bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphocyte populations during tuberculosis infection: V beta repertoires
Authors: Gambón-Deza F , Pacheco Carracedo M , Cerdá Mota T , Montes Santiago J
Published: January 2020

The immune response to Mycobacterium tuberculosis is mediated by T lymphocytes. We studied the changes in lymphocyte populations occurring in peripheral blood, pleural fluid, and ascites during tuberculosis infection. For this purpose, we compared recent-onset patients (newly converted to positive Mantoux reactions) with previously diagnosed patients (individuals with organic lesions). Recent infection was associated with peripheral blood lymphocytosis involving T lymphocytes expressing either T-cell receptor alpha/beta or gamma/delta. Lymphocytosis involved both CD4 and CD8 cells. On the other hand, we detected no changes in the distribution of peripheral blood lymphocyte populations in previously diagnosed patients. No changes were found in the numbers of B lymphocytes or natural killer cells in either recently infected or previously diagnosed patients. The pleural effusion and ascitic fluid samples contained T lymphocytes expressing T-cell receptor alpha/beta, the majority of which were CD4+. These lymphocytes showed an inverted CD45RA-to-CD45RO ratio, and we found high-level expression of the interleukin-2 receptor (CD25) in some patients. The results are compatible with the existence of periods of cell activation in the pleural fluid (which are disclosed by the appearance of the CD25 antigen and the transition of CD45RA expression to CD45RO) together with nonactivation periods (loss of CD25 and persistence of CD45RO expression). We studied a fraction of the V beta repertoire in peripheral blood in both groups and the same fraction of the V beta repertoire in pleural fluid from patients with tuberculous pleuritis, demonstrating that, in recently infected subjects, lymphocytosis was produced by the increase in lymphocytes which expressed some specific V beta subfamilies that differed from one individual to another. In two of five patients studied, we found significant changes in the V beta repertoire between lymphocytes from peripheral blood and the pleural fluid samples.
Parasite strain specificity of precursor cytotoxic T cells in individual animals correlates with cross-protection in cattle challenged with Theileria parva
Authors: Taracha E L , Goddeeris B M , Morzaria S P , Morrison W I
Published: January 2020

Class I major histocompatibility complex-restricted parasite-specific cytotoxic T lymphocytes (CTL) are known to be a major component of the bovine immune response to the protozoan parasite Theileria parva, but formal proof for their role in protection of cattle against infection with T. parva has been lacking. Animals immunized with one stock of T. parva show variations in the degree of protection against heterologous challenge and also in the parasite strain specificity of their CTL responses. The present study investigated the relationship of strain specificity of CTL responses and cross-protection in an effort to verify the role of CTL in protection. The parasite strain specificity of the CTL responses generated in 23 cattle immunized with either of two immunologically distinct parasite populations was examined, and the susceptibility of individual cattle to challenge with the heterologous parasite population was determined. The frequency of stock-specific or cross-reactive CTL precursor cells (CTLp) in individual animals was measured by a limiting-dilution microassay. A proportion of animals immunized with either parasite exhibited cross-reactive CTLp, whereas CTLp detected in the remaining animals were specific for the homologous parasite. On challenge with the heterologous stock, those animals with cross-reactive CTLp were solidly protected while those with strain-specific CTLp showed moderate to severe reactions, although many of them recovered. The finding of a close association between strain specificity of the CTL response and protection against challenge provides strong evidence that CTL are important in mediating immunity.
A Mycoplasma strain F38 growth-inhibiting monoclonal antibody (WM-25) identifies an epitope on a surface-exposed polysaccharide antigen
Authors: Rurangirwa F R , Wambugu A , Kihara S M , McGuire T C
Published: January 2020

Monoclonal antibody (MAb) WM-25 differentiates by in vitro growth inhibition Mycoplasma capricolum subsp. capripneumoniae (Mycoplasma strain F38), which causes contagious carpine pleuropneumonia, from other Mycoplasma spp. (F. R. Rurangirwa, T. C. McGuire, A. J. Musoke, and A. Kibor, Infect. Immun. 55:3219-3220, 1987). The antigen identified by MAb WM-25 was isolated from solubilized Mycoplasma strain F38 organisms by MAb WM-25 affinity chromatography and was stained with Schiff's reagent, but not with Coomassie blue, after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of purified F38 polysaccharide with periodate abolished binding with MAb WM-25, and MAb WM-25 binding was blocked with laminarin, a complex oligosaccharide with beta(1-->3) sugar linkages. Purified F38 polysaccharide blocked both growth inhibition and agglutination of live F38 organisms caused by MAb WM-25 and rabbit antiserum to F38 organisms. The results in this paper demonstrate that MAb WM-25 binds a periodate-sensitive epitope on the F38 polysaccharide which is also exposed on the surface of Mycoplasma strain F38. Because MAb WM-25 also causes in vitro growth inhibition of F38, the reactive polysaccharide epitope may induce protective immune responses.
Cloning and sequence analysis of a protective M-like protein gene from Streptococcus equi subsp. zooepidemicus
Authors: Timoney J F , Walker J , Zhou M , Ding J
Published: January 2020

