bluuebunny/crossref_metadata_2025 huggingface dataset:
Opsonic antibodies to outer membrane protein P2 of nonencapsulated Haemophilus influenza are strain specific
Authors: Troelstra A , Vogel L , van Alphen L , Eijk P , Jansen H , Dankert J
Published: January 2020

The ability of monoclonal antibodies (MAbs) specific for variable and conserved epitopes of outer membrane protein (OMP) P2 (b,c) of nonencapsulated Haemophilus influenza to promote opsonophagocytosis of this bacterium by human polymorphonuclear leucocytes (PMNs) was determined by flow cytometry. MAbs rendering PMNs fluorescent because of association with fluorescein isothiocyanate-labelled bacteria were defined as stimulating opsonophagocytosis. Opsonophagocytosis was dependent on the presence of both antibodies and complement. Of the 14 MAbs directed to the variable parts of OMP P2 (L. van Alphen, P. Eijk, L. Geelen-van den Broek, and J. Dankert, Infect. Immun. 59:247-252, 1991), 9 stimulated opsonophagocytosis. Four of the five nonopsonophagocytic MAbs that were immunoglobulin G1 were unable to cause complement activation. The MAbs promoting opsonophagocytosis included MAbs specific for one or more OMP P2 antigenic variants of H. influenzae strains isolated from patients with chronic bronchitis during persistent infection. MAbs cross-reacting in enzyme-linked immunosorbent assays with nonrelated H. influenzae did not promote opsonophagocytosis of strains from other patients. Opsonophagocytosis was not observed in the presence of three MAbs reacting with OMP P2 epitopes common in H. influenzae. These results indicate that OMP P2-dependent opsonophagocytosis of nonencapsulated H. influenzae is strictly strain specific.
Immunological and molecular characterization of Helicobacter felis urease
Authors: Gootz T D , Perez-Perez G I , Clancy J , Martin B A , Tait-Kamradt A , Blaser M J
Published: January 2020

Urease activity has recently been shown to be an important virulence determinant for Helicobacter pylori, allowing it to survive the low pH of the stomach during colonization. Experimental murine infection with Helicobacter felis is now being used as a model for H. pylori infection to study the effects of vaccines, antibiotics, and urease inhibitors on colonization. However, little information comparing the ureases of H. felis and H. pylori is available. Urease was partially purified from the cell surface of H. felis ATCC 49179 by A-5M agarose chromatography, resulting in an eightfold increase in specific activity over that of crude urease. The apparent Km for urea for the partially purified urease was 0.4 mM, and the enzyme was inhibited in a competitive manner by flurofamide (50% inhibitory concentration = 0.12 microM). Antiserum to whole cells of H. pylori recognized both H. pylori and H. felis urease B subunits. Antiserum raised against H. felis whole cells recognized the large and small autologous urease subunits and the cpn60 heat shock molecule in both H. felis and H. pylori. However, this antiserum showed only a weak reaction with the B subunit of H. pylori urease. Two oligomeric DNA sequences were used as probes to evaluate the relatedness of H. felis and H. pylori urease gene sequences. One 30-mer from the ureA sequence, which had been shown previously to be specific for H. pylori, failed to hybridize to H. felis genomic DNA. A probe to the putative coding sequence for the active site of the H. pylori ureB subunit hybridized at low intensity to a 2.8-kb fragment of BamHI-HindIII-digested H. felis DNA, suggesting that the sequences were homologous but not identical, a result confirmed from the recently published sequences of ureA and ureB from H. felis.
CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles
Authors: Brown W C , Davis W C , Dobbelaere D A , Rice-Ficht A C
Published: January 2020

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.
Polyclonal expansion of peripheral gamma delta T cells in human Plasmodium falciparum malaria
Authors: Ho M , Tongtawe P , Kriangkum J , Wimonwattrawatee T , Pattanapanyasat K , Bryant L , Shafiq J , Suntharsamai P , Looareesuwan S , Webster H K
Published: January 2020

