100-fold more potent at inducing tumor necrosis factor secretion than LAM from the virulent Erdman strain, thus leading us to hypothesize that the structure of LAM from a given mycobacterial isolate may directly influence its ability to elicit, or avoid, cytokine-mediated mechanisms of host resistance.
Inhibition of Aspergillus fumigatus elastase with monoclonal antibodies produced by using denatured elastase as an immunogen
Authors: Frosco M , Fahed C , Chase T , Macmillan J D
Published: January 2020

In preparing monoclonal antibodies to the elastase from Aspergillus fumigatus, we found that the enzyme was weakly immunogenic in BALB/c mice. Antiserum titers were only 1:1,000 to 1:5,000, and hybridomas secreted nonspecific immunoglobulin M (IgM). Denaturing the elastase in 0.5% sodium dodecyl sulfate at 80 degrees C for 10 min prior to injection increased titers of antiserum against the nondenatured (native) enzyme 10-fold. Of eight hybridomas selected following immunization with the denatured enzyme, seven produced IgG reactive with the native enzyme and one produced nonspecific IgM. The nondenatured immunogen tested again yielded mainly IgM producers. Immunoblots and enzyme-linked immunosorbent assay showed that the IgG monoclonal antibodies were reactive with both the denatured and nondenatured fungal elastases; none cross-reacted with human neutrophil elastase, porcine pancreatic elastase, or Pseudomonas elastase. Elastase-specific polyclonal antibody produced in mice inhibited elastase activity beginning at a molar ratio (antibody to elastase) of 4:1, and activity was completely inhibited at 14.5:1. Some individual monoclonal antibodies partially inhibited elastase, but certain pairs, at a molar ratio of each antibody to elastase of 5.4:1, acted synergistically to inhibit the activity completely.
Lipopolysaccharide-tumor necrosis factor-glucocorticoid interactions during cecal ligation and puncture-induced sepsis in mature versus senescent mice
Authors: Hyde S R , McCallum R E
Published: January 2020

Previous work in our laboratory demonstrated increased sensitivity of senescent (24-month-old) mice to cecal ligation and puncture (CLP) sepsis compared with that of mature (12-month-old) mice. In this study the median lethal dose of the strain of Escherichia coli most frequently isolated during CLP sepsis was determined. No significant age-associated difference in the mean lethal dose or the mean survival time was noted; however, sham surgery before injection of E. coli decreased the mean lethal dose by at least 100-fold. With surgical manipulation, the average time to death after bacterial injection simulated more closely that observed after CLP surgery. Host responses to CLP sepsis were investigated by measuring the levels of corticosterone, glucose, and tumor necrosis factor (TNF) in the sera of mature and senescent mice at 2-h intervals after surgery. Corticosterone levels increased gradually during the course of sepsis in mature mice; however, senescent mice demonstrated a pronounced elevation in hormone levels at 2 and 4 h after surgery. At subsequent sampling intervals the corticosterone levels remained elevated, although they were similar for both ages. At all sampling intervals, the glucose levels in serum were lower in senescent mice than in mature mice. Pronounced hypoglycemia (less than 80 mg/dl) was observed in senescent mice at 8 h postsurgery. TNF was detected in serum within a narrow time frame in both age groups at 6, 8, and 10 h postsurgery. Although elevated TNF levels in serum were not seen in every mouse in each group (approximately 50%), the data hinted that senescent animals produced larger quantities of TNF during CLP sepsis than did mature animals. E. coli lipopolysaccharide (1 mg/kg) was injected intraperitoneally, and the TNF levels in serum and peritoneal lavage fluid were measured at 30, 60, and 90 min. Senescent mice demonstrated a level of TNF in serum at 90 min after lipopolysaccharide treatment that was 20-fold higher than that of mature mice (299,877 pg/ml versus 15,594 pg/ml). The amount of TNF produced locally in the peritoneum was also substantially higher in senescent mice than in mature animals (1,716 pg/ml versus 776 pg/ml). The increased production of TNF in senescent animals, despite elevated circulating corticosterone levels, suggested an age-related defect in glucocorticoid-directed downregulation of TNF production. This was confirmed in lipopolysaccharide-treated animals given exogenous dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)
Mouse model for Pneumocystis carinii pneumonia that uses natural transmission to initiate infection
Authors: Powles M A , McFadden D C , Pittarelli L A , Schmatz D M
Published: January 2020