Streptococcus equi subsp. zooepidemicus, a Lancefield group C streptococcus, is a frequently isolated opportunist pathogen from a variety of animal hosts, including the horse. Previous studies have indicated that equine strains carry antigens with characteristics of the antiphagocytic M proteins on the Lancefield groups A and G streptococci. We have cloned a protective M-like protein gene (SzPW60) of an equine strain of S. equi subsp. zooepidemicus W60 and determined its sequence. This gene encodes a protein with a molecular weight of 40,123 which protects mice against subsp. zooepidemicus but not subsp. equi, stimulates antibodies which opsonize subsp. zooepidemicus but not equi, and reacts with antiserum to the protein of the parent strain. The predicted amino acid structure shows significant homology with the carboxy termini of groups A and G M proteins but no other homology. The M-like protein, although showing an extensive region of alpha helix, lacks the A, B, and C repeats found in group A M proteins and has a shorter signal sequence. A proline-rich region upstream from the LPSTGE motif contains 20 repeats of the tetrapeptide PEPK. The presence of this repeat region may account for the slow migration of the M-like protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Paracoccidioides brasiliensis-stimulated human gamma/delta T cells support antibody production by B cells
Authors: Munk M E , Fazioli R A , Calich V L , Kaufmann S H
Published: January 2020

Paracoccidioidomycosis patients show hyperactive humoral immune responses. Consequently, we investigated whether cytokines in supernatants from Paracoccidioides brasiliensis-stimulated gamma/delta T cells support B-cell activation. We detected proliferation of B cells and increased immunoglobulin M (IgM) and IgG production. Thus, gamma/delta T cells may participate in polyclonal B-cell activation during paracoccidioidomycosis.
Interleukin-8 response of gastric epithelial cell lines to Helicobacter pylori stimulation in vitro
Authors: Sharma S A , Tummuru M K , Miller G G , Blaser M J
Published: January 2020

Gastric infection with Helicobacter pylori activates a mucosal inflammatory response by mononuclear cells and neutrophils that includes expression of cytokines interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, and IL-8. In this study, we analyzed the IL-8 response of human gastric cancer cell lines (Kato III, AGS, and MKN28) to H. pylori infection in vitro. IL-8 mRNA expression was detected by reverse transcription-PCR amplification of RNA extracted from epithelial cells after incubation with different H. pylori wild-type and mutant strains, and IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Exposure to viable H. pylori induced IL-8 mRNA and protein synthesis in all three gastric cell lines but not in nongastric epithelial cell lines. Heat-killed H. pylori and a crude cytotoxin preparation did not induce significant IL-8 secretion. IL-8 mRNA peaked between 2 and 4 h postinfection, and IL-8 protein production was maximal 24 h postinfection. Exposure of gastric carcinoma cells to other gastrointestinal bacteria, such as Pseudomonas aeruginosa, Campylobacter jejuni, and Escherichia coli, but not Campylobacter fetus, induced IL-8 synthesis. Wild-type strains that expressed the vacuolating cytotoxin (Tox+) and a cytotoxin-associated gene (cagA) product (CagA+) induced significantly more IL-8 than did CagA- Tox- strains. However, there was no decrease in IL-8 induction by isogenic mutants of CagA-, Tox-, or Cag- Tox- strains or by a mutant lacking the urease subunits. These results indicate that exposure to H. pylori and other gram-negative organisms that do not colonize the gastric mucosa induces IL-8 production by gastric carcinoma cells in vitro. Although the CagA+ Tox+ phenotype of H. pylori is associated with enhanced IL-8 production by gastric cell lines, other bacterial constituents are clearly essential.
Interleukin-5 is the predominant cytokine produced by peripheral blood mononuclear cells in alveolar echinococcosis
Authors: Sturm D , Menzel J , Gottstein B , Kern P
Published: January 2020

