bluuebunny/crossref_metadata_2025 huggingface dataset:
Deviant Expression of Rab5 on Phagosomes Containing the Intracellular PathogensMycobacterium tuberculosisandLegionella pneumophilaIs Associated with Altered Phagosomal Fate
Authors: Clemens Daniel L. , Lee Bai-Yu , Horwitz Marcus A.
Published: July 2002

The intracellular human pathogensLegionella pneumophilaandMycobacterium tuberculosisreside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. TheL. pneumophilaphagosome exists completely outside the endolysosomal pathway, and theM. tuberculosisphagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development ofL. pneumophilaandM. tuberculosisphagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-typeL. pneumophilanever acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutantL. pneumophila(which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast,M. tuberculosisphagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads,M. tuberculosis, and avirulentL. pneumophilabut not wild-typeL. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-typeL. pneumophilaphagosomes, functional Rab5 persists onM. tuberculosisphagosomes. The absence of Rab5 on theL. pneumophilaphagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on theM. tuberculosisphagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.
Role of Manganese-Containing Superoxide Dismutase in Oxidative Stress and Virulence of
Streptococcus pneumoniae
Authors: Yesilkaya Hasan , Kadioglu Aras , Gingles Neill , Alexander Janet E. , Mitchell Tim J. , Andrew Peter W.
Published: July 2002

Streptococcus pneumoniae
was shown to contain two types of superoxide dismutase, MnSOD and FeSOD. Levels of MnSOD increased during growth in an aerobic environment. The
sodA
gene, encoding MnSOD, of virulent
S. pneumoniae
type 2 strain D39 was inactivated to give mutant D39HY1. Aerobically, D39HY1 had a lower growth rate than the wild type and exhibited susceptibility to the redox-active compound paraquat, but anaerobic growth of D39HY1 was identical to that of the wild type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than that of mice infected with the wild-type pneumococcus. In contrast to the wild type, D39HY1 did not multiply in lungs during the first 24 h but thereafter grew at the same rate as the wild type. Appearance in the bloodstream was also delayed, but growth in the blood was unimpaired by the
sodA
mutation. The pattern of inflammation in lungs infected with D39HY1 differed from that in wild-type-infected mice. After infection with D39HY1, neutrophils were densely packed around bronchioles, in contrast to the wild-type infection, where neutrophils were more diffusely localized.
Role of Gamma Interferon and Interleukin-4 in Host Defense against the Human Filarial ParasiteBrugia malayi
Authors: Babu Subash , Ganley Lisa M. , Klei Thomas R. , Shultz Leonard D. , Rajan T. V.
Published: July 2002

We have investigated the roles of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense againstBrugia malayi. Our data suggest that the lack of either IFN-γ or IL-4 prolongs the time required to achieve sterile immunity, suggesting that both canonical type 1 and type 2 responses are involved in the clearance of infection.
Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44
Authors: Kim Hyung-Yong , Rikihisa Yasuko
Published: July 2002

Human granulocytic ehrlichiosis (HGE) is an emerging febrile systemic disease caused by the HGE agent, an obligatory intracellular bacterium of granulocytes. The pathogenicity- and immunity-related mechanisms of HGE are unknown. In this study, several cytokines generated in human peripheral blood leukocytes (PBLs) incubated with the HGE agent or a recombinant 44-kDa major surface protein (rP44) of the HGE agent were examined by reverse transcription-PCR and a capture enzyme-linked immunosorbent assay. The HGE agent induced expression of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 mRNAs and proteins in PBLs in a dose-dependent manner to levels as high as those resulting fromEscherichia colilipopolysaccharide stimulation. The kinetics of induction of these three cytokines in PBLs by rP44 and by the HGE agent were similar. Proteinase K treatment of the HGE agent or rP44 eliminated the ability to induce these three cytokines. Induction of these cytokine mRNAs was not dependent on superoxide generation. These results suggest that P44 proteins have a major role in inducing the production of proinflammatory cytokines by PBLs. Expression of IL-8, IL-10, gamma interferon, transforming growth factor β, and IL-2 mRNAs in response to the HGE agent was not remarkable. Among PBLs, neutrophils and lymphocytes expressed IL-1β mRNA but not TNF-α or IL-6 mRNA in response to the HGE agent, whereas monocytes expressed all three of these cytokine mRNAs. These observations suggest that induction of proinflammatory-cytokine gene expression by the major outer membrane protein of the HGE agent in monocytes, which are not the primary host cells of the HGE agent, contributes to HGE pathogenesis and immunomodulation.
Characterization of an Anticryptococcal Protein Isolated from Human Serum
Authors: Sridhar Sridevi , Ahluwalia Mala , Brummer Elmer , Stevens David A.
Published: July 2002