Animal models for Pneumocystis carinii, for the most part, have been limited to immunosuppressed rats and ferrets, while a dependable mouse model has been more difficult to develop. A P. carinii mouse model has now been established with several strains of mice, including C3Heb/FeJ, C3HeN, BALB/c, DBA/2N, and BALB/c nu/nu (athymic). In lieu of using invasive methods for initiating P. carinii infections, mice harboring P. carinii transmitted the disease to mice without latent infection via short-term cohabitation. After the exposure period, the seed mice were sacrificed to confirm the presence of acute P. carinii pneumonia. Acute infections in recipient mice developed at approximately 7 to 8 weeks, while control unseeded littermates remained uninfected. All recipient mice and their littermates were maintained in isolation hoods to eliminate the possibility of exposure to other sources of P. carinii. This approach allows investigators to consistently transmit P. carinii to mice and to select the strain of mouse desired for use in a particular study. The results presented here suggest that more attention should be given to the potential for patient-to-patient transmission of P. carinii in immunocompromised patients such as those with AIDS.
Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice
Authors: Doyle A G , Halliday W J , Barnett C J , Dunn T L , Hume D A
Published: January 2020

Brucella abortus injected into CBA mice replicated primarily in the spleen and liver, reaching a peak bacterial count in both organs about 7 days postinfection. The organism was eliminated from the liver but declined to a chronic phase in the spleen. The infection caused hepatosplenomegaly. An influx of macrophages into the two organs was monitored by quantitative Northern (RNA blot) analysis of the macrophage-specific marker lysozyme mRNA. Lysozyme mRNA was detectable in spleen and increased three- to fourfold during infection. In liver, lysozyme mRNA was initially undetectable, but at about the peak of infection it reached a level comparable to that in the spleen. Macrophage colony-stimulating factor 1 (CSF-1) has been reported to be elevated in the circulation of animals infected with B. abortus and is known to stimulate monocytopoiesis. To investigate the role of CSF-1 in pathogenesis, we studied the effect of further increasing the CSF-1 concentration by administration of recombinant human CSF-1. Since the infection is characterized by several distinct phases, recombinant human CSF-1 was administered at defined times relative to these phases. Pronounced effects were observed only when CSF-1 administration was begun during the developing acute phase. The consequences were decreased bacterial numbers in the spleen but an increase in the liver, reduced antibody generation, and increased hepatosplenomegaly. A feature of many chronic intracellular infections is immunosuppression. B. abortus caused a substantial diminution of responsiveness of spleen cells to T-cell mitogens, particularly concanavalin A. This action was mimicked by CSF-1 treatment of the animals prior to spleen cell isolation. The results suggest that CSF-1 plays a role in macrophage recruitment in brucellosis and that recruited macrophages contribute to the immunopathology and immunosuppression.
Separation of T-cell-stimulating activity from streptococcal M protein
Authors: Fleischer B , Schmidt K H , Gerlach D , Köhler W
Published: January 2020

The superantigenic properties of M protein type 5 of Streptococcus pyogenes have been implicated as an important pathogenicity factor in streptococcal autoimmune diseases. Here we show that after a single purification step by affinity chromatography on immobilized albumin or fibrinogen, M protein has no mitogenic activity for T cells. We demonstrate that the superantigenicity of M proteins of type 5 and type 1 is due to contamination with the highly potent pyrogenic exotoxins of S. pyogenes in the range of 0.1 to 0.01%. These results raise a general caveat for work with these extremely active T-cell mitogens, because the mitogenicity of other streptococcal or staphylococcal proteins could be due to similar minute contamination with potent superantigens that are undetectable by any biochemical method but extremely effective in stimulating sensitive T cells.
Inactivation of human serum bactericidal activity by a trypsinlike protease isolated from Porphyromonas gingivalis
Authors: Grenier D
Published: January 2020

