bluuebunny/crossref_metadata_2025 huggingface dataset:
Leishmania pifanoiPathogenesis: Selective Lack of a Local Cutaneous Response in the Absence of Circulating Antibody
Authors: Colmenares María , Constant Stephanie L. , Kima Peter E. , McMahon-Pratt Diane
Published: November 2002

Recently, a role for B cells in the pathogenesis associated with infection byLeishmania(Leishmania mexicanacomplex andL. donovani) has been established. In the case ofL. mexicanacomplex parasites (L. mexicana,L. pifanoi, andL. amazonensis), a critical role for immunoglobulin G-mediated mechanisms for the amastigote stage in the host is evident; however, the immunological mechanisms involved remain to be established. In vitro analysis of the kinetics of parasite uptake by macrophages failed to indicate a major effect of antibody opsonization. Given the importance of CD4+T cells in the development of disease caused by these parasites, the possibility that the lack of pathogenesis was due to the lack of development of an immune response at the local site (draining lymph node and/or cutaneous site) was explored. Interestingly, the level of CD4+-T-cell activation (proliferation and cytokine) in draining lymph nodes from mice lacking circulating antibody (resistant) was found to be comparable to that in nodes from wild-type mice (susceptible) at 2, 5, and 10 weeks postinfection. However, antibody-deficient animals had markedly reduced numbers of monocytes and lymphocytes recruited or retained at the site of cutaneous infection in comparison to wild-type mice, indicating a selective impairment in the local cutaneous immune response. In vitro antigen presentation studies employing tissue-derived (opsonized) amastigotes demonstrated thatL. pifanoi-infected FcR−/−macrophages, in contrast to comparably infected wild-type cells, failed to activateLeishmaniaantigen-specific T lymphocytes. These data, taken together, suggest that one possible mechanism for the role of antibody in pathogenesis may be to mediate parasite uptake and regulate the immune response at the local cutaneous site of infection.
Immunization with a Combination of Merozoite Surface Proteins 4/5 and 1 Enhances Protection against Lethal Challenge withPlasmodium yoelii
Authors: Kedzierski Lukasz , Black Casilda G. , Goschnick Matthew W. , Stowers Anthony W. , Coppel Ross L.
Published: November 2002

It is widely believed that subunit vaccines composed of multiple components will offer greater protection against challenge by malaria, and yet there is little experimental evidence to support this view. We set out to test this proposition in thePlasmodium yoeliichallenge system in rodents by comparing the degree of protection conferred by immunization with a mixture of merozoite surface proteins to that conferred by single proteins. We therefore examined a defined protein mixture made of the epidermal growth factor-like domains ofP. yoellimerozoite surface protein 1 (MSP1) and MSP4/5, the homologue ofP. falciparumMSP4 and MSP5. In the present study we demonstrate that this combination of recombinant proteins dramatically enhances protection against lethal malaria challenge compared to either protein administered alone. Many mice immunized with the MSP4/5 plus MSP119combination did not develop detectable parasitemia after challenge. Combined immunization with MSP119and yMSP4/5, a product characterized by lower protective efficacy, also greatly enhanced protection by reducing peak parasitemias and increasing the numbers of survivors. In some combination trials, levels of antibodies to MSP119were elevated compared to the MSP119alone group; however, improved protection occurred regardless of whether boosting of the anti-MSP119response was observed. Boosting of anti-MSP119did not appear to be due to contaminating endotoxin in the EcMSP4/5 material since enhanced protection was observed in C3H/HeJ mice, which are endotoxin insensitive. Collectively, these experiments show that multiantigen combinations offer enhanced levels of protection against asexual stage infection and suggest that combinations of MSP1, MSP4, and MSP5 should be evaluated further for use in humans.
Dynamics of Gamma Interferon, Interleukin-12 (IL-12), IL-10, and Transforming Growth Factor β mRNA Expression in PrimaryMycobacterium bovisBCG Infection in Guinea Pigs Measured by a Real-Time Fluorogenic Reverse Transcription-PCR Assay
Authors: Kawahara Mamoru , Nakasone Tadashi , Honda Mitsuo
Published: November 2002