Human serum at low concentrations inhibits the growth of
Cryptococcus neoformans
in vitro. Fractionation of serum yielded a purified inhibitory protein with a molecular mass of ∼81.8 kDa, a pI of ∼6.2, and an amino acid sequence that matched that of human transferrin. The inhibitory activity and that of apotransferrin and 5% human serum were reversed by 10 μM freshly prepared FeCl
3
.
Differential Infectivity and Division ofToxoplasma gondiiin Human Peripheral Blood Leukocytes
Authors: Channon Jacqueline Y. , Seguin Rosanne M. , Kasper Lloyd H.
Published: July 2002

When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils or lymphocytes were infected. Although tachyzoites were able to divide in each of these cell types, monocytes and dendritic cells were more permissive to rapid tachyzoite division than neutrophils or lymphocytes.
Staphylococcus aureus
RN6390 Replicates and Induces Apoptosis in a Pulmonary Epithelial Cell Line
Authors: Kahl Barbara C. , Goulian Mark , van Wamel Willem , Herrmann Mathias , Simon Sanford M. , Kaplan Gilla , Peters Georg , Cheung Ambrose L.
Published: July 2002

Staphylococcus aureus
frequently colonizes the airways of patients with compromised airway defenses (e.g., cystic fibrosis [CF] patients) for extended periods. Persistent and relapsing infections may be related to live
S. aureus
bacteria actively residing inside epithelial cells. In this study, we infected a respiratory epithelial cell line, which was derived from a CF patient, with
S. aureus
RN6390. Internalization of
S. aureus
was found to be time and dose dependent and could be blocked by cytochalasin D. Transmission electron microscopy revealed that internalized bacteria resided within endocytic vacuoles without any evidence of lysosomal fusion in a 24-h period. The results of internalization experiments and time-lapse fluorescence microscopy of epithelial cells infected with green fluorescent
S. aureus
indicate that, after an initial lag period of 7 to 9 h, intracellular bacteria began to replicate, with three to five divisions in a 24-h period, leading to apoptosis of infected cells. Induction of apoptosis required bacterial internalization and is associated with intracellular replication. The slow and gradual replication of
S. aureus
inside epithelial cells hints at the role of host factors or signals in bacterial growth and further suggests possible cross talk between host cells and
S. aureus.
Direct Evidence for Involvement of NF-κB in Transcriptional Activation of Tumor Necrosis Factor by a Spirochetal Lipoprotein
Authors: Udalova Irina A. , Vidal Vincent , Scragg Ian G. , Kwiatkowski Dominic
Published: July 2002

Variable major lipoprotein (Vmp) is a major tumor necrosis factor (TNF)-inducing component ofBorrelia recurrentis, the agent of louse-borne relapsing fever.B. recurrentisVmp rapidly stimulates nuclear translocation of NF-κB and proinflammatory cytokine gene expression in the human monocyte-like cell line MonoMac 6. By overexpressing disabled mutant IκBα in MonoMac 6 cells cotransfected with a reporter gene, we provide evidence that NF-κB is essential for the transcriptional activation of TNF in this system.
Salmonella
Pathogenicity Island 2 Influences Both Systemic Salmonellosis and
Salmonella
-Induced Enteritis in Calves
Authors: Bispham J. , Tripathi B. N. , Watson P. R. , Wallis T. S.
Published: July 2002

We have used signature-tagged mutagenesis to identify mutants of the host-specific
Salmonella enterica
serotype Dublin which were avirulent in calves and/or BALB/c mice. A mutant with a transposon insertion in the
sseD
gene of
Salmonella
pathogenicity island 2 (SPI-2), which encodes a putative secreted effector protein, was identified. This mutant was recovered from the bovine host but not from the murine host following infection with a pool of serotype Dublin mutants. However, a pure inoculum of the
sseD
mutant was subsequently shown to be attenuated in calves following infection either by the intravenous route or by the oral route. The
sseD
mutant was fully invasive for bovine intestinal mucosa but was subsequently unable to proliferate to the same numbers as the parental strain in vivo. Both the
sseD
mutant and a second SPI-2 mutant, with a transposon insertion in the
ssaT
gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that SPI-2 is required by serotype Dublin for the induction of both systemic and enteric salmonellosis in calves.
Enhancement of Innate Immunity againstMycobacterium aviumInfection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase
Authors: Hayashi Tomoko , Rao Savita P. , Takabayashi Kenji , Van Uden John H. , Kornbluth Richard S. , Baird Stephen M. , Taylor Milton W. , Carson Dennis A. , Catanzaro Antonino , Raz Eyal
Published: July 2002

Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course ofMycobacterium aviuminfection.M. aviumgrowth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, andM. aviumgrowth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-α/β), and IFN-γ. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.
Phosphorylcholine-Dependent Cross-Reactivity between Dental Plaque Bacteria and Oxidized Low-Density Lipoproteins
Authors: Schenkein Harvey A. , Berry Collin R. , Purkall Donald , Burmeister John A. , Brooks Carol N. , Tew John G.
Published: July 2002

Antibodies reactive with phosphorylcholine (PC) are ubiquitous in human sera, but the antigens stimulating their production and their function are not clear. Previous studies have shown that a significant proportion of dental plaque bacteria contain PC as determined by reactivity with PC-specific mouse myeloma proteins and monoclonal antibodies. Additionally, serum antibody concentrations of immunoglobulin (IgG) G anti-PC are higher in sera of individuals who have experienced periodontal attachment loss than those who are periodontally healthy. These data implicate the oral microflora as a source of antigen-stimulating anti-PC responses. Recent data also indicate that antibodies with specificity for PC are elevated in ApoE-deficient mice, a model for studies of athersclerosis, and that such antibodies bound oxidized low-density lipoproteins (LDL) (oxLDL) in atherosclerotic plaques. These data prompted the hypothesis that human anti-PC could bind to both oral bacteria and human oxLDL, and that these antigens are cross-reactive. We therefore examined the ability of human anti-PC to bind to PC-bearing strains of oral bacteria using enzyme-linked immunosorbent inhibition assays and by assessment of direct binding of affinity-purified human anti-PC to PC-bearingActinobacillus actinomycetemcomitans. Our results indicated that PC-bearing strains ofStreptococcus oralis,Streptococcus sanguis,Haemophilus aphrophilus,Actinomyces naeslundii,Fusobacterium nucleatum, andA. actinomycetemcomitans, as well as a strain ofStreptococcus pneumoniae, absorbed up to 80% of anti-PC IgG antibody from human sera. Furthermore, purified anti-PC bound to a PC-bearing strain ofA. actinomycetemcomitansbut only poorly to a PC-negative strain. OxLDL also absorbed anti-PC from human sera, and oxLDL but not LDL reacted with up to 80% of the anti-PC in human sera. Furthermore, purified anti-PC bound directly to oxLDL but not to LDL. The data indicate that PC-containing antigens on a variety of common oral bacteria are cross-reactive with neoantigens expressed in oxLDL. We propose that PC-bearing dental plaque microorganisms may induce an antibody response to PC that could influence the inflammatory response associated with atherosclerosis.
Effects of Alum Adjuvant or a Booster Dose on Immunogenicity during Clinical Trials of Group B Streptococcal Type III Conjugate Vaccines
Authors: Paoletti L. C. , Rench M. A. , Kasper D. L. , Molrine D. , Ambrosino D. , Baker C. J.
Published: July 2002

Phase 1 and 2 clinical trials of group B streptococcal (GBS) capsular polysaccharide (CPS)-protein conjugate vaccines in healthy adults have demonstrated their safety and improved immunogenicity compared with uncoupled CPSs. Two recent trials sought to determine (i) whether adsorption of conjugate vaccine to aluminum hydroxide would improve immunogenicity and (ii) whether the CPS-specific immunoglobulin G (IgG) response could be boosted by administration of a second dose. Adsorption of GBS type III CPS-tetanus toxoid (III-TT) conjugate vaccine to alum did not improve the immune response to a 12.5-μg dose in healthy adult recipients. Four weeks after vaccination, the geometric mean antibody concentrations (GMCs) for the 15 recipients of III-TT with or without alum were 3.3 and 3.6 μg/ml, respectively. In the second trial, 36 healthy adults vaccinated previously with GBS III-TT conjugate were given a second 12.5-μg dose 21 months later. At 4 weeks after the second dose, the GMCs of type III CPS-specific IgG were similar to those measured 4 weeks after the primary vaccination, suggesting a lack of a booster response. However, 8 (22%) of the 36 participants who had undetectable III CPS-specific IgG (<0.05 μg/ml) before the first dose of III-TT conjugate exhibited a booster response to the second dose, with a fourfold-greater GMC of type III CPS-specific IgG than after the initial immunization. These results suggest that prior natural exposure to type III GBS or a related antigen may be responsible for the brisk IgG response to CPS noted in most adults after vaccination. However, a second dose of GBS III-TT conjugate vaccine may be required for adults whose initial CPS-specific IgG concentrations are very low and would also restore the initial peak-specific III CPS-IgG in responders to previous vaccination.
Potential Role for a Carbohydrate Moiety in Anti-CandidaActivity of Human Oral Epithelial Cells
Authors: Steele Chad , Leigh Janet , Swoboda Rolf , Ozenci Hatice , Fidel Paul L.
Published: July 2002