Plasmodium falciparum malaria in humans is associated with an increase in the percentage and absolute number of gamma delta T cells in the peripheral blood. This increase begins during the acute infection phase and persists for at least 4 weeks during convalescence. In the present study, 25 to 30% of the gamma delta T cells expressed HLA-DR antigens in vivo and in some patients they proliferated in response to further stimulation by purified human interleukin 2 in vitro. However, there was no in vitro proliferative response to various malarial antigens, including a 75-kDa heat shock protein and a 72-kDa glucose-regulated protein of P. falciparum during the acute infection phase. Cytofluorographic studies showed that although an increase of V delta 1- gamma delta T cells was largely responsible for the expansion of the total number of gamma delta T cells, there was also a proportional increase in V delta 1+ cells. These results were confirmed with anchored PCR and by DNA sequencing to characterize at the molecular level the set of T-cell receptor (TCR) delta mRNAs expressed in the peripheral blood of two patients with high levels of gamma delta T cells. In each case, most of the TCR delta mRNA transcripts corresponded to nonproductively rearranged delta genes (unrearranged J delta or near J delta spliced to C delta). In those sequences which did represent productively rearranged genes, most of the transcripts originated from a V delta 2/J delta 1 joining, as in normal individuals. A minority of transcripts originated from a V delta 1/J delta 1 rearrangement, and one originated from a V alpha 4/J delta 1 rearrangement. Polyclonal activation of gamma delta T cells was inferred from the extensive junctional diversity seen in the delta mRNAs analyzed. Expansion of a heterogeneous set of both V delta 1(-)- and V delta 1(+)-bearing T cells suggests that the elevated levels of gamma delta T cells seen during acute P. falciparum malaria arose from immune responses to multiple distinct parasite antigens or unidentified host factors.
Oral administration of polymeric immunoglobulin A prevents colonization with Vibrio cholerae in neonatal mice
Authors: Lee C K , Weltzin R , Soman G , Georgakopoulos K M , Houle D M , Monath T P
Published: January 2020

A simple animal model was used to demonstrate passive protection by immunoglobulin A (IgA) against a mucosal pathogen, Vibrio cholerae. Oral administration of a monoclonal IgA directed against a lipopolysaccharide component of the vibrio protected neonatal mice against oral challenge, as measured by reduced intestinal colonization. A single dose of 0.1 microgram of polymeric monoclonal IgA given 1 h prior to challenge reduced the number of recoverable vibrios by at least 100-fold. An additional dose 3 h before challenge or 1 h after challenge did not enhance protection. A 10-fold-higher concentration of monomeric IgA was required to achieve the same level of protection as that conferred by polymeric IgA. Polymeric IgA digested with trypsin or human duodenal aspirates to lower-molecular-weight fragments retained most of its ability to protect mice against challenge.
Immunoglobulin A (IgA), IgA1, and IgA2 antibodies to Candida albicans in whole and parotid saliva in human immunodeficiency virus infection and AIDS
Authors: Coogan M M , Sweet S P , Challacombe S J
Published: January 2020

Human immunodeficiency virus (HIV)-infected individuals are predisposed to recurrent oral candidiasis, and, although it has been assumed that this is because of deficient mucosal immune responses, this has not been properly established. The present study aimed to compare the concentrations and secretion rates of immunoglobulin A (IgA) and IgA subclass antibodies to Candida albicans in whole and parotid saliva samples from HIV-infected patients, AIDS patients, and control subjects. Levels of IgA antibody to Candida species in whole saliva were higher in the HIV group than in the controls and were highest in the AIDS group (P < 0.05). In parotid saliva, the mean antibody levels were significantly greater in HIV-positive patients than in controls (P < 0.05) but fell to lower levels in the AIDS group. The secretion rates of Candida antibodies in parotid saliva were reduced in AIDS patients compared with HIV patients. The specific activities of the IgA antibodies and both subclasses were significantly higher in the HIV and AIDS patients than in the controls in both whole and parotid saliva (P < 0.05). Antibody levels were significantly correlated with the numbers of Candida organisms isolated from saliva (P < 0.05). These results suggest clear differences in salivary antibody profiles among HIV-infected. AIDS, and control subjects and are indicative of a response to antigenic challenge by infecting Candida species. No obvious defect in the mucosal immune response in the HIV or AIDS groups that might account for the increased prevalence of candidiasis was apparent.
lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity
Authors: Toder D S , Ferrell S J , Nezezon J L , Rust L , Iglewski B H
Published: January 2020

The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced. Its function and significance have been studied by genetic approaches (D. S. Toder, M. J. Gambello, and B. H. Iglewski, Mol. Microbiol. 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J. E. Peters and D. R. Galloway, J. Bacteriol. 172:2236-2240, 1990). To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1. Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity. Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present. Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity. We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64. Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB.
Tumor necrosis factor alpha (TNF-alpha) and TNF-beta and their receptors in experimental cutaneous leishmaniasis
Authors: de Kossodo S , Grau G E , Louis J A , Müller I
Published: January 2020