A protease was isolated from an outer membrane vesicle preparation of Porphyromonas gingivalis ATCC 33277 and assessed for its ability to inactivate the bactericidal activity of normal human serum. The enzyme, which was activated by reducing agents, was found to be a trypsinlike protease with a molecular mass of approximately 80 kDa. Prior to being tested in the bactericidal assay, pooled human serum was preincubated with the partially purified enzyme. Under conditions in which the trypsinlike protease was activated, a strong reduction of the serum bactericidal activity against Capnocytophaga ochracea was noted. On the other hand, no reduction of the bactericidal action of serum was observed when the serum-protease mixture was preincubated in the presence of an inhibitor of the enzyme. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease was shown to degrade immunoglobulins G and M as well as complement factor C3. This study confirms the previous hypothesis that the proteases of P. gingivalis can interfere with the protective action of human serum.
Further evidence of the toxic effect of ammonia produced by Helicobacter pylori urease on human epithelial cells
Authors: Mégraud F , Neman-Simha V , Brügmann D
Published: January 2020

Former studies have shown that Helicobacter pylori can induce vacuolation of vacuolation of epithelial cells in vitro and possibly in vivo, either by direct action of a cytotoxin or by the action of its strong urease, which breaks down the urea physiologically present in the stomach into cytotoxic ammonia. We have developed a test using HEp2 cells with adherent H. pylori bacteria in order to compare the effects of an H. pylori urease-negative variant with those of its urease-positive parent strain in the presence of 10 mM urea. The level of ammonia production as well as cell vacuolation and viability were monitored for 72 h. The ammonia produced (20 mM) was found to be the essential determinant of the degree of cell vacuolation and viability of HEp2 cells. However, the addition of acetohydroxamic acid (200 mg/liter), a potent urease inhibitor which inhibits ammonia production, did not completely restore cell growth, suggesting the difficulty of neutralizing the ammonia in the vicinity of the cells. Antibodies directed against H. pylori did not neutralize the urease activity. When H. mustelae was tested in the same manner, the detrimental effects were not observed because a lower quantity of ammonia (5 mM) was generated. This was due to a lower urease activity, although the adherence properties of H. mustelae were different from those of H. pylori both quantitatively (greater adherence) and qualitatively (localized instead of diffuse adherence). We conclude that H. pylori-induced ammonia is an essential determinant of its cell toxicity as well as its adherence properties, which allow a high concentration of ammonia at the cellular level.
Recognition of Leishmania antigens by T lymphocytes from nonexposed individuals
Authors: Kemp M , Hansen M B , Theander T G
Published: January 2020

Crude antigen preparations of Leishmania promastigote sonicates were found to induce in vitro proliferation and gamma interferon production in peripheral blood mononuclear cells (PBMC) from individuals without known exposure to the parasite. The proliferating cells were mainly CD2-positive T cells. The proliferative response was maximal after more than 6 days of incubation with the antigens in contrast to the proliferation induced by the mitogenic lectin phytohemagglutinin (PHA), which peaked after 3 to 5 days. Judged by limiting dilution analysis, the frequencies of antigen-reactive precursor cells were less than 1:10,000 and varied considerably between individuals. Depletion of CD45R0-positive (memory) cells from the PBMC abolished proliferative responses induced by Leishmania antigen and by tetanus toxoid. In cell populations depleted of CD45RA-positive (naive) cells, only a small reduction in response was observed. Cell populations depleted of either CD45R0-positive cells or CD45RA-positive cells both responded to PHA. We conclude that presumably unexposed individuals have a low number of Leishmania-reactive T cells in their circulatory systems. The Leishmania-reactive T cells in these individuals are most likely memory cells recognizing antigens present both in the Leishmania preparations and in the environment.
Laminin enhances binding of Toxoplasma gondii tachyzoites to J774 murine macrophage cells
Authors: Furtado G C , Slowik M , Kleinman H K , Joiner K A
Published: January 2020

