bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrapulmonary response to Histoplasma capsulatum in gamma interferon knockout mice
Authors: Allendoerfer R , Deepe G S
Published: January 2020

We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (IFN-gamma) knockout mice (GKO mice). Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 x 10(6) yeasts, whereas all wild-type (WT) mice survived for 45 days. Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4. To determine if IFN-gamma is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10(4) yeasts intranasally and given amphotericin B for 3 weeks. Six weeks later, mice were rechallenged with 2.5 x 10(6) yeasts. All GKO mice died by day 6, whereas all WT mice survived for 45 days. Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-alpha and GM-CSF levels but not IL-12 or IL-4. Thus, IFN-gamma is requisite for control of pulmonary histoplasmosis in naive and reexposed mice.
Regulation of anthrax toxin activator gene (atxA) expression in Bacillus anthracis: temperature, not CO2/bicarbonate, affects AtxA synthesis
Authors: Dai Z , Koehler T M
Published: January 2020

Anthrax toxin gene expression in Bacillus anthracis is dependent on the presence of atxA, a trans-acting regulatory gene located on the resident 185-kb plasmid pXO1. In atxA+ strains, expression of the toxin genes (pag, lef, and cya) is enhanced by two physiologically significant signals: elevated CO2/bicarbonate and temperature. To determine whether increased toxin gene expression in response to these signals is associated with increased atxA expression, we monitored steady-state levels of atxA mRNA and AtxA protein in cells cultured in different conditions. We purified histidine-tagged AtxA [AtxA(His)] from Escherichia coli and used anti-AtxA(His) serum to detect AtxA in protein preparations from B. anthracis cells. AtxA was identified as a protein with an apparent size of 56 kDa in cytoplasmic fractions of B. anthracis cells. Our data indicate that atxA expression is not influenced by CO2/bicarbonate levels. However, the steady-state level of atxA mRNA in cells grown in elevated CO2/bicarbonate at 37 degrees C is five- to sixfold higher than that observed in cells grown in the same conditions at 28 degrees C. A corresponding difference in AtxA protein was also seen at the different growth temperatures. When atxA was cloned on a multicopy plasmid in B. anthracis, AtxA levels corresponding to the atxA gene copy number were observed. However, this strain produced significantly less pag mRNA and protective antigen protein than the parental strain harboring atxA in single copy on pXO1. These results indicate that increased AtxA expression does not lead to a corresponding increase in pag expression. Our data strongly suggest that an additional factor(s) is involved in regulation of pag and that the relative amounts of such a factor(s) and AtxA are important for optimal toxin gene expression.
Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies
Authors: Pupo G M , Karaolis D K , Lan R , Reeves P R
Published: January 2020

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.
Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential
Authors: Wiedmann M , Bruce J L , Keating C , Johnson A E , McDonough P L , Batt C A
Published: January 2020

A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently. These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed. The clinical history of the L. monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages. Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III. Animal isolates were found in all three lineages. We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay. Strains from lineage III also seem to form larger plaques than strains from lineage II. A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L. monocytogenes subspecies. None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.
Effects of neutrophil, natural killer cell, and macrophage depletion on murine Clostridium piliforme infection
Authors: Van Andel R A , Hook R R , Franklin C L , Besch-Williford C L , van Rooijen N , Riley L K
Published: January 2020

Clostridium piliforme infection (Tyzzer's disease) induces enterohepatic disease in many domestic and laboratory animals. Murine susceptibility to Tyzzer's disease varies with host strain, age, and immune status However, little is known about the role of the immune system in control of this disease. To investigate the role of host immunity in Tyzzer's disease, mice were depleted of either neutrophils, natural killer cells, or macrophages by antibody administration or chemotherapy. After depletion, DBA/2 mice, which are naturally susceptible to C. piliforme, or naturally resistant C57BL/6 mice were inoculated intravenously with C. piliforme. Animals were euthanized 3 days postinoculation and evaluated for gross and histologic lesions and hepatic bacterial load. In juvenile DBA/2 or C57BL/6 mice, depletion of either neutrophils or natural killer cells increased severity of disease. In adult mice, depletion of natural killer cells significantly increased severity of Tyzzer's disease in the resistant (C57BL/6) but not in the susceptible (DBA/2) strain. Macrophage depletion did not alter the course of infection in either mouse strain. These studies indicate an important role for neutrophils and natural killer cells in the pathogenesis of murine Tyzzer's disease. The role of macrophages in murine C. piliforme infection will require further evaluation.
Cloning and characterization of a Prevotella melaninogenica hemolysin
Authors: Allison H E , Hillman J D
Published: January 2020