Experimental infection of BALB/c mice with Leishmania major leads to lesions which progress without healing and visceralization, reproducing the most severe forms of human leishmaniasis, while resistant mice like CBA spontaneously resolve lesions and develop protective immunity. Given the conflicting data pertaining to the role of tumor necrosis factor alpha (TNF) in Leishmania infection, we analyzed the expression of TNF, tumor necrosis factor beta (lymphotoxin), and TNF receptor type I (TNF-RI) and type II (TNF-RII) genes in vivo and correlated TNF gene expression in vivo with the production of biologically active TNF by lymphoid cells in vitro. No significant difference in the expression of TNF mRNA was found between susceptible and resistant strains of mice during the course of infection. The depletion of CD4+ T cells in vitro did not change the level of TNF mRNA in BALB/c lymph node cells but led to the total disappearance of TNF mRNA in CBA mice. Unprimed spleen cells did not produce detectable amounts of TNF, whereas 1 week after infection, TNF bioactivity was detected and increased in both strains of mice until 5 weeks of infection. While neutralization of TNF activity in vivo did not alter the course of infection in BALB/c mice, in CBA mice it led to an increase in lesion size and a delay in the healing process but did not interfere significantly with the outcome of infection. Finally, no significant difference in the levels of lymphotoxin, TNF-RI, or TNF RII mRNA expression was found between both strains. The information resulting from these investigations supports the notion that, in vivo, TNF is not the decisive factor responsible for the resistant versus susceptible phenotype in leishmania infection.
Humoral immune response to class 1 outer membrane protein during the course of meningococcal disease
Authors: Guttormsen H K , Wetzler L M , Solberg C O
Published: January 2020

We have determined the amounts of specific anti-class 1 outer membrane protein antibodies in sera from 25 patients during the course of systemic meningococcal disease, using purified class 1 protein as the sensitizing antigen in an enzyme-linked immunosorbent assay. The class 1 protein was obtained from a variant of strain 44/76 (B:15:P1.7,16) lacking class 3 and class 4 outer membrane proteins. Specific anti-class 1 (serosubtype P1.7,16) outer membrane protein immunoglobulin G (IgG) antibody levels increased significantly in 12 patients (12 of 25; 48%), regardless of the serotype of the infecting strain, indicating that the antibodies reacted in part with epitopes not determined by the monoclonal antibodies used for serotyping. Most patients had low levels of anti-class 1 IgG antibodies during the acute illness. The antibody levels peaked during the second week of disease and returned to near baseline levels in sera collected 6 weeks to 12 months after the onset of the disease. The majority of the specific anti-class 1 IgG antibodies bound to surface-exposed epitopes on whole bacteria and belonged to the IgG1 and IgG3 subclasses. Anti-class 1 IgA and IgM antibodies were not detected in any of the patient sera. Prior to disease, seven patients had been immunized with a meningococcal outer membrane vesicle vaccine developed from strain 44/76 (P1.7,16). None of these patients was infected with meningococcal strains containing class 1 protein homologous or partly homologous to that of the vaccine strain, indicating serosubtype-specific protection. The highest anti-class 1 IgG antibody peak levels were seen in immunized patients infected with strains of heterologous serotype, suggesting an anamnestic response. However, these patients were not protected from meningococcal disease after immunization.
Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity
Authors: Lane T E , Otero G C , Wu-Hsieh B A , Howard D H
Published: January 2020

The antihistoplasma activity of recombinant murine gamma interferon (rMuIFN-gamma)-treated macrophages of the RAW 264.7 cell line depends on the generation of nitric oxide (NO.) from L-arginine. Macrophages of the P388D1 cell line treated with rMuIFN-gamma do not produce NO. or inhibit the intracellular growth of Histoplasma capsulatum. NO. is generated by the inducible enzyme nitric oxide synthase (iNOS) formed by stimulated macrophages. Northern (RNA) blot analysis of RAW 264.7 cells revealed the expression of iNOS mRNA after exposure to rMuIFN-gamma. In contrast, rMuIFN-gamma-treated P388D1 cells did not produce detectable levels of iNOS. These data suggest that the failure of P388D1 cells to generate NO. and to restrict the intracellular growth of H. capsulatum is due to a lack of expression of iNOS following treatment with rMuIFN-gamma.
Microtiter plate adherence assay and receptor analogs for Mycoplasma hyopneumoniae
Authors: Zhang Q , Young T F , Ross R F
Published: January 2020

A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by use of purified swine tracheal cilia which contained receptors for the mycoplasmas. M. hyopneumoniae bound specifically to plates coated with solubilized cilia. The binding was dependent on both the concentration of cilia and the number of mycoplasmas. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited the binding of the mycoplasmas. The six inhibitors also disrupted the adherence of the mycoplasmas to intact ciliated cells. Preincubation with either mycoplasmas or cilia indicated that heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesive molecules on the surface of the mycoplasmas, while laminin blocked the receptors in cilia. The basis for the inhibition induced by dextran sulfate was unknown. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas, whereas treatment of cilia with sodium metaperiodate decreased binding. These results indicate that receptors for M. hyopneumoniae in the ciliated epithelium of the respiratory tract of pigs are glycoconjugate in nature.
Transgene-encoded antiphosphorylcholine (T15+) antibodies protect CBA/N (xid) mice against infection with Streptococcus pneumoniae but not Trichinella spiralis
Authors: Lim P L , Choy W F , Chan S T , Leung D T , Ng S S
Published: January 2020