We investigated the effects of the extracellular matrix proteins laminin and fibronectin on the attachment of tachyzoites of Toxoplasma gondii to the murine macrophage cell line J774. Laminin but not fibronectin increased parasite attachment to J774 cells in a dose-dependent fashion. Cyclic YIGSR, a laminin-derived peptide which inhibits laminin binding to the 32/67-kDa laminin-binding protein on host cells, blocked the laminin-mediated enhancement of parasite attachment. An antiserum to the 32/67-kDa laminin-binding protein also inhibited binding of parasites to J774 cells. These results, in conjunction with our previous observations (G. C. Furtado, F. L. Collins, and K. A. Joiner, submitted for publication), demonstrate that tachyzoites bearing surface laminin bind to multiple laminin receptors in attaching to different target cells.
Identification and initial characterization of five Cryptosporidium parvum sporozoite antigen genes
Authors: Petersen C , Gut J , Leech J H , Nelson R G
Published: January 2020

Cryptosporidium parvum, an Apicomplexan parasite of gastrointestinal epithelial cells, causes severe disease in persons with AIDS and is a common cause of self-limited diarrhea in children, animal handlers, and residents of developing countries. No approved therapy exists; in research studies, however, hyperimmune bovine colostrum raised to Cryptosporidium oocysts and sporozoites has eradicated disease or decreased parasite burden in some AIDS patients. Although the protective antigens recognized by bovine hyperimmune colostrum have not been defined, protective antigens of other Apicomplexan parasites frequently have been associated with two unique structures of invasive forms, the trilaminar pellicle and the apical complex. In order to identify immunogenic Cryptosporidium proteins that may be protective antigens for use as recombinant immunogens in passive and/or active immunotherapy, we screened two genomic DNA expression libraries with polyspecific anti-Cryptosporidium antibodies. We used an approach to cloning apical complex and pellicle protein antigens that succeeded despite the lack of large numbers of organisms that would be necessitated for conventional biochemical approaches requiring organelle or membrane purification. We report here the molecular cloning of five C. parvum genes and the characterization of the cognate sporozoite proteins having molecular masses of greater than 500, 68/95, 45, 23, and 15/35 kDa. The light microscopic immunofluorescence pattern of antibodies recognizing these protein antigens suggest that they are located in the pellicle or apical complex of Cryptosporidium sporozoites.
Isolation and antigenicity of a 45-kilodalton Paracoccidioides brasiliensis immunodominant antigen
Authors: Ferreira-da-Cruz M F , Galvão-Castro B , Daniel-Ribeiro C
Published: January 2020

In the present study, we analyzed human antibody responses to Paracoccidioides brasiliensis cellular antigens by the immunoblot technique to identify specific cellular components and to investigate the existence of antigen profile differences among serological responses of paracoccidioidomycosis (PCM) patients. Among the 64 PCM serum samples analyzed, a relatively homogeneous immunoglobulin G response to P. brasiliensis antigens was observed. The polypeptide with a mass of 45 kDa was the most clinically important, since antibody to this antigen was detectable in 90.6% of PCM patients studied and the six individuals who did not produce antibody were either at the end of treatment or in the posttherapy period and had shown clinical recovery. These facts suggested that the presence of this antibody may be an indicator of active disease. The 45-kDa antigen was also the most specific antigen of the PCM humoral immune response, since it reacted with only 2 of 79 (2.5%) heterologous serum samples tested: 1 histoplasmosis case and 1 tuberculosis case. This polypeptide was isolated from gels by electroelution and, when tested by an immunoradiometric assay and immunoblotting, maintained its reactivity with PCM sera and also with anti-P. brasiliensis polyclonal antibodies raised in rabbits at the same sensitivity levels as those obtained in immunoblotting with a crude antigen. Since in our assays the 45-kDa polypeptide was the major P. brasiliensis antigen and seemed to be specific for PCM, its use in alternative diagnostic methods is promising, especially in patients suspected of having the juvenile clinical form of PCM often associated with negative double-immunodiffusion results.
Immunogenicity and antigenic heterogeneity of a human transferrin-binding protein in Neisseria meningitidis
Authors: Ferron L , Ferreiros C M , Criado M T , Pintor M
Published: January 2020