An involvement of cellular immunity in alveolar echinococcosis is strongly suggested by the intense granulomatous infiltrations observed around the hepatic parasite lesions. However, the basis of cellular immunoregulation in patient with alveolar echinococcosis is poorly understood. The present report shows a comparative analysis of lymphoid cell function in peripheral blood mononuclear cells (PBMC) of 16 patients with alveolar echinococcosis and of healthy individuals. Our in vitro restimulation studies with crude Echinococcus multilocularis antigen demonstrated that PBMC from patients with alveolar echinococcosis were responsive to challenge with parasitic antigen as measured by lymphoid cell proliferation. In this system, we also evaluated cytokine expression at the gene and protein levels after stimulation with E. multilocularis antigen. Analysis of cytokine mRNA expression revealed distinct patterns of cytokine expression in patients and normal donors. By using reverse transcriptase PCR, we could demonstrate that the TH1 cytokine transcripts interleukin-2 (IL-2) and gamma interferon (IFN-gamma) are present in PBMC from patients with alveolar echinococcosis. Moreover, it was found that stimulation with E. multilocularis antigen induced or enhanced the expression of the TH2 cytokine IL-3, IL-4, IL-10, and especially IL-5 mRNAs in PBMC from 13 of 16 patients with alveolar echinococcosis. Two patients who were examined after radical surgery, as well as another patient with a stable course of the disease under continuous chemotherapy, were not able to generate the same pattern of cytokine response and had no evidence of IL-5 mRNA synthesis. In contrast to the frequent expression of TH2 cytokine mRNAs observed in patients with alveolar echinococcosis, PBMC cultures from normal donors showed prominent IL-2 and IFN-gamma mRNA expression but weak IL-3, IL-4, and IL-10 mRNA expression. Most interestingly, IL-5 mRNA was substantially absent in PBMC from healthy individuals. In accordance with the mRNA studies, it was found that E. multilocularis antigen induced the secretion of large amounts of IL-5 and intermediate amounts of IFN-gamma in patients with alveolar echinococcosis, whereas large amounts of IFN-gamma and no or threshold amounts of IL-5 were detected in supernatants from healthy individuals. Collectively, the present study provides the first evidence that a TH2 immune response is gradually activated during the course of E. multilocularis infection, indicating a critical role for IL-5 in the manifestation of human alveolar echinococcosis.
Endobronchial inflammation following Pseudomonas aeruginosa infection in resistant and susceptible strains of mice
Authors: Morissette C , Skamene E , Gervais F
Published: January 2020

The early endobronchial inflammation induced by Pseudomonas aeruginosa infection varies in resistant and susceptible strains of mice. Mice of the DBA/2 strain are severely afflicted by the infection, with a high bacterial burden accumulating rapidly following inoculation and a high mortality rate occurring. Mice of the BALB/c strain are resistant to infection and clear the bacteria within 3 to 7 days. Infection of (BALB/c x DBA/2)F1 hybrid mice showed that the resistance to lung P. aeruginosa infection is inherited as a dominant trait. Mice of the A/J and C57BL/6 strains were found to have an intermediate phenotype to Pseudomonas aeruginosa infection when compared with BALB/c and DBA/2 strains. The decrease in the bacterial load seen early after infection coincided with a steady and strong recruitment of inflammatory cells to the bronchoalveolar spaces of mice of the resistant BALB/c strain. On the other hand, the recruitment of inflammatory cells to the lungs of mice of the susceptible DBA/2 strain was deficient, resulting in the failure to control bacterial multiplication. Chemotactic factors, proinflammatory cytokines, and the number and function of recruited inflammatory cells may play major roles in the determination of the genetic resistance to lung infection with P. aeruginosa in a normal immunocompetent host.
Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice
Authors: Aguirre K , Havell E A , Gibson G W , Johnson L L
Published: January 2020

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.
Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774 macrophages
Authors: Tsolis R M , Bäumler A J , Heffron F
Published: January 2020

The Salmonella typhimurium gene for Mn-cofactored superoxide dismutase (sodA) was cloned by complementation of an Escherichia coli sodA sodB mutant for growth on minimal medium. Sequence analysis revealed an open reading frame of 618 bp encoding a polypeptide with 97% identity to E. coli SodA. A S. typhimurium sodA mutant was created by allelic exchange and tested for the ability to survive in the murine macrophage-like cell line J774. Growth of bacteria under iron-limiting conditions, inactivation of the Fur repressor, or expression of sodA from a plasmid resulted in increased resistance to early killing by J774 cells, which was abolished in the sodA mutant. These results suggest that resistance to the early oxygen-dependent microbicidal mechanisms of phagocytes involves the SodA gene product. The S. typhimurium sodA mutant was not significantly attenuated in mice, however, which suggests that resistance to early oxygen-dependent microbicidal mechanisms in vivo may play only a minor role in Salmonella pathogenesis.
Local Th1-like responses are induced by intravaginal infection of mice with the mouse pneumonitis biovar of Chlamydia trachomatis
Authors: Cain T K , Rank R G
Published: January 2020