Candida albicansis both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candidahost defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of severalCandidaspecies in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candidaactivity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candidaactivity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candidaactivity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence ofC. albicansto oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candidaactivity occurs exclusively with viable epithelial cells through contact withC. albicansby an as-yet-undefined carbohydrate moiety.
Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of
Escherichia coli
Authors: Sandt Carol H. , Hill Charles W.
Published: July 2002

The
eib
genes of
Escherichia coli
encode surface-exposed proteins which bind immunoglobulins (Ig) such as the Fc fragment of human IgG (IgG Fc) in a nonimmune manner. The Eib proteins belong to a family which includes YadA of
Yersinia
, UspA2 of
Moraxella
, and DsrA of
Haemophilus ducreyi.
This family of surface-exposed proteins shares several features, such as the ability to impart resistance to human serum complement and a tendency to exist as stable multimers. Four genes,
eibA
,
eibC
,
eibD
and
eibE
, were previously identified and cloned from ECOR-9, a strain from the
E. coli
reference collection. EibC, -D, and -E bind human serum IgA in addition to IgG, but no IgA binding has been observed for EibA. Here, we report the cloning of a new
eib
gene,
eibF
, from a second strain of
E. coli
, ECOR-2. The product, EibF, has a relatively strong preference for IgA. Like the other
eib
genes,
eibF
attenuates serum sensitivity, occurs as a stable multimer, and is associated with a prophage. By subcloning portions of the
eibA
and
eibF
genes, we have identified distinct sequence segments sufficient to cause Ig binding, multimerization, and discrimination between IgA and IgG. The ability to multimerize is associated with a sequence close to the C terminus that is homologous to other family members such as YadA. Binding of IgG Fc is associated with a sequence that is highly conserved among all Eib proteins but otherwise unique. Binding of IgA is associated with a sequence of EibF that is not similar to any EibA sequence.
Role of the Classical Pathway of Complement Activation in Experimentally Induced Polymicrobial Peritonitis
Authors: Celik Ilhan , Stover Cordula , Botto Marina , Thiel Steffen , Tzima Sotiria , Künkel Dieter , Walport Mark , Lorenz Wilfried , Schwaeble Wilhelm
Published: July 2002

The complement system and the natural antibody repertoire provide a critical first-line defense against infection. The binding of natural antibodies to microbial surfaces opsonizes invading microorganisms and activates complement via the classical pathway. Both defense systems cooperate within the innate immune response. We studied the role of the complement system in the host defense against experimental polymicrobial peritonitis using mice lacking either C1q or factor B and C2. The C1q-deficient mice lacked the classical pathway of complement activation. The factor B- and C2-deficient mice were known to lack the classical and alternative pathways, and we demonstrate here that these mice also lacked the lectin pathway of complement activation. Using inoculum doses adjusted to cause 42% mortality in the wild-type strain, none of the mice deficient in the three activation routes of complement (factor B and C2 deficient) survived (mortality of 100%). Mortality in mice deficient only in the classical pathway of complement activation (C1q deficient) was 83%. Application of further dilutions of the polymicrobial inoculum showed a dose-dependent decrease of mortality in wild-type controls, whereas no changes in mortality were observed in the two gene-targeted strains. These results demonstrate that the classical activation pathway is required for an effective antimicrobial immune defense in polymicrobial peritonitis and that, in the infection model used, the remaining antibody-independent complement activation routes (alternative and lectin pathways) provide a supporting line of defense to gain residual protection in classical pathway deficiency.
Role of Gamma Interferon in the Pathogenesis of Severe Schistosomiasis in Interleukin-4-Deficient Mice
Authors: La Flamme Anne Camille , Patton Elisabeth A. , Pearce Edward J.
Published: July 2002