Immunodeficient CBA/N (xid) mice are highly susceptible to Streptococcus pneumoniae infection. Previous studies indicated that this susceptibility may be attributed to the lack of antibodies to phosphorylcholine (PC) in the circulation of these animals. We now provide direct proof that when these mice are genetically manipulated to produce significant amounts of circulating anti-PC immunoglobulin G antibodies of the T15 idiotype, they can be protected against a lethal challenge with S. pneumoniae. Transgenic mice were also used to investigate whether the transgene-encoded antibodies could protect the animals against another PC-bearing microorganism, Trichinella spiralis; in this case, there was no protection. These results were further supported by experiments with CFW mice which had been immunized to produce high levels of anti-PC antibodies but which were found to be just as susceptible to T. spiralis infection as nonimmunized animals.
Evidence for adhesin activity in the acid-stable moiety of the phosphomannoprotein cell wall complex of Candida albicans
Authors: Kanbe T , Cutler J E
Published: January 2020

Previously, we showed that Candida albicans hydrophilic yeast cells adhere specifically to mouse splenic marginal-zone macrophages. The adhesins are part of the yeast cell wall phosphomannoprotein complex, and one adhesin site, which reacts with the monoclonal antibody 10G, was identified as a beta-1,2-linked tetramannose in the acid-labile portion of the complex. We report here that the acid-stable part of the complex, which has not been reported previously to have adhesin activity, is in large part responsible for yeast cell binding to the splenic marginal zone. The phosphomannoprotein complex, termed Fr.II, was isolated from C. albicans serotype B yeast cells by beta-mercaptoethanol extraction and concanavalin A-agarose affinity chromatography. Fr.II is devoid of the serotype A-specific antigen factor 6, which functions in yeast cell attachment to epithelial cells. The acid-stable part of Fr.II (i.e., Fr.IIS) was obtained by mild acid hydrolysis and size exclusion fractionation. Fr.IIS was further fractionated into four fractions, Fr.IIS1, Fr.IIS2, Fr.IIS3, and Fr.IIS4, by concanavalin A-agarose column chromatography and elution with a linear gradient of alpha-methyl-D-mannopyranoside. Adhesin activity of these fractions was determined by their ability to block yeast cell binding to the splenic marginal zone. Fr.IIS1 and Fr.IIS2 yielded more material and stronger adhesin activity than either Fr.IIS3 or Fr.IIS4. Only Fr.IIS1 did not react with antibodies (anti-factor 5 and monoclonal antibody 10G) specific for the acid-labile beta-1,2-linked oligosaccharides. Fr.IIS1-coated latex beads attached specifically to the marginal zone in a pattern identical to that of yeast cell binding. Furthermore, Fr.IIS1-latex bead attachment was inhibited by soluble Fr.II or Fr.IIS. Initial chemical analyses indicate that the adhesin site on Fr.IIS1 is a carbohydrate because adhesin activity was destroyed by periodate oxidation but not by proteinase K digestion.
Variable region sequences and idiotypic expression of a protective human immunoglobulin M antibody to capsular polysaccharides of Neisseria meningitidis group B and Escherichia coli K1
Authors: Azmi F H , Lucas A H , Raff H V , Granoff D M
Published: January 2020

We determined the heavy (H)- and light (L)-chain variable (V) region nucleotide and translated amino acid sequences of the human immunoglobulin M(kappa) monoclonal antibody (MAb) 5E1, which is specific for the polysaccharide capsule of Escherichia coli K1 and Neisseria meningitidis group B (poly[alpha(2-->8)-N-acetylneuraminic acid]) and which is protective in animal models of infection. The 5E1 VH gene is a member of the VHIIIb family and is 97% homologous to the 9.1 germ line gene. The 5E1 VL gene is a member of the kappa I subgroup and is 98% homologous to the germ line gene, 15A, also known as KLO12. The VL and/or VH genes used by 5E1 are highly homologous to the V genes encoding antibodies to the Haemophilus influenzae type b polysaccharide and to antibodies reactive with self-antigens such as erythrocyte "i," DNA, and thyroid peroxidase. We also produced three murine anti-idiotype (Id) MAbs against 5E1. All three anti-Ids recognize a minor subset of antimeningococcal B polysaccharide antibodies present in serum from normal adults. Two of the anti-Ids define distinct Ids associated with antibodies having kappa I-15A V regions. These 15A-associated Ids are expressed by some heterologous human antimeningococcal B polysaccharide MAbs, and they also are independently expressed by two human MAbs that are specific for either the H. influenzae b polysaccharide or the i erythrocyte antigen and that utilize the kappa I-15A V region. Taken together, these data indicate that the 5E1 antibody uses V regions that recur in the human antibody repertoires to this polysaccharide and to structurally dissimilar polysaccharides and autoantigens. Thus, the poor immunogenicity of poly[alpha(2-->8)-N-acetylneuraminic acid] cannot be explained by the unavailability of certain critical VH and VL genes required for generation of antibody response.
Isolation and characterization of Pseudomonas pseudomallei flagellin proteins
Authors: Brett P J , Mah D C , Woods D E
Published: January 2020