A critical role for cell-mediated immunity (CMI) has been demonstrated for effecting the resolution of genital infections of mice infected intravaginally with the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn). However, little is known about expression of CMI in the murine genital tract. The mouse MoPn model was used to examine CMI responses in the genital tract and associated lymph nodes during the course of infection. MoPn-specific lymphocytes were present in the genital mucosa, with the maximum level of proliferation in response to MoPn at 3 weeks postinfection. MoPn-stimulated cells secreting gamma interferon were also detected in the cells from the genital mucosa, but few interleukin-4-secreting cells were seen at any time postinfection, indicating the induction of a Th1-like response in the cells of the genital mucosa. The iliac node draining the genital tract was the major node stimulated as a result of a genital infection and exhibited a predominant Th1-like pattern of cytokine secretion as well. Mesenteric lymph node cells demonstrated poor proliferative responses to MoPn and few antigen-stimulated cytokine-secreting cells after the primary infection. However, 7 days after a second infection administered 50 days following the primary infection, there was a marked increase in both proliferative responses and the frequencies of MoPn-stimulated gamma interferon- and interleukin-4-secreting cells. These studies provided information regarding the local CMI response to MoPn in mice which may prove valuable in the development of vaccination strategies for the prevention of chlamydial genital infections.
Differential susceptibility of yeast and hyphal forms of Candida albicans to macrophage-derived nitrogen-containing compounds
Authors: Blasi E , Pitzurra L , Puliti M , Chimienti A R , Mazzolla R , Barluzzi R , Bistoni F
Published: January 2020

Candida albicans is a dimorphic fungus capable of transition from the yeast form (Y-Candida) to the hyphal form (H-Candida). Both Y-Candida and H-Candida are known to be growth inhibited by murine macrophages (M phi) in vitro. In the present report, we demonstrate that M phi exposed to interferon gamma (IFN-gamma) plus lipopolysaccharide (LPS) show enhanced anti-Y-Candida and anti-H-Candida activities. To further investigate the phenomenon, Y-Candida and H-Candida were assessed for susceptibilities to M phi-derived supernatants. Only the growth of H-Candida, and not that of Y-Candida, is impaired by cell-free supernatants from M phi treated with IFN-gamma plus LPS. In contrast, no H-Candida growth inhibition occurs when supernatants from M phi exposed to IFN-gamma plus LPS in the presence of NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthesis, are employed. Finally, supernatants from M phi incubated with sodium nitroprusside, an NO-generating agent, also show anti-H-Candida activity. In conclusion, these results indicate that H-Candida but not Y-Candida is susceptible to extracellular antifungal mechanisms employed by M phi, which likely involve stable nitrogen-containing compounds.
Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii
Authors: Holmes A R , Gopal P K , Jenkinson H F
Published: January 2020

Candida albicans ATCC 10261 and CA2 bound to cells of the oral bacteria Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguis when these bacteria were immobilized onto microtiter plate wells, but they did not bind to cells of Streptococcus mutans or Streptococcus salivarius. Cell wall polysaccharide was extracted with alkali from S. gordonii NCTC 7869, the streptococcal species to which C. albicans bound with highest affinity, and was effective in blocking the coaggregation of C. albicans and S. gordonii cells in the fluid phase. When fixed to microtiter plate wells, the S. gordonii polysaccharide was bound by all strains of C. albicans tested. The polysaccharide contained Rha, Glc, GalNAc, GlcNAc, and Gal and was related compositionally to previously characterized cell wall polysaccharides from strains of S. oralis and S. sanguis. The adherence of yeast cells to the immobilized polysaccharide was not inhibitable by a number of saccharides. Antiserum raised to the S. gordonii NCTC 7869 polysaccharide blocked adherence of C. albicans ATCC 10261 to the polysaccharide. The results identify a complex cell wall polysaccharide of S. gordonii as the coaggregation receptor for C. albicans. Adherent interactions of yeast cells with streptococci and other bacteria may be important for colonization of both hard and soft oral surfaces by C. albicans.
Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures
Authors: Nishihara T , Ueda N , Amano K , Ishihara Y , Hayakawa H , Kuroyanagi T , Ohsaki Y , Nagata K , Noguchi T
Published: January 2020

The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Furthermore, a correlation between IL-1 alpha and prostaglandin E2 in the osteoclast recruitment induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide is discussed.
Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein
Authors: Malik A , Houghten R , Corradin G , Buus S , Berzofsky J A , Hoffman S L
Published: January 2020

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.
Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae
Authors: Berry A M , Alexander J E , Mitchell T J , Andrew P W , Hansman D , Paton J C
Published: January 2020