Hemolysins have been proven to be important virulence factors in many medically relevant pathogenic organisms. Their production has also been implicated in the etiology of periodontal disease. Hemolytic strain 361B of Prevotella melaninogenica, a putative etiologic agent of periodontal disease, was used in this study. The cloning, sequencing, and characterization of phyA, the structural gene for a P. melaninogenica hemolysin, is described. No extensive sequence homology could be identified between phyA and any reported sequence at either the nucleotide or amino acid level. As predicted from sequence analysis, this gene produces a 39-kDa protein which has hemolytic activity as measured by zymogram analysis. Unlike many Ca2+-dependent bacterial hemolysins, both the cloned and native PhyA proteins were enhanced by the presence of EDTA in a dose-dependent fashion with 40 mM EDTA allowing maximum activity. Ca2+ and Mg2+ were found to be inhibitory. The hemolytic activity also was found to have a dose-dependent endpoint. Through recovery of hemolytic activity from a spent reaction, this endpoint was shown to be the result of end product inhibition. This is the first report describing the cloning and sequencing of a gene from P. melaninogenica.
Analysis of toxicity of streptococcal pyrogenic exotoxin A mutants
Authors: Roggiani M , Stoehr J A , Leonard B A , Schlievert P M
Published: January 2020

Streptococcal pyrogenic exotoxin A (SPE A) is secreted by some strains of Streptococcus pyogenes and is strongly associated with streptococcal toxic shock syndrome (STSS), a severe and often fatal illness. SPE A possesses a number of biological properties, some of which are shared with a group of exotoxins of streptococcal and staphylococcal origins, the pyrogenic toxin superantigens (PTSAgs). SPE A's most extensively studied property is superantigenicity. Superantigenic activation of T cells and monocytes stimulates the release of cytokines such as tumor necrosis factors alpha and beta, interleukin 1, and gamma interferon. These endogenous mediators are considered to be the primary cause of capillary leak, hypotension, and shock, the most severe manifestations of STSS. However, several studies have suggested that other properties of SPE A, such as ability to greatly enhance host susceptibility to endotoxin and ability to interact directly with endothelial cells, may play substantial roles in the syndrome. In this work we generated single- and double-site mutations of SPE A at residues K16, N20, C87, C90, C98, K157, S195, N20/C98, and N20/K157. The mutant SPE A's were analyzed in vivo for their lethal activity and in vitro for their superantigenic ability. Our results indicate that SPE A's ability to induce lethality and endotoxin enhancement does not require superantigenicity, and conversely superantigenicity does not necessarily lead to lethality. Thus, these properties and their relative contributions to the onset of hypotension and shock may be separable. Furthermore, evidence is presented that certain mutant toxins may be suitable for use as vaccine toxoids.
Anti-Ehrlichia chaffeensis antibody complexed with E. chaffeensis induces potent proinflammatory cytokine mRNA expression in human monocytes through sustained reduction of IkappaB-alpha and activation of NF-kappaB
Authors: Lee E H , Rikihisa Y
Published: January 2020

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages and is the etiologic agent of human ehrlichiosis in the United States. Our previous studies showed that the exposure of human monocytes to E. chaffeensis induces the expression of interleukin-1beta (IL-1beta), IL-8, and IL-10 genes in vitro but not the expression of tumor necrosis factor alpha (TNF-alpha) and IL-6 mRNAs. In this study, the effect of anti-E. chaffeensis antibody complexed with E. chaffeensis on the expression of major proinflammatory cytokines in human monocytes was examined. Human monocytic cell line THP-1 was treated with E. chaffeensis which had been preincubated with human anti-E. chaffeensis serum for 2 h, and the levels of cytokine mRNAs were evaluated by competitive reverse transcription-PCR. Anti-E. chaffeensis antibody complexed with E. chaffeensis significantly enhanced mRNA expression of IL-1beta in THP-1 cells. The expression of TNF-alpha and IL-6 mRNAs was also induced. The levels of secreted IL-1beta, TNF-alpha, and IL-6 during 24 h of stimulation were comparable to those induced by Escherichia coli lipopolysaccharide at 1 microg/ml. Fab fragment of anti-E. chaffeensis immunoglobulin G complexed with E. chaffeensis did not induce any of these three cytokines, indicating that ehrlichial binding is required for IL-1beta mRNA expression and that binding of the immune complex to the Fc gamma receptor is required for TNF-alpha and IL-6 mRNA expression and enhanced IL-1beta mRNA expression. Furthermore, prolonged degradation of IkappaB-alpha and activation of NF-kappaB were demonstrated in THP-1 cells exposed to anti-E. chaffeensis serum and E. chaffeensis. This result implies that development of anti-E. chaffeensis antibody in patients can result in the production of major proinflammatory cytokines, which may play an important role in the pathophysiology of ehrlichiosis and immune responses to it.
A monoclonal antibody directed against the 97-kilodalton gonococcal hemin-binding protein inhibits hemin utilization by Neisseria gonorrhoeae
Authors: Lee B C , Levesque S
Published: January 2020