Growing Neisseria meningitidis on an iron restriction medium induces the synthesis of new outer membrane proteins, some of them true iron-regulated outer membrane proteins (IROMPs) and others synthesized because of the stress produced by the iron restriction. Some of these proteins are antigenic and can be considered for the development of vaccines; this is especially desirable in the case of N. meningitidis serogroup B, for which polysaccharide vaccines are not efficient. The antigenicity of N. meningitidis 37- and 70-kDa IROMPs has been studied previously; in this work, we studied the immunogenicity and antigenic heterogeneity of another IROMP, the human transferrin-binding protein 2 (TBP2), which seems to be indispensable for meningococcal growth inside the host. Mice were inoculated with purified outer membrane vesicles (blebs) from 5 selected N. meningitidis strains, and the five serum samples obtained were analyzed for anti-TBP2 antibodies by using the homologous strain and for cross-reactivity with the TBP2 of the 4 other selected strains and another 35 heterologous N. meningitidis strains. The TBP2s of the 5 strains tested were all immunogenic in mice to various degrees depending on the strain, and all five TBP2s shared one or more epitopes with heterologous strains (as shown by the cross-reactivities of the five serum samples), although the number of cross-reacting strains was very variable, ranging from 2 for strain V002 to 35 for strain P391. This suggests that the TBP2 epitopes of different strains differ in nature or in their accessibility to the immune system. Under the iron restriction conditions used, all strains synthesized a non-TBP2 antigenic 56-kDa protein thought to be a stress protein.
Localization and biochemical characterization of the ORF6 gene product of the Mycoplasma pneumoniae P1 operon
Authors: Layh-Schmitt G , Herrmann R
Published: January 2020

ORF6 represents one of the two open reading frames flanking the P1 attachment protein gene of Mycoplasma pneumoniae in the order ORF4-P1-ORF6 (J.M. Inamine, T.P. Denny, S. Loechel, U. Schaper, C.H. Huang, K.F. Bott, and P.C. Hu, Gene 64:217-219, 1988; J.M. Inamine, S. Loechel, and P.C. Hu, Gene 73:175-183, 1988; C.J. Su, V.V. Tryon, and J.B. Baseman, Infect. Immun. 55:3023-3029, 1987), indicating an operonlike organization. As described previously, we identified two proteins with molecular masses of 40 and 90 kDa (B. Sperker, P.C. Hu, and R. Herrmann, Mol. Microbiol. 5:299-306, 1991) which might represent two cotranslational cleavage fragments of the ORF6 gene product. To determine the site of the putative cotranslational cleavage, the first 10 amino acids of the N terminus of the isolated 90-kDa protein were sequenced. The data are consistent with the DNA-deduced amino acid sequence between amino acid positions 455 and 465 (RAGNSSETDAL). Thus, the cleavage site was identified at amino acid position 455 (R). In this study, the two proteins were localized and biochemically characterized. Both proteins are part of the insoluble fraction of M. pneumoniae as shown by immunoblots of supernatants and pellets of mechanically disrupted cells subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Surface proteolysis followed by SDS-PAGE and Western blot (immunoblot) analysis, covalent labelling of surface-exposed proteins with [125I]iodide and subsequent immunoprecipitation of both radiolabelled proteins, immunofluorescence studies with formalized and living M. pneumoniae, and immunoadsorption experiments provided strong evidence that the 40- and 90-kDa proteins are membrane-associated proteins expressing surface-exposed regions.
Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants
Authors: Byrd W , Kadis S
Published: January 2020

Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP.
Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages
Authors: van't Wout J , Burnette W N , Mar V L , Rozdzinski E , Wright S D , Tuomanen E I
Published: January 2020