Flagellin proteins from several different strains of Pseudomonas pseudomallei have been isolated and purified to homogeneity by mechanical shearing and differential centrifugation techniques. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded flagellin monomer protein bands with an estimated M(r) of 43,400. No lipopolysaccharide contamination of the purified protein preparations was detectable by silver staining of flagellin displayed on polyacrylamide gels and by Western immunoblotting with P. pseudomallei antilipopolysaccharide monoclonal antibody. NH2-terminal amino acid sequence analysis of the flagellin protein of P. pseudomallei 319a revealed significant homology with flagellins from Proteus mirabilis, Bordetella bronchiseptica, and Pseudomonas aeruginosa PAK. Rabbit polyclonal antiserum raised against the 319a flagellin protein reacted with 64 of 65 P. pseudomallei strains tested. The polyclonal antiserum proved effective in inhibiting the motility of these organisms in motility agar plates. Passive immunization studies demonstrated that 319a flagellin-specific antiserum was capable of protecting diabetic rats from challenge with a heterologous P. pseudomallei strain.
Production of tumor necrosis factor alpha in human leukocytes stimulated by Cryptococcus neoformans
Authors: Levitz S M , Tabuni A , Kornfeld H , Reardon C C , Golenbock D T
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of inflammation and may promote human immunodeficiency virus replication in latently infected cells. Since cryptococcosis often is associated with aberrations in the host inflammatory response and occurs preferentially in persons with AIDS, we defined the conditions under which human leukocytes produce TNF-alpha when stimulated by Cryptococcus neoformans. Peripheral blood mononuclear cells (PBMC) produced comparable amounts of TNF-alpha following stimulation with C. neoformans and lipopolysaccharide. Detectable TNF-alpha release in response to C. neoformans occurred only when fungi with small-sized capsules were used and complement-sufficient serum was added. Fractionation of PBMC established that monocytes were the predominant source of TNF-alpha. TNF-alpha gene expression and release occurred significantly later in PBMC stimulated with C. neoformans than in PBMC stimulated with LPS. C. neoformans was also a potent inducer of TNF-alpha from freshly isolated bronchoalveolar macrophages (BAM). Upon in vitro culture, BAM and monocytes bound greater numbers of fungal cells, yet their capacity to produce TNF-alpha following cryptococcal stimulation declined by 74 to 100%. However, this decline was reversed if the BAM and monocytes were cultured with gamma interferon. These data establish that C. neoformans can potently stimulate TNF-alpha release from human leukocytes. However, several variables profoundly affected the amount of TNF-alpha released, including the type of leukocyte and its state of activation, the size of the cryptococcal capsule, and the availability of opsonins.
Monoclonal antibodies to the circumsporozoite protein repeats of a Plasmodium vivax-like human malaria parasite and Plasmodium simiovale
Authors: Udhayakumar V , Qari S H , Patterson P , Collins W E , Lal A A
Published: January 2020

We have recently described a Plasmodium vivax-like human malaria parasite. The circumsporozoite protein of this parasite is identical to that of a simian malaria parasite, P. simiovale, but different from two known types of P. vivax. Here, we describe the production of two monoclonal antibodies, Pam 172 and Pam 135, specific for the circumsporozoite protein repeat sequence APGANQEGGAA of the P. vivax-like malaria parasite. These two monoclonal antibodies recognized air-dried sporozoites of P. simiovale but not other human, simian, or rodent malaria parasites tested.
Specific interaction of Aspergillus fumigatus with fibrinogen and its role in cell adhesion
Authors: Coulot P , Bouchara J P , Renier G , Annaix V , Planchenault C , Tronchin G , Chabasse D
Published: January 2020

Interaction between Aspergillus fumigatus conidia and different proteins known to mediate the attachment of malignant tumor cells or microorganisms to the host tissues was studied in vitro. Flow cytometry using fluorescein isothiocyanate-conjugated fibrinogen confirmed that binding of human fibrinogen to the conidia was dose dependent and specific. Binding was inhibited by unlabeled fibrinogen and by basement membrane laminin. Moreover, the expression of fibrinogen receptors at the surfaces of conidia seemed to be related to the maturation of the conidia. Binding sites appeared to be located in the D domains of the fibrinogen molecule. However, the peptide sequence recognized by the fungus could not be identified but was different from the classical adhesive recognition sequences, RGDS and fibrinogen gamma-chain dodecapeptide. In addition, an assay of adherence to proteins immobilized onto microtiter plates allowed us to establish the role of these interactions in fungal adhesion. Conidia strongly adhered to human fibrinogen and to laminin but not to fibronectin. Adhesion to fibrinogen substrates was specific, since it was inhibited by soluble fibrinogen and by specific antibodies, and seemed to be mediated by the D domains of the molecule. Study of the adhesion of numerous strains or clinical isolates to various mammalian fibrinogens did not reveal any particular affinity of strains for some animal species. Finally, by cultivation of the fungus in the presence of 125I-human fibrinogen and analysis of the radiolabeled material bound to the surface of the fungus, we were able to specify the sequence of events allowing its installation within the host. The interactions identified here may play an important role in governing fungal adherence and host tissue invasion.
Effect of salicylate, bismuth, osmolytes, and tetracycline resistance on expression of fimbriae by Escherichia coli
Authors: Kunin C M , Hua T H , Guerrant R L , Bakaletz L O
Published: January 2020