In the absence of interleukin-4 (IL-4), infection withSchistosoma mansonileads to a severe fatal disease rather than the chronic survivable condition that occurs in wild-type (WT) mice. Because the sustained production of NO most closely correlates to weight loss and fatality in infected IL-4−/−mice and because gamma interferon (IFN-γ) is an important inducer of inducible NO synthase, infected IL-4−/−mice were treated with anti-IFN-γ antibodies to determine the role of IFN-γ during schistosomiasis in WT and IL-4−/−animals. When IFN-γ was neutralized, Th2 responses were enhanced and NO production was reduced in both WT and IL-4−/−mice. The decreased NO production correlated with a rescue of proliferation in splenocytes from infected IL-4−/−mice. Furthermore, the neutralization of IFN-γ in vivo improved the gross appearance of the liver and led to a reduction in granuloma size in infected IL-4−/−but not WT mice. However, the neutralization of IFN-γ in vivo did not affect the development of severe disease in infected IL-4−/−mice. These results suggest that while the increased production of IFN-γ does lead to some of the pathology observed in infected IL-4−/−mice, it is not ultimately responsible for cachexia and death.
The Inducible Nitric Oxide Synthase Locus Confers Protection against Aerogenic Challenge of Both Clinical and Laboratory Strains ofMycobacterium tuberculosisin Mice
Authors: Scanga Charles A. , Mohan Vellore P. , Tanaka Kathryn , Alland David , Flynn JoAnne L. , Chan John
Published: July 2002

Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) againstMycobacterium tuberculosisinfection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense againstM. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous,M. tuberculosisinfection of mice. Furthermore, in vitro studies have demonstrated that different strains ofM. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates ofM. tuberculosisfollowing both aerogenic and intravenous challenges. Three recently isolated and uniqueM. tuberculosisstrains were used to infect both wild-type (wt) C57BL/6 andNOS2gene-disrupted mice. Regardless of the route of infection, NOS2−/−mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain ofM. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2−/−mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2−/−mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains ofM. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.
Temporal and Spatial Arrangement of Lymphocytes within Lung Granulomas Induced by Aerosol Infection withMycobacterium tuberculosis
Authors: Gonzalez-Juarrero Mercedes , Turner Oliver C. , Turner Joanne , Marietta Peter , Brooks Jason V. , Orme Ian M.
Published: July 2002

The progression of the immune response in the lungs after aerosol infection withMycobacterium tuberculosisis a complex cellular event dominated by macrophages and lymphocytes. Although the phenotype of lymphocytes participating in this response is becoming increasingly well characterized, the dynamic influx of these cells during the infection and their spatial arrangements within the lung tissue are still poorly understood. This study shows that in the first month after aerosol infection withM. tuberculosisthere was a steady increase in the percentages of total CD3+, CD3+CD4+and CD3+CD8+cells, with consistently larger numbers of CD3+CD4+cells than of CD3+CD8+cells. As granuloma formation continued, the granuloma was found to consist of macrophages, CD4, and CD8 T cells, as well as a smaller number of B cells. Whereas CD4 T cells formed organized aggregates, CD8 T cells were fewer and more scattered and tended to be more prominent toward the periphery of the granulomas. The possible ramifications of the juxtapositions of these two major T-cell subsets are discussed.
Expression of Hemin Receptor Molecule ChuA Is Influenced by RfaH in Uropathogenic
Escherichia coli
Strain 536
Authors: Nagy Gábor , Dobrindt Ulrich , Kupfer Maren , Emödy Levente , Karch Helge , Hacker Jörg
Published: July 2002

The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic
Escherichia coli
strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic
E. coli
strain 536. In an
rfaH
mutant, the
chuA
transcript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within the
chuA
gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of the
chuA
determinant and their distribution in
E. coli
pathogroups are described.
Legionella pneumophilaSuppresses Interleukin-12 Production by Macrophages
Authors: Matsunaga Kazuto , Klein Thomas W. , Newton Catherine , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