Neisseria gonorrhoeae expresses two hemin-binding proteins (HmBPs) of 97,000 and 44,000 in molecular weight. A murine monoclonal antibody (MAb) produced against the 97-kDa HmBP from N. gonorrhoeae PID543 specifically inhibited in a concentration-dependent manner the ability of hemin to promote growth. The anti-97-kDa HmBP MAb competitively inhibited binding of the 97-kDa HmBP to a hemin-agarose affinity column. In Western immunoblots, the MAb recognized the 97-kDa homologs from a limited survey of clinical gonococcal isolates. These results support the contention that the 97-kDa HmBP is involved in the gonococcal hemin acquisition pathway.
Successful therapy of chronic, nonhealing murine cutaneous leishmaniasis with sodium stibogluconate and gamma interferon depends on continued interleukin-12 production
Authors: Li J , Sutterwala S , Farrell J P
Published: January 2020

Treatment of nonhealing forms of human leishmaniasis with antimonial drugs in combination with gamma interferon (IFN-gamma) may promote healing more effectively than conventional drug therapy. Although the natures of immune responses in patients prior to treatment are often unclear, it is generally assumed that such therapy also promotes a switch from a Th2-type response to a dominant Th1-type response. We have examined the efficacy of IFN-gamma therapy, in combination with drug therapy, to promote healing and a Th2-to-Th1 switch in highly susceptible BALB/c mice infected with Leishmania major. Short-term treatment with the antileishmanial drug sodium stibogluconate failed to significantly alter the course of disease or the immune response when it was given during the third and fourth weeks of infection. IFN-gamma therapy, administered over the same time period, also failed to induce cure or a Th1 dominant response. In contrast, mice treated with a combination of drug and IFN-gamma therapy resolved their infections and developed Th1-type responses. However, administration of an antibody to interleukin 12 (IL-12) reversed the therapeutic effects of therapy with drug plus IFN-gamma, suggesting that IFN-gamma promotes cure through an IL-12-dependent mechanism. Analysis of mRNA levels within parasitized lesions suggests that drug treatment plus IFN-gamma treatment, in addition to reducing parasite numbers, results in reduced levels of IL-4, IL-10, and transforming growth factor beta transcripts but increased levels of transcripts of the p40 chain of IL-12 and inducible nitric oxide synthase, which catalyzes the production of nitric oxide. Together, these results suggest that such immunotherapy may promote the development of a protective Th1-type response in susceptible mice by a mechanism which involves both suppression of regulatory cytokines and enhancement of IL-12 and nitric oxide production.
Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages
Authors: Salkowski C A , Detore G R , Vogel S N
Published: January 2020

Monophosphoryl lipid A (MPL) is a nontoxic derivative of the lipid A region of lipopolysaccharide (LPS) that is being developed as both an adjuvant and prophylactic drug for septic shock. We compared the ability of LPS and MPL to induce interleukin-10 (IL-10), IL-12 p35, IL-12 p40, gamma interferon (IFN-gamma), glucocorticoid receptor (GR), IL-1 receptor antagonist (IL-1ra), and inducible nitric oxide synthase mRNA expression in murine peritoneal macrophages. These genes were chosen for their ability to positively or negatively regulate the host immune response and thus for their potential involvement in MPL-induced adjuvanticity or in its ability to protect against sepsis. LPS was a more potent inducer of IL-12 p35, IL-12 p40, and IFN-gamma mRNA, as well as of IL-12 protein, than MPL. In contrast, MPL induced higher levels of IL-10 mRNA than did LPS from 1 to 1,000 ng/ml. In general, MPL was not a more potent inducer of negative regulatory genes, since MPL and LPS induced similar levels of GR and IL-1ra mRNA. Addition of anti-IL-10 antibody to cultures increased the induction of MPL-induced IL-12 p35, IL-12 p40, and IFN-gamma mRNA, suggesting that the enhanced production of IL-10 by MPL-stimulated macrophages contributes to decreased production of mRNA for IL-12 (p35 and p40) and IFN-gamma. Conversely, the addition of exogenous IL-10 to LPS-treated macrophages reduced the mRNA expression of these cytokine genes. These studies suggest that enhanced production of IL-10 by MPL-stimulated macrophages may contribute to the reduced toxicity of MPL through its negative action on induction of cytokines shown to enhance endotoxicity.
Infection with Salmonella typhimurium modulates the immune response to Schistosoma mansoni glutathione-S-transferase
Authors: Comoy E E , Vendeville C , Capron A , Thyphronitis G
Published: January 2020