The thiol-activated toxin pneumolysin is a known pneumococcal virulence factor, with both cytotoxic (hemolytic) and complement activation properties. Copies of the pneumolysin gene carrying defined point mutations affecting either or both of these properties were introduced into the chromosome of Streptococcus pneumoniae D39 by insertion-duplication mutagenesis. The virulences of these otherwise isogenic strains were then compared. There was no significant difference in either the median survival time or overall survival rate between mice challenged with D39 derivatives producing the wild-type toxin and those expressing a pneumolysin gene with an Asp-385-->Asn mutation, which abolishes the complement activation property. However, mice challenged with strains carrying either His-367-->Arg or Trp-433-->Phe plus Cys-428-->Gly mutations, which reduce hemolytic activity to approximately 0.02 and 0.0001% of the wild-type level, respectively, had significantly greater median survival times and overall survival rates than mice challenged with D39 derivatives expressing a wild-type pneumolysin gene. No additional reduction in virulence was observed when mice were challenged with a D39 derivative carrying Trp-433-->Phe, Cys-428-->Gly, and Asp-385-->Asn, rather than Trp-433-->Phe and Cys-428-->Gly, mutations in the pneumolysin gene. Thus, it appears that in the intraperitoneal challenge model, the contribution of pneumolysin to virulence is largely attributable to its hemolytic (cytotoxic) properties rather than to its capacity to activate complement. Interestingly, however, the amount of pneumolysin required for full virulence may be very small, as D39 derivatives carrying the Trp-433-->Phe mutation (which reduces hemolytic activity to 0.1% of the wild-type level) had intermediate virulence.
Discrimination of virulent and avirulent Streptococcus suis capsular type 2 isolates from different geographical origins
Authors: Quessy S , Dubreuil J D , Caya M , Higgins R
Published: January 2020

In an effort to relate the protein profile to virulence, proteins from the cellular fractions and from culture supernatants of Streptococcus suis capsular type 2 strains from different geographical origins were compared by using Western blots (immunoblots). The protein profiles of the cellular fractions were similar for the majority of virulent and avirulent isolates studied, with the exception of three virulent Canadian strains for which a 135-kDa protein was not detected. Examination of the culture supernatants revealed the presence of a 135-kDa protein in all strains except the same three virulent Canadian isolates. In addition, a 110-kDa protein was present in 14 of 16 virulent strains and not in avirulent isolates. When injected into mice, the 110-kDa protein induced an immunoglobulin G response and protected against infection with homologous and heterologous virulent strains. Four strains (1330, 0891, TD10, and R75/S2) that were avirulent in the mouse model of infection and four other strains (1591, 999, JL590, and AAH4) that were virulent in the mouse model were injected into pigs. All virulent strains reproduced the disease, and all avirulent strains failed to reproduce the disease (with the exception of transient lameness in one case and fever in another case). The 110-kDa protein therefore appears to be a reliable virulence marker and a good candidate for a subunit vaccine.
Antigens derived from lung-stage larvae of Schistosoma mansoni are efficient stimulators of proliferation and gamma interferon secretion by lymphocytes from mice vaccinated with attenuated larvae
Authors: Mountford A P , Harrop R , Wilson R A
Published: January 2020

Protective immunity in C57BL/6 mice exposed to optimally irradiated larvae of Schistosoma mansoni operates against challenge parasites in the lungs and is dependent upon T-helper 1 (Th1) lymphocytes which secrete abundant gamma interferon (IFN-gamma). As an initial step in the identification of the molecules which mediate this immunity, antigenic materials released by larvae at various times during in vitro culture were compared for the ability to induce proliferation of lymph node cells recovered from mice 4 to 6 days after exposure to attenuated parasites. Cells from mice vaccinated with cercariae proliferated most strongly to larval antigens released soon after transformation. In contrast, cells from mice immunized with lung-stage schistosomula responded poorly to these early secretions but proliferated vigorously to antigens released by older larvae. In further studies on the cytokine profile of the responding lymphocytes, it was observed that the balance between IFN-gamma and interleukin-4 (IL-4) secretion depended on the source of antigen used for restimulation. Thus, material released between days 6 and 8 by in vitro-cultured larvae, and the soluble extracts of whole lung-stage larvae, induced abundant IFN-gamma but little IL-4. This finding implies that an overwhelming proportion of the lymphocytes responsive to lung-stage antigens had the Th1 phenotype. In contrast, antigens from cercariae and skin-stage larvae induced the lowest levels of IFN-gamma but higher levels of IL-4. It appears that a proportion of the cells with specificities for early antigens had the Th2 or Th0 phenotype. Our results emphasize that antigens from lung-stage larvae are an important source of potentially protective molecules.
A recombinant Leishmania chagasi antigen that stimulates cellular immune responses in infected mice
Authors: Wilson M E , Young B M , Andersen K P , Weinstock J V , Metwali A , Ali K M , Donelson J E
Published: January 2020