The guinea pig has been utilized as a model for studying infectious diseases because its reactions closely resemble those of humans biologically and immunologically. However, the cytokine responses in this animal remain to be studied. Initially, we established a quantitative assay using a real-time reverse transcription-PCR (RT-PCR) to measure guinea pig gamma interferon (IFN-γ), interleukin-12 (IL-12), IL-10, and transforming growth factor β (TGF-β) mRNA. By preparing primer-fluorogenic probe sets for these cytokines and standard RNA templates corresponding to the target sequence of each cytokine, we obtained linear standard curves essential for quantitative determination. In guinea pigs immunized by intradermal (i.d.) vaccination with the Tokyo strain ofMycobacterium bovisBCG (0.1 mg) or else hyperimmunized with the same vaccine (10 mg) given intravenously (i.v.), peripheral blood mononuclear cells (PBMCs) at 4 weeks showed an increase in IFN-γ mRNA expression in the latter but not the former animals. However, at week 10, IFN-γ mRNA expression was markedly elevated in PBMCs, spleen cells, and cells in bronchoalveolar lavage fluid in both the i.d.- and the i.v.-immunized animals, the level of expression being 10 times higher in the latter. In contrast, the expression levels of IL-12 mRNA in PBMCs, spleen cells, and BAL cells were not enhanced in either group at 10 weeks postimmunization. The expression of IL-10 and TGF-β increased slightly only in PBMCs. Regardless of differences in the levels of cytokine responses, the magnitudes of the purified protein derivative of tuberculin-specific delayed-type hypersensitivity (DTH) skin reactions for the two groups did not differ significantly at 8 weeks postvaccination. In this study, we quantitatively measured IL-10, IL-12, TGF-β, and IFN-γ mRNA in BCG-immunized guinea pigs and showed that the level of IFN-γ mRNA expression does not necessarily reflect the magnitude of the DTH response, suggesting that there may be an intricate relationship between protective immunity, the level of IFN-γ, and the DTH response. Thus, our quantitative assay would be of use for the development of vaccines using guinea pig models.
Mannoprotein fromCryptococcus neoformansPromotes T-Helper Type 1 Anticandidal Responses in Mice
Authors: Pietrella Donatella , Mazzolla Rosanna , Lupo Patrizia , Pitzurra Lucia , Gomez Maria Jesus , Cherniak Robert , Vecchiarelli Anna
Published: November 2002

We previously demonstrated that mannoprotein (MP) fromCryptococcus neoformans(CnMP) stimulates interleukin-12 production by human monocytes, thus fostering a T-helper type 1 (Th1) protective anticryptococcal response. In this paper we show that CnMP was also able to induce aCandida albicans-directed protective Th1 response. This was demonstrated for mice immunized with CnMP by induction of a delayed-type hypersensitivity (DTH) reaction toC. albicansMP (CaMP) as well as induction of gamma interferon production by CD4+and CD8+splenic T cells stimulated in vitro with CaMP. CnMP-immunized mice were also partially protected from lethal systemic challenge withC. albicans, as shown by prolonged median survival times and decreased fungal burden in the kidney. Much evidence supports the validity of these cross-reactive and functional Th1 responses: (i) a non-cross-reactiveC. albicansantigen, such as enolase, did not produce a DTH response to CaMP; (ii) passive adoptive transfer of T cells primed with CnMP induced a DTH reaction; (iii)C. neoformansextract elicited a DTH response to CaMP; and (iv) a monoclonal antibody (7H6) directed against a major and immunodominant T-cell-stimulatory 65-kDa MP (MP65) ofC. albicansalso recognized discrete 100-kDa constituents ofC. neoformansextracts, as well as secretory constituents of the fungus. These results suggest the presence of common Th1 antigenic determinants in the mannoproteic material ofC. neoformansandC. albicansepitopes, which should be considered in devising common strategies for immunoprophylactic or immunotherapeutic control of the fungi.
Anomalous Role of Tumor Necrosis Factor Alpha in Experimental Enterococcal Infection
Authors: Papasian Christopher J. , Silverstein Richard , Gao Jian Jun , Bamberger David M. , Morrison David C.
Published: November 2002