Immune response polarization is controlled by several factors, including cytokines, antigen-presenting cells, antigen dose, and others. We have previously shown that adjuvants and live vectors play a critical role in polarization. Thus, immunization with the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28-GST) in aluminum hydroxide induced a type 2 cytokine profile and the production of immunoglobulin G1 (IgG1)- and IgE-specific antibodies. In contrast, mice infected with recombinant Salmonella typhimurium expressing Sm28-GST developed a type 1 cytokine profile and produced IgG2a-specific antibodies against Sm28-GST and Salmonella antigens. In this study, to determine if S. typhimurium not expressing Sm28-GST would still influence the type of the response against this antigen, we compared the profiles of the immune responses generated against Sm28-GST administered in alum in mice infected and not infected with S. typhimurium. Infected mice generated both IgG1 and IgG2a antibodies against Sm28-GST, while noninfected mice produced only IgG1 anti-Sm28-GST antibodies. Moreover, interleukin-4 (IL-4) mRNA expression in infected mice was near background levels, while gamma interferon (IFN-gamma) mRNA expression in coinfected mice was significantly higher than in mice immunized with Sm28-GST in alum only. However, after antigen-specific stimulation in vitro with Sm28-GST, levels of IL-4 and IFN-gamma cytokine production were similar in the two groups of mice. These results suggest that (i) the immune milieu produced during an infection may modify the response against an irrelevant antigen and (ii) isotype switching may be influenced by the cytokine environment of a bystander immune response, even though the specific antigen-driven cytokine production is not modified. Thus, the isotypic profile is not always an absolute reflection of the cytokines produced by antigen-specific Th cells.
Complement fragment C5a and inflammatory cytokines in neutrophil recruitment during intramammary infection with Escherichia coli
Authors: Shuster D E , Kehrli M E , Rainard P , Paape M
Published: January 2020

Generation of inflammatory mediators and leukocyte recruitment to infection at an epithelial surface were studied during Escherichia coli-induced mastitis. One uninfected gland of each of eight midlactation cows was challenged with only 30 CFU of E. coli McDonald strain 487, a serum-resistant isolate from a cow with mastitis. Bacteria grew logarithmically during the first 10 to 12 h after challenge, reaching concentrations of more than 10(5) CFU/ml with no detectable host response during this time. An intense inflammatory reaction began approximately 12 h after the challenge and was characterized by a breakdown in the blood-milk permeability barrier followed by pyrexia and a pronounced leukocytic influx. Coincident with the onset of mammary inflammation was the appearance of neutrophil chemotactic activity in the milk from infected glands. Factors able to upregulate CD18 expression on peripheral blood neutrophils also appeared in milk at this time. The lack of appearance of chemotactic and CD18-upregulating activities until 12 h after challenge indicated that delays in neutrophil recruitment resulted from an initial lack of bacterial recognition and inflammatory mediator production. Production of complement fragment C5a, tumor necrosis factor, and interleukin-1 (IL-1) occurred earlier than production of IL-6 or IL-8. The early and intense production of C5a indicates that this chemoattractant may be more important than IL-8 during the initial recruitment and activation of neutrophils to a developing E. coli infection.
Potential role of autoantibodies in the regulation of cytokine responses during bacterial infections
Authors: Bakhiet M , Diab A , Mahamustafa  , Jiezhu  , Lindqvist L , Link H
Published: January 2020