Pertussis toxin (PT) and filamentous hemagglutinin can each mediate the association of Bordetella pertussis with human macrophages. Adherence via filamentous hemagglutinin leads to integrin-mediated entry and survival of the bacteria within the human cell. We determined the contribution of PT to bacterial adherence to human macrophages. Plating macrophages on wells coated with recombinant PT subunit 2 (S2) or S3 decreased PT-dependent bacterial binding by greater than 60%; S1, S4, and S5 were ineffective. S3-dependent adherence was reduced 63% +/- 8% by sialic acid, while S2-dependent adherence was reduced 53% +/- 11% by galactose. Loss of the carbohydrate recognition properties of S2 by deletion of residues 40 to 54 or site-specific mutations at Asn-93, His-47, or Arg-50 eliminated the ability of the subunit protein to competitively inhibit bacterial binding. Peptides corresponding to residues 28 to 45 of S2 and S3 competitively inhibited adherence. Treatment of macrophages with antibodies to Le(a) or Le(x) but not CD14, CD15, CD18, or HLA interfered with PT-mediated binding. Exposure of the macrophages to the B oligomer, S2, or S3 increased binding to the CD11b/CD18 integrin. These results indicate that the carbohydrate recognition domains of both S2 and S3 participate in adherence of B. pertussis to human macrophages. The PT receptor(s), as yet unidentified, appears to carry the Le(a) or Le(x) determinants and is functionally capable of modulating integrin-mediated binding to the macrophage.
Acanthamoebae bind to glycolipids of rabbit corneal epithelium
Authors: Panjwani N , Zhao Z , Baum J , Pereira M , Zaidi T
Published: January 2020

By use of a thin-layer chromatogram (TLC) overlay procedure, 35S-labeled acanthamoebae were shown to bind to seven glycolipids of rabbit corneal epithelium. Corneal epithelial cells were grown in culture and were subjected to Folch extraction to isolate a chloroform-rich lower phase containing neutral glycosphingolipids (NGSL) and an aqueous upper phase containing gangliosides, i.e., sialic acid-containing glycolipids. Thin-layer chromatography of the upper phase revealed the presence of 10 ganglioside components. Acanthamoebae were shown to bind to four of these components, referred to as 2, 3, 6, and 7. On TLC plates, ganglioside components 2 and 3 migrated slightly ahead of the glycolipid standard GD1a, component 7 comigrated with standard GM3, and component 6 migrated a little more slowly than GM3. Likewise, of the 10 NGSL known to be present in the lower phase, acanthamoebae bound to components 1, 5, and 6. NGSL components 1, 5, and 6 migrated on TLC plates with relative mobilities similar to those of standards asialo GM1, asialo GM2, and ceramidetrihexoside, respectively. We propose that one or more of the Acanthamoeba-reactive glycolipids of corneal epithelium identified in this study may play a role in the pathogenesis of Acanthamoeba keratitis by mediating the adherence of the parasites to the cornea.
The Th1/Th2-like switch in syphilitic infection: is it detrimental?
Authors: Fitzgerald T J
Published: January 2020

Organisms that cause chronic diseases have evolved mechanisms to evade those immune defenses that resolve the acute stage of infection (10, 12-14, 21, 22, 32, 35, 37, 38, 40, 42, 45-49, 53). Much is to be learned by specifically identifying the mechanisms underlying these evasive strategies. Important new insights will emerge in terms of immunoregulatory pathways. This in turn will facilitate vaccine development. A good example is leishmania infection. The acute stage of this disease is resolved by DTH-macrophage activation. Leishmanial components preferentially activate Th2 lymphocytes. As a consequence, Th1 effects are minimized and infection is exacerbated leading to chronicity (10, 14, 32). To overcome this negative tendency, leishmanial vaccines are administered in combination with exogenous gamma interferon (42). This selects for Th1 predominance and generates protective immunity. Syphilis exhibits many parallels to the other nine chronic diseases mentioned above. Similarities include an acute localized stage that readily heals, early clearance via DTH-macrophage activation, transient concomitant immunity during acute infection, development of macrophage suppression through PGE2 down-regulation, beneficial effects of exogenous gamma interferon, and elements of autoimmunity. Some of the complexities of immunoregulation during treponemal infection have just begun to be unraveled. It will be important to develop further insight into the Th1/Th2 switch especially as it relates to chronicity. Macrophages seem to be intimately involved in the mechanics of this switch, and their specific role needs further clarification. Whatever is learned about syphilis, as well as other chronic infections will contribute to a better understanding of the generalized pathways of immunoregulation.