In vitro infection of macrophages withLegionella pneumophilainduced interleukin-1α (IL-1α), IL-10, monocyte chemotactic protein 1 (MCP-1), and MCP-3 but not IL-12. The lipopolysaccharide (LPS)-induced production of IL-12 was down-regulated by infection with virulentL. pneumophila, but other cytokines were not affected. In contrast, avirulentL. pneumophilaor UV-killed, virulentL. pneumophiladid not induce any suppression of IL-12. The IL-12 suppression occurred at the level of mRNA accumulation for IL-12 genes in response to LPS stimulation, but the infection induced a marked accumulation of mRNA for both MCP-1 and MCP-3, which are known to suppress IL-12 production in LPS-stimulated macrophages. However, pretreatment of macrophages with MCP-1 did not suppress LPS-induced IL-12 production at the concentrations induced byL. pneumophilainfection. These results suggest thatL. pneumophilaselectively suppresses IL-12 production induced by LPS from macrophages in vitro by an MCP-independent mechanism.
Immunoglobulin A (IgA) Responses and IgE-Associated Inflammation along the Respiratory Tract after Mucosal but Not Systemic Immunization
Authors: Hodge Lisa M. , Marinaro Mariarosaria , Jones Harlan P. , McGhee Jerry R. , Kiyono Hiroshi , Simecka Jerry W.
Published: July 2002

The purpose of the present study was to determine the extent of immunologic responses, particularly immunopathologic responses, within the upper and lower respiratory tracts after intranasal immunization using the mucosal adjuvant cholera toxin (CT). BALB/c mice were nasally immunized with influenza virus vaccine combined with CT. The inclusion of the mucosal adjuvant CT clearly enhanced generation of antibody responses in both the nasal passages and lungs. After nasal immunization, antigen-specific immunoglobulin A (IgA) antibody-forming cells dominated antibody responses throughout the respiratory tract. However, IgG responses were significant in lungs but not in nasal passages. Furthermore, parenteral immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions, characterized by mononuclear cell infiltration, developed within the lungs of mice but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising adjuvancy. Serum IgE responses were also enhanced in a dose-dependent manner by inclusion of CT. In summary, there are differences in the generation of humoral immunity between the upper respiratory tract and the lung. As the upper respiratory tract is in a separate compartment of the immune system from that stimulated by parenteral immunization, nasal immunization is an optimal approach to generate immunity throughout the respiratory tract. Despite the promise of nasal immunization, there is also the potential to develop adverse immunopathologic reactions characterized by pulmonary airway inflammation and IgE production.
Perturbation and Proinflammatory Type Activation of Vδ1+γδ T Cells in African Children withPlasmodium falciparumMalaria
Authors: Hviid Lars , Kurtzhals Jørgen A. L. , Adabayeri Victoria , Loizon Severine , Kemp Kåre , Goka Bamenla Q. , Lim Annick , Mercereau-Puijalon Odile , Akanmori Bartholomew D. , Behr Charlotte
Published: July 2002

γδ T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the γδ T-cell response to malaria in African children, who suffer the large majority of malaria-associated morbidity and mortality. This is unfortunate, since available data suggest that simple extrapolation of conclusions drawn from studies of nonimmune adults ex vivo and in vitro is not always possible. Here we show that both the frequencies and the absolute numbers of γδ T cells are transiently increased following treatment ofPlasmodium falciparummalaria in Ghanaian children and they can constitute 30 to 50% of all T cells shortly after initiation of antimalarial chemotherapy. The bulk of the γδ T cells involved in this perturbation expressed Vδ1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the Vδ1+cell population at the peak of their increase showed that all expressed Vγ chains were used, and CDR3 length polymorphism indicated that the expanded Vδ1 population was highly polyclonal. A very high proportion of the Vδ1+T cells produced gamma interferon, while fewer Vδ1+cells than the average proportion of all CD3+cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-γδ+cells in general or Vδ1+cells in particular. Taken together, our data point to an immunoregulatory role of the expanded Vδ1+T-cell population in this group of semi-immuneP. falciparummalaria patients.
Bactericidal Activity of Human Eosinophilic Granulocytes againstEscherichia coli
Authors: Persson Terese , Andersson Pia , Bodelsson Mikael , Laurell Martin , Malm Johan , Egesten Arne
Published: July 2002