An immunoregulatory mechanism involving release of neutralizing autoantibodies (Aabs) to self cytokines during bacterial infections is presented herein. Intraperitoneal inoculation of Haemophilus influenzae type b into Sprague-Dawley rats resulted in a self-limiting meningitis. High levels of cells expressing mRNA for gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) were detected 12 to 48 h postinoculation (p.i.) in splenocytes, and large numbers of IFN-gamma-secreting cells were present in the spleen on day 3 p.i. These levels were undetectable at days 9 and 14 p.i. Increased titers of Aabs of immunoglobulin G (IgG) isotypes to both cytokines were observed, with a peak at day 7 p.i. and with very low levels at day 30. Upon reinoculation with H. influenzae type b at day 30, regeneration of Aabs was recorded 7 days later (i.e., at day 37). To elucidate their regulatory importance, Aabs dose-dependently inhibited IFN-gamma production by splenocytes, IFN-gamma-induced major histocompatibility complex expression by peritoneal macrophages, and TNF-alpha-induced thymocyte proliferation. To control the specificity of these Aabs, Fab fragments of purified serum Igs from day p.i. exhibited binding and neutralizing effects. Furthermore, preincubation of the sera with a cytokine inhibited the binding and neutralization effects of that particular cytokine, but not those of any other cytokine. Aab-producing B cells were cloned, and their supernatants had similar effects. Our data suggest a role for autoimmunity in cytokine regulation and suggest that a maintained balance of this mechanism may protect from sequelae.
Pathogenesis of tuberculosis in mice exposed to low and high doses of an environmental mycobacterial saprophyte before infection
Authors: Hernandez-Pando R , Pavön L , Arriaga K , Orozco H , Madrid-Marina V , Rook G
Published: January 2020

Mycobacteria are ubiquitous in the environment, but they are not part of the normal human microbial flora. It has been suggested that variable contact with mycobacteria can influence susceptibility to mycobacterial pathogens and the efficacy of subsequent Mycobacterium bovis BCG vaccination. To test this, mice were immunized with high or low doses of an environmental saprophyte, M. vaccae, that is intensely immunogenic as an autoclaved preparation. Two months later, they received an intratracheal challenge with M. tuberculosis H37Rv. Recipients of a low Th1-inducing dose (10(7) organisms) were partially protected and maintained a high ratio of interleukin 2 (IL-2)-positive to IL-4-positive cells in the perivascular, peribronchial, and granulomatous areas of the lung, whereas in unimmunized controls the IL-4-positive cells increased markedly between days 21 and 28. In contrast, recipients of the high dose (10(9) organisms), which primes Th2 as well as Th1 cytokine production, died more rapidly than unimmunized controls and showed massive pneumonia from day 7. The ratio of IL-2-positive to IL-4-positive cells in all compartments of the lung rapidly fell to 1 by day 14 for these animals. These events correlated with cytokine mRNA profiles and with increases in the local toxicity of tumor necrosis factor alpha (TNF-alpha), demonstrable only when a major Th2 component was present. These data indicate that cross-reactive epitopes present in an environmental saprophyte can evoke either protective responses or responses that increase susceptibility to M. tuberculosis. The latter are associated with the presence of a Th2 component and increased sensitivity to TNF-alpha.
Colonization of congenitally immunodeficient mice with probiotic bacteria
Authors: Wagner R D , Warner T , Roberts L , Farmer J , Balish E
Published: January 2020

We assessed the capacity of four probiotic bacteria (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei GG, and Bifidobacterium animalis) to colonize, infect, stimulate immune responses in, and affect the growth and survival of congenitally immunodeficient gnotobiotic beige-athymic (bg/bg-nu/nu) and beige-euthymic (bg/bg-nu/+) mice. The bacteria colonized and persisted, in pure culture, in the alimentary tracts of both mouse strains for the entire study period (12 weeks). Although all adult and neonatal beige-euthymic mice survived probiotic colonization, some infant mortality occurred in beige-athymic pups born to mothers colonized with pure cultures of L. reuteri or L. casei GG. The probiotic bacteria manifested different capacities to adhere to epithelial surfaces, disseminate to internal organs, affect the body weight of adult mice and the growth of neonatal mice, and stimulate immune responses. Although the probiotic species were innocuous for adults, these results suggest that caution and further studies to assess the safety of probiotic bacteria for immunodeficient hosts, especially neonates, are required.
Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system
Authors: Tacket C O , Kelly S M , Schödel F , Losonsky G , Nataro J P , Edelman R , Levine M M , Curtiss R
Published: January 2020

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.
Synthesis, stability, and subcellular distribution of major histocompatibility complex class II molecules in Langerhans cells infected with Leishmania major
Authors: Flohé S , Lang T , Moll H
Published: January 2020