The murined-galactosamine (d-gal) model of tumor necrosis factor alpha (TNF-α) hypersensitization was used as an initial tool to investigate the potential contribution of TNF-α to lethal intraperitoneal (i.p.) infection withEnterococcus faecalis.d-gal sensitized mice to lethalE. faecalisinfection, whereas dexamethasone and neutralizing anti-TNF-α antibody protectedd-gal-treated,E. faecalis-infected mice, implicating TNF-α in the lethal response toE. faecalisinfection ind-gal-treated mice. Circulating TNF-α was undetectable for at least 8 h following i.p.E. faecalisinfection, although low peritoneal levels of TNF-α were detected within 3 h, suggesting that localized TNF-α production contributed to the lethal response toE. faecalisinfection ind-gal-treated mice. Although i.p.E. faecalisinfection failed to induce a detectable systemic TNF-α response, circulating Interleukin-6 (IL-6) was detected within 3 h of infection. IL-6 was also detected in the peritoneum within an hour of infection, prior to the appearance of peritoneal TNF-α. In striking contrast to in vivo results,E. faecalisinduced a potent and rapid TNF-α response from both mouse peritoneal macrophages and the RAW 264.7 cell line in vitro. This led us to hypothesize that TNF-α production in response toE. faecalisinfection is suppressed by IL-6 in vivo. In vitro experiments demonstrated a statistically significant, but modest, inhibitory effect of IL-6 on TNF-α production by RAW cells stimulated withE. faecalis. Collectively, these data indicate that acute, lethalE. faecalisinfection appears to induce an unusual cytokine response that differs in character from that previously described for most other gram-positive and gram-negative bacteria.
Differences in Expression of Toll-Like Receptors and Their Reactivities in Dendritic Cells in BALB/c and C57BL/6 Mice
Authors: Liu Tie , Matsuguchi Tetsuya , Tsuboi Naotake , Yajima Toshiki , Yoshikai Yasunobu
Published: November 2002

We have previously reported that differences in early production of interleukin 12 (IL-12) by dendritic cells (DC) underlies the difference between the susceptibilities toListeria monocytogenesof C57BL/6 and BALB/c mice. To elucidate mechanisms for the different abilities of DC to produce cytokine in C57BL/6 and BALB/c mice, we examined Toll-like receptor (TLR) expression by DC and their responses in vitro to known microbial ligands for TLRs. We found that DC isolated from the spleens of naive C57BL/6 mice preferentially expressed TLR9 mRNA, whereas DC from naive BALB/c mice strongly expressed TLR2, -4, -5, and -6 mRNAs. C57BL/6 DC produced a higher level of IL-12p40 in response to the ligands for TLR4 (lipopolysaccharide), TLR2 (lipoprotein), and TLR9 (CpG), whereas BALB/c DC responded to these ligands by producing a larger amount of monocyte chemoattractant protein 1. C57BL/6 DC expressed higher levels of CD40 and Stat4 than BALB/c DC did, suggesting that naive C57BL/6 mice contained more-mature subsets of DC than naive BALB/c mice. Differences in reactivities of DC to microbial molecules through TLRs may be associated with susceptibility and resistance toListeriainfection in BALB/c and C57BL/6 mice.
EndoS and SpeB fromStreptococcus pyogenesInhibit Immunoglobulin-Mediated Opsonophagocytosis
Authors: Collin Mattias , Svensson Mikael D. , Sjöholm Anders G. , Jensenius Jens C. , Sjöbring Ulf , Olsén Arne
Published: November 2002

The human pathogenStreptococcus pyogenesprimarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival ofS. pyogenesin human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests thatS. pyogenesexpresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system.
Marked Enhancement of the Antigen-Specific Immune Response by Combining Plasmid DNA-Based Immunization with a Schiff Base-Forming Drug
Authors: Charo Jehad , Sundbäck Maria , Wasserman Ken , Ciupitu Anne-Marie T. , Mirzai Babak , van der Zee Ruurd , Kiessling Rolf
Published: November 2002

Although plasmid DNA (pDNA)-based immunization has proven efficacy, the level of immune responses that is achieved by this route of vaccination is often lower than that induced by traditional vaccines, especially for primates and humans. We report here a simple and potent method to enhance pDNA-based vaccination by using two different plasmids encoding viral or bacterial antigens. This method is based on coadministration of low concentrations of a recently described immunopotentiating, Schiff base-forming drug called tucaresol which has led to significant augmentation of antigen-specific humoral and cellular immune responses. Our data suggest that enhancement of the immune response with tucaresol might provide a powerful tool for the further development of pDNA-based immunization for humans.
Counteracting Interactions between Lipopolysaccharide Molecules with Differential Activation of Toll-Like Receptors
Authors: Hajishengallis George , Martin Michael , Schifferle Robert E. , Genco Robert J.
Published: November 2002