Cellular immune mechanisms resulting in gamma interferon production are critical for protection against visceral leishmaniasis. Antigens stimulating T-cell responses are likely present in the intracellular amastigote form of the parasite, since this is the form found in a mammalian host. To identify T-cell antigens of Leishmania chagasi, the parasite causing South American visceral leishmaniasis, we used a double antibody-T-cell technique to screen an amastigote cDNA library. One cDNA selected (Lcr1) encodes an antigen that stimulated proliferation of splenic T lymphocytes from infected mice that were either resistant (C3H.HeJ) or susceptible (BALB/c) to L. chagasi infection. The Lcr1 cDNA contains four highly divergent 201-bp repeats homologous to the 204-bp repeat of a Trypanosoma cruzi flagellar antigen gene. Results are consistent with a single copy of the Lcr1 gene producing an mRNA of > 10 kb and a protein of > 200 kDa. Recombinant Lcr1, cloned adjacent to polyhistidine and purified on a nickel affinity column, stimulated gamma interferon but not interleukin-4 (IL-4), IL-5, or IL-10 secretion by T-cell-enriched splenocytes from either susceptible or resistant mice during L. chagasi infection. Immunization with Lcr1 partially protected BALB/c mice against challenge with L. chagasi, indicating the utility of the double screening approach in selecting relevant T-cell antigens.
A toxic shock syndrome toxin 1 mutant that defines a functional site critical for T-cell activation
Authors: Cullen C M , Blanco L R , Bonventre P F , Choi E
Published: January 2020

Toxic shock syndrome toxin 1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a causative agent of toxic shock syndrome (TSS). This superantigen is a potent stimulator of T cells and macrophages/monocytes, resulting in the release of cytokines that are implicated in the pathogenesis of TSS. This study characterizes a mutant TSST-1, derived by site-directed mutagenesis, that has an alanine substitution at histidine 135 (mutant 135). This single-amino-acid change results in a mutant toxin that has lost mitogenic activity for T cells. In contrast to wild-type TSST-1, this mutant does not induce T cells to express interleukin-2, gamma interferon, or tumor necrosis factor beta (TNF-beta). The inability of mutant 135 to activate T cells is not due to a lack of binding to the class II major histocompatibility complex receptor. In addition, the mutant TSST-1 does not induce expression of TNF-alpha, which plays a role in the development of lethal shock. The lack of TNF-alpha induction by mutant 135 is likely due to its inability to activate T cells. These data suggest that the mutation at histidine 135 in TSST-1 affects toxin interactions with the T-cell receptor rather than the class II major histocompatibility complex receptor.
Characterization of the structural elements in lipid A required for binding of a recombinant fragment of bactericidal/permeability-increasing protein rBPI23
Authors: Gazzano-Santoro H , Parent J B , Conlon P J , Kasler H G , Tsai C M , Lill-Elghanian D A , Hollingsworth R I
Published: January 2020

Both human bactericidal/permeability-increasing protein (BPI) and a recombinant amino-terminal fragment of BPI (rBPI23) have been shown to bind with high affinity to the lipid A region of lipopolysaccharide (LPS) (H. Gazzano-Santoro, J. B. Parent, L. Grinna, A. Horwitz, T. Parsons, G. Theofan, P. Elsbach, J. Weiss, and P. J. Conlon, Infect. Immun. 60:4754-4761, 1992). In the present study, lipid A preparations derived from bacterial LPS as well as synthetic lipid A's and various lipid A analogs were used to determine the structural elements required for rBPI23 binding. rBPI23 bound in vitro to a variety of synthetic and natural lipid A preparations (both mono- and diphosphoryl forms), including lipid A's prepared from Escherichia coli and Salmonella, Neisseria, and Rhizobium species. Binding does not require that the origin of negative charge be phosphate, since rBPI23 bound with high affinity to lipid A's isolated from Rhizobium species that contain carboxylate (Rhizobium trifolii) or sulfate (Rhizobium meliloti) anionic groups and lack phosphate. Lipid A acyl chains are important, since rBPI23 did not bind to four synthetic variants of the beta(1-6)-linked D-glucosamine disaccharide lipid A head group, all devoid of acyl chains. rBPI23 also bound weakly to lipid X, a monosaccharide lipid precursor of LPS corresponding to the reducing half of lipid A. Lipid IVA, a precursor identical to E. coli lipid A except that it lacks the 2' and 3' acyl chains, was the simplest structure identified in this study that rBPI23 bound with high affinity. These results demonstrate that rBPI23 has a binding specificity for the lipid A region of LPS and binding involves both electrostatic and hydrophobic components.
High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi
Authors: Norris S J , Howell J K , Garza S A , Ferdows M S , Barbour A G
Published: January 2020