Eosinophils participate in allergic inflammation and may have roles in the body's defense against helminthic infestation. Even under noninflammatory conditions, eosinophils are present in the mucosa of the large intestine, where large numbers of gram-negative bacteria reside. Therefore, roles for eosinophils in host defenses against bacterial invasion are possible. In a system for bacterial viable counts, the bactericidal activity of eosinophils and the contribution of different cellular antibacterial systems againstEscherichia coliwere investigated. Eosinophils showed a rapid and efficient killing ofE. coliunder aerobic conditions, whereas under anaerobic conditions bacterial killing decreased dramatically. In addition, diphenylene iodonium chloride (DPI), an inhibitor of the NADPH oxidase and thereby of superoxide production, also significantly inhibited bacterial killing. The inhibitor of nitric oxide (NO) productionl-N5-(1-iminoethyl)-ornithine dihydrochloride did not affect the killing efficiency, suggesting that NO or derivatives thereof are of minor importance under the experimental conditions used. To investigate the involvement of superoxide and eosinophil peroxidase (EPO) in bacterial killing, EPO was blocked by azide. The rate ofE. colikilling decreased significantly in the presence of azide, whereas addition of DPI did not further decrease the killing, suggesting that superoxide acts in conjunction with EPO. Bactericidal activity was seen in eosinophil extracts containing granule proteins, indicating that oxygen-independent killing may be of importance as well. The findings suggest that eosinophils can participate in host defense against gram-negative bacterial invasion and that oxygen-dependent killing, i.e., superoxide acting in conjunction with EPO, may be the most important bactericidal effector function of these cells.
Helicobacter hepaticus-Induced Colitis in Interleukin-10-Deficient Mice: Cytokine Requirements for the Induction and Maintenance of Intestinal Inflammation
Authors: Kullberg Marika C. , Rothfuchs Antonio Gigliotti , Jankovic Dragana , Caspar Patricia , Wynn Thomas A. , Gorelick Peter L. , Cheever Allen W. , Sher Alan
Published: July 2002

We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted withHelicobacter hepaticusdevelop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology followingH. hepaticusinfection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with establishedH. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS) mRNA levels, andH. hepaticus-specific IFN-γ secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3+T cells and significantly reduced frequencies ofHelicobacter-reactive CD4+Th1 cells in MLN. In contrast, anti-IFN-γ and/or anti-TNF-α had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-γ double-deficient mice, we further show that IFN-γ is not required for the development of colitis follwingH. hepaticusinfection. MLN cells from infected IL-10/IFN-γ KO animals secreted elevated amounts of IL-12 and TNF-α following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.
CdtA, CdtB, and CdtC Form a Tripartite Complex That Is Required for Cytolethal Distending Toxin Activity
Authors: Lara-Tejero Marı́a , Galán Jorge E.
Published: July 2002

Campylobacter jejuniencodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G2/M transition phase of the cell cycle. Highly related toxins are also produced by other important bacterial pathogens. CDT activity requires the function of three genes:cdtA, cdtB, andcdtC. Recent studies have established that CdtB is the active subunit of CDT, exerting its effect as a nuclease that damages the DNA and triggers cell cycle arrest. Microinjection of CdtB into target cells led to G2/M arrest and cytoplasmic distention, in a manner indistinguishable from that caused by CDT treatment. Despite this progress, nothing is known about the composition of the CDT holotoxin or the function of CdtA and CdtC. We show here that, when applied individually, purified CdtA, CdtB, or CdtC does not exhibit toxic activity. In contrast, CdtA, CdtB, and CdtC when combined, interact with one another to form an active tripartite holotoxin that exhibits full cellular toxicity. CdtA has a domain that shares similarity with the B chain of ricin-related toxins. We therefore proposed that CDT is a tripartite toxin composed of CdtB as the enzymatically active subunit and of CdtA and CdtC as the heterodimeric B subunit required for the delivery of CdtB.
Immune Response to Chlamydial 60-Kilodalton Heat Shock Protein in Tears from Nepali Trachoma Patients
Authors: Hessel Tracey , Dhital S. P. , Plank Rebeca , Dean Deborah
Published: July 2002