Adherence of Escherichia coli is facilitated by fimbriae and several outer membrane proteins (OMPs). Hypertonic conditions, salicylate, and Mar mutations are known to reduce OmpF expression. We speculated that OMPs involved in export or assembly of fimbrial subunits might be similarly affected. To explore this hypothesis, E. coli expressing P, type 1, S, colonization factor antigen I (CFA/I), or CFA/II fimbriae was grown in the presence of salicylate, bismuth salts, NaCl, and nonfermented sugars. Tetracycline-resistant clones were derived from several P-fimbriated strains. The bacteria were tested for the ability to agglutinate erythrocytes, yeast cells, and alpha-D-Gal(-4)-beta-D-Gal-bonded latex (Gal-Gal) beads and were examined for fimbriae by electron microscopy. Hyperosmolar conditions decreased fimbrial expression for all strains. Expression of P fimbriae by pyelonephritic strains, all of which were OmpF+, was reversibly repressed by salicylate and bismuth salts. CFA strains were similarly affected. Tetracycline-resistant P-fimbriated strains were OmpF deficient, were unable to agglutinate erythrocytes and Gal-Gal beads, and lacked fimbriae as observed by electron microscopy. Strains with plasmid-encoded P-fimbrial genes did not demonstrate OmpF on polyacrylamide gel electrophoresis profiles and were not affected by salicylate. The type 1-fimbriated phenotype was not affected by salicylate or bismuth unless the strains also expressed P fimbriae. S-fimbriated strains were not affected. The mechanism by which salicylates, bismuth salts, and tetracycline resistance inhibit or modulate the expression of P fimbriae may be mediated through OmpF and other OMPs.
Entrance and survival of Salmonella typhimurium and Yersinia enterocolitica within human B- and T-cell lines
Authors: Verjans G M , Ringrose J H , van Alphen L , Feltkamp T E , Kusters J G
Published: January 2020

Lymphocytes, located within the Peyer's patches, might be involved in the dissemination of enteropathogenic Salmonella typhimurium and Yersinia enterocolitica bacteria. To test this hypothesis, we have investigated the susceptibility of human B- and T-cell lines to bacterial adhesion and invasion. The two S. typhimurium strains analyzed were highly invasive, while the two Y. enterocolitica (O:8) strains adhered to the B- and T-cell lines but did not enter the cell lines in significant amounts. We hypothesize that the incapability of the Y. enterocolitica (O:8) strains to enter the human B- and T-cell lines is most probably due to the bacterial inability to induce the internalization process upon adhesion to both cell lines. Although immortalized B- and T-cell lines were used in this study, the results presented suggest the possibility that both cell types could play a role in the dissemination of intracellularly residing S. typhimurium in vivo.
Mycoplasma arthritidis-derived superantigen induces proinflammatory monokine gene expression in the THP-1 human monocytic cell line
Authors: al-Daccak R , Mehindate K , Hébert J , Rink L , Mecheri S , Mourad W
Published: January 2020

Soluble factors produced by Mycoplasma arthritidis play an important role in the pathology of arthritis in rodents, which closely resembles human rheumatoid arthritis. At least one of the products of these microorganisms, M. arthritidis-T cell mitogen (MAM), has biological activities in common with superantigens. These superantigens activate T cells in a V beta-restricted fashion, and this response is strictly dependent on the presence of major histocompatibility complex (MHC) class II-positive cells. In the present study, we have examined the ability of MAM to induce proinflammatory monokine (interleukin 1 beta [IL-1 beta] and tumor necrosis factor alpha [TNF-alpha]) gene expression in the THP-1 monocytic cell line. Treatment of these cells (which express a very low level of HLA-DR molecules) with gamma interferon (INF-gamma) induced HLA-DR, -DQ, and -DP molecules and enabled them to respond to MAM in a dose-dependent manner, resulting in an increase in the level of steady-state mRNA for IL-1 beta and TNF-alpha. Stimulation of the U937 monocytic cell line (MHC class II-negative even after INF-gamma treatment) with MAM did not induce either IL-1 beta or TNF-alpha transcription. Moreover, MAM adsorption on Raji (MHC class II-positive) cells resulted in the loss of its cytokine-inducing activity to induce monokine gene expression. These findings demonstrate clearly that MAM induces monokine gene expression following interaction with MHC class II molecules. Pretreatment of INF-gamma-treated THP-1 cells with the transcription inhibitor actinomycin D prevented the induction of monokine mRNA, whereas cycloheximide superinduced mRNA after stimulation with MAM. Finally, our results, obtained with protein tyrosine kinase inhibitors and antiphosphotyrosine Western blotting (immunoblotting), indicate that protein tyrosine kinase is involved in MAM-induced IL-1 beta and TNF-alpha gene expression in the THP-1 monocytic cell line. The capacity of MAM to induce proinflammatory cytokine transcription in monocytes via MHC class II molecules can be one pathway of MAM contribution to autoimmune diseases.
Cloning and sequence analysis of a gene encoding a 67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with class II major histocompatibility antigens
Authors: Kil K S , Cunningham M W , Barnett L A
Published: January 2020