Borrelias that cause Lyme disease lose the ability to infect and cause disease in laboratory animals following 10 to 16 passages of in vitro culture. In this study, clonal populations of the Sh-2-82 (Sh2) and B31 strains of Borrelia burgdorferi were isolated by subsurface plating on BSK-II agar plates and examined for infectivity in the C3H/HeN mouse model. Mice were injected intradermally with 10(5) B. burgdorferi organisms, and the tibiotarsal joint, heart, and bladder were cultured 2 to 4 weeks postinfection to determine whether viable organisms were present. Clones exhibited either a high-infectivity phenotype, in which cultures were consistently positive at all organ sites, or a low-infectivity phenotype, in which a low proportion of cultures were positive (5 of 40 in a representative experiment). In an Sh2 population that had undergone five in vitro passages, 7 of 10 clones were of the high-infectivity phenotype, and the remaining clones were of the low-infectivity phenotype. The proportion of high-infectivity clones decreased with continued in vitro passage, with only 1 of 10 clones exhibiting the high-infectivity phenotype after 10 passages and 0 of 10 clones yielding positive cultures after 20 passages. Representative high- and low-infectivity clones from passage 5 Sh2 cultures had 50% infectious doses of 1.8 x 10(2) and 1 x 10(5), respectively. Subclones consistently reflected the same infectivity phenotypes as those of the parent clones. The protein profiles and plasmid contents of the high- and low-infectivity clones were compared and exhibited few discernible differences. On the basis of these results, the loss of infectivity during in vitro culture results from the outgrowth of low-infectivity clones and begins to occur within the first five in vitro passages. Further examination of clonal populations may lead to the identification of genetic and protein factors important in the virulence and pathogenicity of Lyme disease borrelias.
Plasmodium falciparum rosetting is associated with malaria severity in Kenya
Authors: Rowe A , Obeiro J , Newbold C I , Marsh K
Published: January 2020

Rosette formation in 154 fresh Plasmodium falciparum isolates from Kenyan children with mild (n = 54), moderate (n = 64), or severe (n = 36) malaria was studied to determine whether the ability to form rosettes in vitro is correlated with malaria severity. There was a wide distribution of rosette frequencies within each clinical category; however, a clear trend towards higher rosette frequency with increasing severity of disease was seen, with the median rosette frequency of the mild-malaria group (1%; range, 0 to 82%) being significantly lower than those of the moderate-malaria group (5%; range, 0 to 45%; Mann-Whitney U test, P < 0.02) and the severe-malaria group (7%; range, 0 to 97%; Mann-Whitney U test, P < 0.003). Within the severe-malaria category there was no difference in rosetting among isolates from cerebral malaria patients or those with other forms of severe malaria. We also examined the ABO blood groups of the patients from whom isolates were obtained and found that isolates from group O patients (median rosette frequency, 2%; range 0 to 45%) rosetted less well than those from group A (median, 7%; range 0 to 82%; Mann-Whitney U test, P < 0.01) or group AB (median, 11%; range 0 to 94%; Mann-Whitney U test, P < 0.03). We therefore confirm that rosetting is associated with severe malaria and provide further evidence that rosetting is influenced by ABO blood group type. Whether rosetting itself plays a direct role in the pathogenesis of severe malaria or is a marker for some other causal factor remains unknown.
Synthetic peptides representing two protective, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa elicit whole-cell-reactive antibodies that are functionally pseudomonad specific
Authors: Gilleland L B , Gilleland H E
Published: January 2020

Peptide 9 (TDAYNQKLSERRAN) and peptide 10 (NATAEGRAINRRVE) represent surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Rats immunized with four intramuscular inoculations on days 0, 14, 28, and 42 with either peptide 9 or peptide 10 conjugated to keyhole limpet hemocyanin were afforded protection against pulmonary lesions when examined 7 days subsequent to challenge (day 56) via intratracheal inoculation of P. aeruginosa-containing agar beads. Peptide 9 shares considerable homology with other OmpA-related outer membrane proteins in various bacteria, whereas peptide 10 displays little homology with these other proteins. Antisera directed to peptide 9 reacted weakly with cell envelope proteins from the various other OmpA-associated bacteria upon immunoblot analysis. However, antisera directed to peptide 10 reacted only with Neisseria gonorrhoeae cell envelope proteins upon immunoblot analysis. Antisera to both peptides 9 and 10 reacted at minimal titers with whole cells of the various other bacteria in a whole-cell enzyme-linked immunosorbent assay (ELISA) but antisera to each of the peptides reacted at high titers when various strains of P. aeruginosa were used as the ELISA antigen. Antibodies to peptides 9 and 10 were protective, reactive to all strain of P. aeruginosa tested except for a protein F-deficient mutant, and functionally specific against pseudomonads.
Porphyromonas gingivalis fimbria-stimulated bone resorption in vitro is inhibited by a tyrosine kinase inhibitor
Authors: Hanazawa S , Kawata Y , Murakami Y , Naganuma K , Amano S , Miyata Y , Kitano S
Published: January 2020