Protozoan parasites of the genus Leishmania exist as obligatory intracellular amastigotes and invade macrophages and Langerhans cells, the dendritic cells of the skin. Langerhans cells are much more efficient in presenting Leishmania major antigen to T cells than macrophages are and have the unique ability to retain parasite antigen in immunogenic form for prolonged periods. To analyze the mechanisms that are responsible for this potency, we defined the synthesis, turnover, conformation, and localization of major histocompatibility complex (MHC) class II molecules in Langerhans cells. Hence, Langerhans cells were pulse-labeled; immunoprecipitation of MHC class II molecules and gel electrophoresis followed. In addition, the subcellular distribution of MHC class II molecules in L. major-infected Langerhans cells was analyzed by confocal microscopy. The results show that (i) newly synthesized MHC class II molecules are required for L. major antigen presentation by Langerhans cells, (ii) MHC class II-peptide complexes in Langerhans cells are long-lived, (iii) phagocytosis of L. major modulates MHC class II biosynthesis by reducing its downregulation during Langerhans cell differentiation, and (iv) newly synthesized MHC class II molecules are associated with the parasitophorous vacuole of infected Langerhans cells. These findings support the conclusion that the traits of MHC class II expression correspond to the highly specialized functions of Langerhans cells in the immunoregulation of cutaneous leishmaniasis.
Specificity and functional activity of anti-Burkholderia pseudomallei polysaccharide antibodies
Authors: Ho M , Schollaardt T , Smith M D , Perry M B , Brett P J , Chaowagul W , Bryan L E
Published: January 2020

The lipopolysaccharide (LPS) of Burkholderia pseudomallei, the causative agent of melioidosis, consists of two O-antigenic polysaccharides designated O-PS I and O-PS II. In this study, the O-PS specificity and functional activity of a protective polyclonal antiserum and an immunoglobulin M (IgM) monoclonal antibody were determined. The polyclonal antiserum recognized both O-PS I and O-PS II, while the monoclonal antibody was O-PS II specific. Both mediated phagocytic killing of B. pseudomallei by polymorphonuclear leukocytes. Patients acutely infected with B. pseudomallei also produced antibodies to the two O-PSs, but these antibodies were not produced by asymptomatic individuals from an area of endemicity who were seropositive by an indirect hemagglutination test using sonicated heat-killed whole organisms as antigen. IgM antibodies were detected only in patients with localized infection. IgG antibodies were detected in all acutely infected patients, but there was no significant difference in antibody levels among patients with localized infection, patients who survived septicemic illness, and patients who died from septicemic illness. Further analysis of the IgG response revealed production of IgG1 and IgG2 antibodies by all patient groups, while an IgG3 response was seen only in survivors of septicemic infection. IgG4 was not detectable even when a fivefold-lower serum dilution was used. Patient sera also mediated phagocytic killing by polymorphonuclear leukocytes, and the killing effect was enhanced by complement. These results suggest that antibodies to the LPS O-polysaccharides of B. pseudomallei are protective by promoting phagocytic killing. The antibodies develop during human infection and may facilitate clearance of the organisms, as seen in a diabetic rat model of B. pseudomallei infection.
Evidence for ethnic tropism of Helicobacter pylori
Authors: Campbell S , Fraser A , Holliss B , Schmid J , O'Toole P W
Published: January 2020

Helicobacter pylori infection in humans is linked to gastritis, gastric and duodenal ulcers, and gastric cancer. Peptic ulcer disease, as distinct from chronic asymptomatic infection, is strongly associated with expression of bacterial virulence markers, including a major antigen, CagA, and the vacuolating cytotoxin VacA. We have previously described significant differences in colonization rates, independent of socioeconomic status, among ethnic groups in New Zealand. To evaluate relative risks for peptic ulcer disease, we examined the frequency of two virulence markers in H. pylori strains infecting these ethnic groups. Although these markers occurred significantly more frequently in strains isolated from Polynesians than in strains from Europeans, this frequency was not reflected in the incidence of peptic ulcer disease in the two groups. DNA fingerprinting of the urease gene showed that Polynesians are more frequently infected by a group of strains which are genetically distinct from those affecting European New Zealanders. Our data suggest that separate bacterial lineages may have evolved in parallel with race-specific specialization.
Interaction of Mycobacterium avium with environmental amoebae enhances virulence
Authors: Cirillo J D , Falkow S , Tompkins L S , Bermudez L E
Published: January 2020

Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium.
Streptococcus sanguis expresses a 150-kilodalton two-domain adhesin: characterization of several independent adhesin epitopes
Authors: Gong K , Herzberg M C
Published: January 2020