The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were found. Mouse anti-IA sera reacted with the recombinant 67-kDa protein about five times more strongly than normal mouse sera in the enzyme-linked immunosorbent assay. Southern hybridization experiments using a probe for the gene encoding the 67-kDa protein showed that the gene was present and conserved among pathogenic groups A, C, and G of streptococci. These data suggest that the streptococcal protein, which is distinct from the M protein, may have structural features in common with the beta chain of the class II antigens, as well as myosin, and may play an important role in the pathogenesis of streptococcal infections.
Identification of a novel antigen from Corynebacterium pseudotuberculosis that protects sheep against caseous lymphadenitis
Authors: Walker J , Jackson H J , Eggleton D G , Meeusen E N , Wilson M J , Brandon M R
Published: January 2020

A 40-kDa protein antigen from Corynebacterium pseudotuberculosis has been identified by application of a strategy that employs locally derived antibody-secreting cells (ASC). ASC probes generated by culture of ASC obtained from the lymph node draining the site of infection showed a specificity restricted to a 40-kDa antigen. Analysis of immunoblots with sequential serum samples taken from sheep during the course of experimental primary infection with C. pseudotuberculosis also revealed the 40-kDa antigen as an early immunodominant antigen. Sheep vaccinated with two 100-micrograms doses of a 40-kDa antigen preparation in aluminium hydroxide adjuvant were protected against infection with C. pseudotuberculosis, with an 82% reduction in the proportion of infected sheep and a 98% reduction in lung lesions. Sera from vaccinated sheep exhibited a strong response only to the 40-kDa antigen on immunoblots. These results strongly suggest that the 40-kDa antigen plays a major role in immunity to caseous lymphadenitis.
Relationship of tumor necrosis factor alpha, the nitric oxide synthase pathway, and lipopolysaccharide to the killing of gamma interferon-treated macrophage-like RAW264.7 cells by Rickettsia prowazekii
Authors: Turco J , Winkler H H
Published: January 2020

Macrophage-like RAW264.7 cells are killed by the combination of gamma interferon (IFN-gamma) treatment and infection with Rickettsia prowazekii. The roles of tumor necrosis factor alpha (TNF-alpha), the nitric oxide synthase pathway, and lipopolysaccharide (LPS) in this killing were investigated. R. prowazekii, both the Breinl and Madrid E strains, induced RAW264.7 cells to produce TNF-alpha. However, dead rickettsiae (which cannot kill the IFN-gamma-treated RAW264.7 cells) induced the production of as much TNF-alpha as viable rickettsiae. Inhibition of the production of TNF-alpha (by the addition of actinomycin D or emetine during the rickettsial infection) or neutralization of TNF-alpha (by the addition of polyclonal rabbit anti-mouse TNF-alpha serum both during the IFN-gamma treatment and during the rickettsial infection) did not inhibit the killing of the RAW264.7 cells. Addition of polymyxin B (which inhibits many effects of LPS) during the IFN-gamma treatment did not inhibit the ability of IFN-gamma to prepare the RAW264.7 cells to be killed by R. prowazekii. Suppression of nitrite production by addition of the nitric oxide synthase inhibitor aminoguanidine both during the IFN-gamma treatment and during the rickettsial infection also did not inhibit the killing of the RAW264.7 cells. R. prowazekii-mediated killing of the RAW264.7 cells was dramatically suppressed in cultures treated with IFN-gamma plus LPS compared with that in cultures treated with IFN-gamma alone, and inhibition of nitric oxide synthase restored the rickettsia-induced killing of the RAW264.7 cells in cultures treated with IFN-gamma plus LPS. These data indicate that (i) TNF-alpha, LPS, and the nitric oxide synthase pathway are not required in order for IFN-gamma to prepare RAW264.7 cells to be killed by R. prowazekii; (ii) neither TNF-alpha nor the nitric oxide synthase pathway is responsible for the killing of the IFN-gamma-treated RAW264.7 cells by R. prowazekii; and (iii) in cultures treated with IFN-gamma plus LPS and then incubated with rickettsiae, a nitric oxide synthase pathway-dependent mechanism inhibits the killing of the RAW264.7 cells.
Comparison of the sensitivities of Salmonella typhimurium oxyR and katG mutants to killing by human neutrophils
Authors: Papp-Szabò E , Firtel M , Josephy P D
Published: January 2020