Although the host immune response to the 60-kDa chlamydial heat shock protein (hsp60) has been implicated in trachoma pathogenesis, no studies have examined mucosal immune responses to hsp60 in populations for which chlamydia is endemic. Tears and sera from Nepali villagers were reacted against hsp60 fusion proteins, whole hsp60, and the major outer membrane protein (MOMP). Tears from villagers without disease were anti-hsp60 immunoglobulin G (IgG) reactive in 6 (38%) of 16 villagers compared with 36 (90%) of 40 with follicular trachoma (TF) (
P
< 0.001); 47 (89%) of 53 with inflammatory trachoma (TI) (
P
< 0.001); and 31 (84%) of 37 with conjunctival scarring (TS) (
P
= 0.002). By multivariate analysis, odds ratios for tear hsp60 IgG immunoreactivity in villagers with TF, TI, and TS were 49.2 (confidence interval [CI], 2.7 to 898), 22.6 (CI, 3 to 170), and 13.6 (CI, 1.4 to 133), respectively. There were no significant differences for tear IgA or secretory IgA (sIgA) reactivity to hsp60 or for tear sIgA and IgG reactivity to MOMP. Serum anti-hsp60 IgG immunoreactivity was associated with TI only. These data suggest that anti-hsp60 IgG immunoreactivity represents largely locally derived antibodies, which may promote disease pathology. In contrast, nonspecific high rates of anti-hsp60 sIgA antibodies suggest chronic or repeat stimulation from an endemic source of organisms.
Dendritic Cells Can Initiate Protective Immune Responses against Malaria
Authors: Bruña-Romero Oscar , Rodriguez Ana
Published: July 2002

An understanding of the antigen presentation mechanisms that mediate induction of protective immune responses against malaria is essential for the development of successful immunization approaches. Here we show that dendritic cells presentingPlasmodium yoeliisporozoite antigens are able to activate specific CD4+and CD8+T cells and initiate protective immune responses against malaria in mice.
Filarial Antigens Impair the Function of Human Dendritic Cells during Differentiation
Authors: Semnani Roshanak Tolouei , Sabzevari Helen , Iyer Ramesh , Nutman Thomas B.
Published: July 2002

The antigen-specific T-cell unresponsiveness seen in lymphatic filariasis is mediated, in part, by diminished antigen-presenting cell function and is most specific for microfilariae (MF), the parasite stage found in large numbers in the peripheral circulation. We investigated the effect of MF antigen (MFAg) on dendritic cells (DC) in both their differentiation process from monocyte precursors and also after they have developed into DC. When MFAg was added to cultures of monocytes during their differentiation process to immature DC, the production of interleukin 12 (IL-12) p40, p70 protein, and IL-10 was significantly (P< 0.03) inhibited in response toStaphylococcus aureusCowan (SAC) and SAC-gamma interferon (IFN-γ) (60% to 80% inhibition). IL-10 was also inhibited (P= 0.04) in response to CD40 ligand–IFN-γ. Moreover, MFAg inhibited the mRNA expression of IL-12 p40 and IL-10 as assessed by RNA protection assays. This effect was antigen specific, as another parasite antigen (solubleToxoplasma gondiiantigen) did not inhibit the production of these cytokines. This effect was also not a result of diminished cell viability nor of an alteration in surface expression of most costimulatory surface molecules, including major histocompatibility complex class I and class II. In contrast to exposure throughout the differentiation process, MFAg added to immature DC had no effect on DC cytokine expression. Although MF-differentiated DC were capable of inducing an allogeneic mixed lymphocyte reaction, they did so to a significantly lesser degree than DC without antigen exposure. These data collectively suggest that once DC are differentiated from their precursor cells, they become resistant to changes by MFAg.
Host Defense Functions of Proteolytically Processed and Parent (Unprocessed) Cathelicidins of Rabbit Granulocytes
Authors: Zarember Kol A. , Katz Seth S. , Tack Brian F. , Doukhan Laurence , Weiss Jerrold , Elsbach Peter
Published: July 2002

Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulatedEscherichia coli(90% inhibitory concentration, ∼600 nM) but it was ∼50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p (∼20 nM) or BPI (∼50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide.
Intracellular Survival ofLeishmania majorin Neutrophil Granulocytes after Uptake in the Absence of Heat-Labile Serum Factors
Authors: Laufs Helmut , Müller Kerstin , Fleischer Jens , Reiling Norbert , Jahnke Nicole , Jensenius Jens C. , Solbach Werner , Laskay Tamás
Published: July 2002

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasiteLeishmaniais poorly understood. In the present study, the interaction of human PMN withLeishmania majorpromastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake ofLeishmania. However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalizeLeishmaniaeven under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake ofLeishmania. Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority ofLeishmaniaparasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response toL. majorinfection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand,Leishmaniacan survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival ofLeishmaniawithin the first hours or days after infection.