We investigated counteracting interactions between the lipopolysaccharides (LPS) fromEscherichia coli(Ec-LPS) andPorphyromonas gingivalis(Pg-LPS), which induce cellular activation through Toll-like receptor 4 (TLR4) and TLR2, respectively. We found that Ec-LPS induced tolerance in THP-1 cells to subsequent tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) induction by Pg-LPS, though the reverse was not true, and looked for explanatory differential effects on the signal transduction pathway. Cells exposed to Pg-LPS, but not to Ec-LPS, displayed persisting expression of IL-1 receptor-associated kinase without apparent degradation, presumably allowing prolonged relay of downstream signals. Accordingly, cells pretreated with Pg-LPS, but not with Ec-LPS, were effectively activated in response to subsequent exposure to either LPS molecule, as evidenced by assessing nuclear factor (NF)-κB activity. In fact, Pg-LPS primed THP-1 cells for enhanced NF-κB activation and TNF-α release upon restimulation with the same LPS. This was a dose-dependent effect and correlated with upregulation of surface TLR2 expression. Furthermore, we observed inhibition of NF-κB-dependent transcription in a reporter cell line pretreated with Ec-LPS and restimulated with Pg-LPS (compared to cells pretreated with medium only and restimulated with Pg-LPS), but not when the reverse treatment was made. Although Pg-LPS could not make cells tolerant to subsequent activation by Ec-LPS, Pg-LPS inhibited Ec-LPS-induced TNF-α and IL-6 release when the two molecules were added simultaneously into THP-1 cell cultures. Pg-LPS also suppressedP. gingivalisFimA protein-induced NF-κB-dependent transcription in the 3E10/huTLR4 reporter cell line, which does not express TLR2. This rules out competition for common signaling intermediates, suggesting that Pg-LPS may block component(s) of the TLR4 receptor complex. Interactions between TLR2 and TLR4 agonists may be important in the regulation of inflammatory reactions.
Fibronectin-Facilitated Invasion of T84 Eukaryotic Cells by
Campylobacter jejuni
Occurs Preferentially at the Basolateral Cell Surface
Authors: Monteville Marshall R. , Konkel Michael E.
Published: November 2002

Previous studies have indicated that the ability to bind to fibronectin is a key feature in successful cell invasion by
Campylobacter jejuni
. Given the spatial distribution of fibronectin and the architecture of the epithelium, this suggests the possibility that
C. jejuni
cell invasion might preferentially occur at the basolateral cell surface. To test this hypothesis, we examined the interaction of
C. jejuni
with T84 human colonic cells. When grown under the appropriate conditions, T84 cells form a polarized cell monolayer.
C. jejuni
translocation of a T84 cell monolayer appeared to occur via a paracellular (extracellular) route as opposed to a transcellular (intracellular) route based on the finding that a
C. jejuni
noninvasive mutant translocated as efficiently as its isogenic parent. Additional studies revealed that two distinct
C. jejuni
wild-type isolates could compete with one another for host cell receptors, whereas a
C. jejuni
fibronectin-binding-deficient mutant could not compete with a wild-type isolate for host cell receptors. Further,
C. jejuni
adherence and internalization were significantly inhibited by antifibronectin antibodies but only when cells were first treated with EGTA to expose basolateral cell surfaces. Together, these results support the theory that
C. jejuni
invasion occurs preferentially at the basolateral surface of eukaryotic cells.
Transgenic Mice Expressing Human Interleukin-10 in the Antigen-Presenting Cell Compartment Show Increased Susceptibility to Infection withMycobacterium aviumAssociated with Decreased Macrophage Effector Function and Apoptosis
Authors: Feng Carl G. , Kullberg Marika C. , Jankovic Dragana , Cheever Allen W. , Caspar Patricia , Coffman Robert L. , Sher Alan
Published: November 2002

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected withMycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers ofM. aviumand succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice toM. aviuminfection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-γ) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.
Analysis of Ebh, a 1.1-Megadalton Cell Wall-Associated Fibronectin-Binding Protein of
Staphylococcus aureus
Authors: Clarke Simon R. , Harris Llinos G. , Richards R. Geoff , Foster Simon J.
Published: November 2002