Streptococcus sanguis binds to saliva-coated hydroxylapatite (sHA), an in vitro model of the enamel pellicle. To learn if more than one adhesin functions during adhesion, 12 reactive monoclonal antibodies (MAbs) were isolated by screening against both adhesive and nonadhesive strains. Two of these MAbs, 1.1 and 1.2, inhibited adhesion in a dose-dependent fashion, although maximum inhibition with either was only 37%. When these two MAbs plus a polyclonal antibody to P1-like adhesin were combined, the inhibition was additive to about 82%. These data indicated that there were at least three distinct, functional adhesion epitopes on the surface of S. sanguis. Western blot analyses of S. sanguis surface macromolecules showed antigens at 36 and 56 (with MAb 1.2), 87 and 150 (with both MAb 1.1 and MAb 1.2), and 100, 130, and 170 kDa (with anti-P1 antibody). The antigens were eluted from gels. Isolated antigens and corresponding antibodies inhibited adhesion similarly. Additivity experiments suggested the distinct epitopes were in three groups: (i) 36/56 kDa, (ii) 87/150 kDa, and (iii) 100/130/170 kDa. The 150-kDa antigen reacting with both MAbs was isolated from gels and digested with trypsin. The digestion revealed a series of tryptic bands. A band at 38 kDa reacted with MAb 1.1 whereas a band at 54 kDa reacted with MAb 1.2 in Western blot analysis, indicating two distinct adhesive epitopes on the 150-kDa antigen. These data strongly suggest that S. sanguis adhesion to sHA is maximized when several adhesin epitopes are coexpressed on surface antigens of different sizes.
Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells
Authors: Hoppe H C , de Wet B J , Cywes C , Daffé M , Ehlers M R
Published: January 2020

The molecular basis for the binding of Mycobacterium tuberculosis to nonphagocytic cells, which are readily infected in vitro, and the in vivo significance of this interaction are incompletely understood. Of six cell types tested, we found that only two, Chinese hamster ovary (CHO) fibroblasts and primary porcine aortic endothelial cells, were able to bind M. tuberculosis H37Rv efficiently in vitro. Binding to both CHO and endothelial cells was markedly (three- to fivefold) enhanced by 10 to 20% human or bovine serum, suggesting that the bacteria were coated by a serum opsonin. Preincubation with individual candidate opsonins revealed that recombinant human mannose-binding protein (rMBP), fibronectin, and transferrin were each able to enhance binding threefold. Preincubation of bacteria in serum depleted of mannan-binding lectins or in genetic MBP-deficient serum resulted in enhancements that were only approximately 60 and 58%, respectively, of that produced by preincubation in control serum. In contrast, serum depleted of fibronectin or transferrin retained its opsonizing capacity, suggesting that the latter two are not significant opsonins in whole serum. Binding of M. tuberculosis and Mycobacterium smegmatis to both CHO and endothelial cells in the presence or absence of serum was blocked (60 to 70%) by a monoclonal antibody, MAb 1D1, selected for recognition of intact bacilli. The 1D1 antigen was purified from mycobacterial cell walls and chemically identified as a polar phosphatidylinositol mannoside (PIM). Latex beads coated with purified 1D1 antigen bound to CHO cells, which was enhanced threefold by serum and abolished by periodate treatment, suggesting a requirement for the PIM mannoses in opsonic adhesion. This was likely mediated, at least in part, by serum MBP, as rMBP bound strongly to 1D1 antigen in both thin-layer chromatography overlay and plate binding assays, the latter in a mannan-inhibitable manner. This is the first demonstration that mycobacterial PIMs can function as adhesins for binding to nonphagocytic cells, both directly and after opsonization with serum proteins, including MBP.
Use of cellular depletion analysis to examine circulation of immune effector function between the vagina and the periphery
Authors: Fidel P L , Luo W , Chabain J , Wolf N A , Van Buren E
Published: January 2020

Results from an animal model of vaginal candidiasis suggest that Candida-specific cell-mediated immunity in the systemic circulation does not mediate protection against vaginitis. The present study used cellular depletion analysis to examine the circulation of immune effector function between the vagina and the periphery. Results showed that anti-Thy-1.2 antibodies given intravenously to mice depleted Thy-1+ T lymphocytes in the systemic compartment but not in the vaginal mucosa, while the same antibodies injected intravaginally significantly reduced Thy-1+ T cells in both the vaginal and systemic compartments. These results support a lack or low level of circulation of immune effector function from the periphery to the vaginal mucosa.
A new restriction fragment length polymorphism from Cryptosporidium parvum identifies genetically heterogeneous parasite populations and genotypic changes following transmission from bovine to human hosts
Authors: Carraway M , Tzipori S , Widmer G
Published: January 2020