The respiratory burst of neutrophils is believed to kill bacteria by generating oxidative species, such as superoxide anion, hydrogen peroxide, and oxidized halogen species. The oxyR gene of Salmonella typhimurium controls a regulon induced by oxidative stress, such as exposure to hydrogen peroxide. Some researchers have suggested that oxyR may play a key role in bacterial survival following phagocytosis. We have tested this possibility by comparing the survival, following exposure to human neutrophils, of isogenic strains bearing different oxyR alleles. Neither inactivation of the oxyR gene nor constitutive overexpression of the oxyR-regulated proteins (oxyR1 allele) greatly alters bacterial resistance to neutrophils. The katG gene, encoding the oxyR-regulated enzyme hydroperoxidase I, was also without effect on survival following exposure to neutrophils. We conclude that the oxyR response does not play a significant role in the resistance of S. typhimurium to phagocytic killing in vitro.
Tumor necrosis factor alpha has a protective role in a murine model of systemic candidiasis
Authors: Louie A , Baltch A L , Smith R P , Franke M A , Ritz W J , Singh J K , Gordon M A
Published: January 2020

The role of tumor necrosis factor alpha (TNF-alpha) in host defense against systemic Candida albicans infection was evaluated in a murine model of systemic candidiasis in which uniform death occurred between 5 and 6 days after infection. TNF-alpha was first detected at 16 h postinfection and progressively increased thereafter. Peak levels (700 to 900 pg/ml) were measured in mice near death. Administration of 0.5 to 1.0 mg of polyclonal immunoglobulin G (IgG) TNF-alpha antibody (TNF-alpha Ab) to mice 2 h preinfection neutralized serum TNF-alpha for up to 30 h. However, this regimen shortened survival from a mean of 5.5 days for IgG controls to 3.4 days (P = 1.9 x 10(-12)). Semiquantitative cultures of spleen, lung, liver, and kidney conducted at 1, 2, and 3 days postinfection found colony counts of spleen and kidney to be significantly higher for TNF-alpha Ab recipients but only for the first 48 h. Administration of 1.5 and 1.0 mg of TNF-alpha Ab at 2 h before and 48 h after fungal injection, respectively, shortened the mean survival from 4.9 to 2.3 days (P = 5.2 x 10(-8)). This regimen neutralized serum TNF-alpha throughout infection. With this regimen, colony counts of all organs were significantly higher in TNF-alpha Ab recipients at 1, 2, and 3 days postinfection. Histopathologic studies showed an increase in the number and size of C. albicans foci in tissues. Peripheral leukocyte counts and inflammatory response in tissue were similar for TNF-alpha Ab and IgG sham recipients. In vitro, incubation of C. albicans with four to eight times the peak serum levels of TNF-alpha for up to 24 h did not inhibit the rate of germ tube or pseudohypha formation. Thus, TNF-alpha that was produced during infection with C. albicans augmented host resistance against this organism and prolonged survival. The protective effect of TNF-alpha was not mediated by increased leukocytes in blood or tissues nor by a direct anticandidal effect of TNF-alpha. This study suggests that the administration of exogenous TNF-alpha may enhance host resistance against systemic C. albicans infection and may improve host survival.
Development of destructive arthritis in vaccinated hamsters challenged with Borrelia burgdorferi
Authors: Lim L C , England D M , DuChateau B K , Glowacki N J , Creson J R , Lovrich S D , Callister S M , Jobe D A , Schell R F
Published: January 2020

We present the first direct evidence that adverse effects, particularly severe destructive arthritis, can develop in vaccinated hamsters after challenge with Borrelia burgdorferi sensu lato isolates. Hamsters were vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant. A severe destructive arthritis was readily evoked in vaccinated hamsters challenged with the homologous B. burgdorferi sensu stricto isolate C-1-11 before high levels of protective borreliacidal antibody developed. Once high levels of C-1-11 borreliacidal antibody developed, hamsters were protected from homologous challenge and development of arthritis. Vaccinated hamsters, however, still developed severe destructive arthritis when challenged with other isolates of the three genomic groups of B. burgdorferi sensu lato (B. burgdorferi sensu stricto isolate 297, Borrelia garinii isolate LV4, and Borrelia afzelii isolate BV1) despite high levels of C-1-11 specific borreliacidal antibody. Vaccines that contained whole spirochetes in adjuvant induced destructive arthritis, but this effect was not dependent on the isolate of B. burgdorferi sensu lato or the type of adjuvant. These studies demonstrate that caution is necessary when employing whole spirochetes in adjuvant for vaccination to prevent Lyme borreliosis. Additional studies are needed to identify the antigen(s) responsible for the induction and activation of arthritis and to define the immune mechanisms involved.