Our previous study (Y. Kawata, S. Hanazawa, S. Amano, Y. Murakami, T. Matsumoto, K. Nishida, and S. Kitano, Infect. Immun. 62:3012-3016, 1994) showed that Porphyromonas gingivalis fimbriae stimulate bone resorption in vitro. Since it has recently been demonstrated that tyrosine kinase encoded by the c-src gene plays an important role in osteoclastic bone resorption, in the present study we examined the effect of a tyrosine kinase inhibitor on the fimbria-stimulated bone resorption. Genistein, a potent inhibitor of tyrosine kinase, markedly inhibited bone resorption stimulated by the fimbriae. Genistein also inhibited induction of several tyrosine-phosphorylated proteins in the fimbria-treated calvarial bone cells from mouse embryos.
Characterization of a Tn4351-generated hemin uptake mutant of Porphyromonas gingivalis: evidence for the coordinate regulation of virulence factors by hemin
Authors: Genco C A , Simpson W , Forng R Y , Egal M , Odusanya B M
Published: January 2020

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. We report here on the isolation and characterization of a transpositional insertion mutant of P. gingivalis A7436 (designated MSM-3) which is defective in the utilization and transport of hemin. P. gingivalis MSM-3 was selected on the basis of its nonpigmented phenotype on anaerobic blood agar following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplied with hemin as a sole source of iron; however, growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown in either hemin-replete or hemin-depleted conditions bound and transported less [14C]hemin or [59Fe]hemin than did the parent strain. At 4 h, P. gingivalis MSM-3 grown in hemin-replete conditions transported only 10,000 pmol of hemin per mg of protein, or 14% of the amount transported by P. gingivalis A7436. Unlike P. gingivalis A7436, hemin binding and transport by P. gingivalis MSM-3 were not tightly regulated by hemin or iron. Examination of P. gingivalis MSM-3 cultures by electron microscopy revealed an overproduction of membrane vesicles, and determination of the dry weight of purified vesicles indicated that P. gingivalis MSM-3 produced twice as much membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 also were found to express increased hemolytic and trypsin-like protease activities compared with the parent strain. When inoculated into subcutaneous chambers implanted in mice, P. gingivalis MSM-3 was highly infectious and more invasive than the parent strain, as indicated by secondary lesion formation and death. Taken together, these results indicate that the decreased transport of hemin by P. gingivalis MSM-3 results in the increased expression of several virulence factors which may be coordinately regulated by hemin.
Expression of adhesion molecules on human granulocytes after stimulation with Helicobacter pylori membrane proteins: comparison with membrane proteins from other bacteria
Authors: Enders G , Brooks W , von Jan N , Lehn N , Bayerdörffer E , Hatz R
Published: January 2020

Type B gastritis in its active form is characterized by a dense infiltration of the lamina propria with granulocytes. Since the bacterium Helicobacter pylori does not invade the epithelial barrier, a signaling pathway chemoattractive for granulocytes must exist across this mucosal boarder. One possible mechanism tested was whether granulocytes are directly activated by water-soluble membrane proteins (WSP) from H. pylori. These findings were compared with the effects of WSP from other bacteria (Helicobacter felis, Campylobacter jejuni, Escherichia coli, and Staphylococcus aureus). A unique activation pattern by H. pylori WSP was found. Like all other WSP tested, they induced an upregulation of CD11b but had no influence on CD11c and, most strikingly, CD62L expression. In contrast, E. coli WSP, e.g., not only induce a strong CD11b and CD11c expression but also lead to a loss in surface CD62L. The lack of CD62L shedding conserves rolling of granulocytes along the endothelium, creating a favorable precondition for granulocytes to stick more readily to activated endothelium after H. pylori stimulation via CD11b-CD54 receptor-counterreceptor interaction. This may explain why H. pylori infection is a very strong stimulus for granulocyte infiltration. The active fraction for the induction of CD11b on granulocytes is a heat- and protease-sensitive protein with a molecular mass between 30 and 100 kDa. One activation step involved may be the binding of WSP to CD15 determinants on granulocytes with subsequent induction of CD11b.