Length and restriction site polymorphism within a 2.8-kb threonine-rich open reading frame from Cryptosporidium parvum was identified and used to determine the genotypes of isolates from calves and humans. In agreement with observations of other genetic loci, all calf isolates were identical at this locus. In contrast, human isolates showed two profiles, one found exclusively in humans and one a superposition of both profiles, which were indicative of heterogeneous parasite populations. PCR fingerprints were consistent with a change in the genetic profile of C. parvum isolates following transmission from bovine to human hosts.
Arbitrarily primed polymerase chain reaction as a rapid method to differentiate crossed from independent Pseudomonas cepacia infections in cystic fibrosis patients
Authors: Bingen E H , Weber M , Derelle J , Brahimi N , Lambert-Zechovsky N Y , Vidailhet M , Navarro J , Elion J
Published: January 2020

We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiological investigation of 23 Pseudomonas cepacia isolates obtained from 11 cystic fibrosis (CF) patients attending our CF center. This approach was compared with ribotyping, pulsed-field gel electrophoresis (PFGE), and conventional phenotypic typing. AP-PCR and ribotyping were identical in resolving power, since the two methods generated four different profiles and identified the same group of strains. Six patients on the one hand and four on the other harbored strains of the same genotype, thus raising the possibility of either patient-to-patient transmission or acquisition from a common hospital environmental source. PFGE results were in good agreement with those of the other two methods, but PFGE seems more discriminative since it generated a fifth profile for a single strain in a group of four. Our results show in vivo stability for the three methods during a period extending from 3 to 41 months. These genotypic techniques are particularly promising for clinical laboratories to help to clarify the epidemiology of P. cepacia in CF patients. The AP-PCR method constitutes an easier alternative to the well-established ribotyping method. AP-PCR provides the quickest results with minimal technical complexity. However, our results suggest that it is less discriminative than the labor-intensive PFGE method.
Comparison of commercial diagnostic tests for Helicobacter pylori antibodies
Authors: Schembri M A , Lin S K , Lambert J R
Published: January 2020

A number of serological tests measuring the presence of Helicobacter pylori-specific serum immunoglobulin G (IgG) are now commercially available. The aim of this study was to evaluate the clinical accuracy of five commercial H. pylori antibody tests: GAP-IgG (Biomerica), HELpTEST (AMRAD, Kew, Victoria, Australia), HELICO-G (Porton Cambridge), Pyloriset (Orion Diagnostica), and ROCHE (Roche Diagnostics). A total of 162 subjects presenting for routine upper endoscopy were studied. H. pylori was diagnosed if culture, histology, or both were positive. Ten milliliters of venous blood was collected at the time of endoscopy for serological assessment. The sensitivity and specificity of each test (GAP-IgG, HELpTEST, HELICO-G, Pyloriset, and ROCHE) were as follows: 83 and 79%, 92 and 77%, 86 and 65%, 89 and 56%, and 98 and 69%, respectively. Positive and negative predictive values were 97 and 83%, 90 and 91%, 76 and 83%, 68 and 84%, and 86 and 97%, respectively. The specificity of most tests increased by approximately 10% when sera from subjects less than 45 years old were examined. The number of sera falling into the grey zone for each test (an indeterminate result with respect to H. pylori status) varied between 2.5 and 19%. This study highlights the need for all serological kits to be independently evaluated on the population to be studied by testing against a microbiologically defined panel of H. pylori-positive and -negative sera.
Quantitative evaluation of hepatitis C virus RNA in patients with concurrent human immunodeficiency virus infections
Authors: Sherman K E , O'Brien J , Gutierrez A G , Harrison S , Urdea M , Neuwald P , Wilber J
Published: January 2020

Quantitation of the hepatitis C virus (HCV) provides a powerful epidemiologic and therapeutic method for the evaluation of infected patients. In this study semiquantitative reverse transcriptase polymerase chain reaction (PCR) is compared with a new branched DNA signal amplification methodology. Samples from HCV-infected patients as well as from human immunodeficiency virus-infected patients were evaluated. Reverse transcriptase PCR correlated well with the branched DNA assay (r = 0.7036, P < 0.05). HCV RNA was found to occur at significantly higher titers (P < 0.05) in patients coinfected with the human immunodeficiency virus compared with titers in those infected with HCV alone. Immune status as defined by the CD4+ count was not associated with the observed difference in viral titer.