In order for
Staphylococcus aureus
to adhere to host extracellular matrix (ECM) substrates, it elicits a wide range of surface proteins. We have characterized a novel ∼1.1-MDa protein in
S. aureus
, termed Ebh (for ECM-binding protein homologue), which has homology to other ECM-binding proteins. Ebh consists of several domains, including a large central region with 44 imperfect repeats of 126 amino acids. Expression analysis revealed
ebh
to be growth phase regulated and repressed by
agr
. A fragment of the central repeat region of Ebh was cloned, overexpressed, and used in ligand-binding studies to determine Ebh function. The recombinant protein was found to specifically bind human fibronectin. Ebh is produced during human infection since serum samples taken from patients with confirmed
S. aureus
infections were found to contain anti-Ebh antibodies. Localization studies revealed Ebh to be cell envelope associated and is proposed to form a specialized surface structure involved in cellular adhesion.
Innate Immune Responses of Human Neonatal Cells to Bacteria from the Normal Gastrointestinal Flora
Authors: Karlsson Helen , Hessle Christina , Rudin Anna
Published: November 2002

The hygiene hypothesis postulates that the prevalence of allergy has increased due to decreased microbial stimulation early in life, leading to delayed maturation of the immune system. The aim of this study was to examine the cytokine pattern produced from cord blood mononuclear cells relative to adult cells after stimulation with bacterial strains from the normal flora. Mononuclear cells from cord and adult blood samples were stimulated with the following bacteria:Bifidobacterium adolescentis,Enterococcus faecalis,Lactobacillus plantarum,Streptococcus mitis,Corynebacterium minutissimum,Clostridium perfringens,Bacteroides vulgatus,Escherichia coli,Pseudomonas aeruginosa,Veillonella parvula, andNeisseria sicca. The levels of interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α), IL-10, and IL-6 were measured by enzyme-linked immunosorbent assay. The TNF-α production was also analyzed after blocking CD14, Toll-like receptor 2 (TLR-2), and TLR-4 prior to stimulation with bacteria. The levels of IL-12 and TNF-α were similar in cord and adult cells. Gram-positive bacteria induced considerably higher levels of IL-12 and TNF-α than gram-negative bacteria in both cord and adult cells. The levels of IL-6 were significantly higher in newborns than in adults, whereas the levels of IL-10 were similar in newborns and adults. Gram-negative and gram-positive bacteria induced similar levels of IL-6 and IL-10 in cord cells.L. plantarumbound or signaled through CD14, TLR-2, and TLR-4, whereasE. coliacted mainly through CD14 and TLR-4. These results indicate that the innate immune response in newborns to commensal bacteria is strong and also suggest that different bacterial strains may have differential effects on the maturation of the immune system of infants.
Characterization ofLeishmania donovaniAntigens Encapsulated in Liposomes That Induce Protective Immunity in BALB/c Mice
Authors: Afrin Farhat , Rajesh Ravindran , Anam Khairul , Gopinath Meenakshisundram , Pal Swati , Ali Nahid
Published: November 2002

Leishmania donovanipromastigote membrane antigens (LAg) encapsulated in positively charged liposomes have been found to induce very significant levels of protection against experimental visceral leishmaniasis. The protectively immunized animals exhibited profound delayed-type hypersensitivity and antibody responses. The extent of protection induced by the same antigens, however, varied depending on the charge of the vesicles, with maximum induction by positively charged liposomes, followed by neutral liposomes and last negatively charged liposomes. Characterization of LAg and LAg entrapped in liposomes of different charges by Western blot analysis revealed the immunodominance of gp63 in all three vaccine preparations. The strong reactivity of antigens in a restricted antigen profile that included, in addition to gp63, 72-, 52-, 48-, 45-, 39-, and 20-kDa components in neutral and positively charged liposomes contrasted with the reactivity of a greater number of LAg components in negatively charged liposomes. Resistance to visceral leishmaniasis appears to depend on the immunity induced by gp63 and a few select antigens in association with the right liposomes. A striking similarity between the immunogenic profile of partially purified soluble antigens and that of LAg in neutral and positively charged liposomes suggests the potentiality of these antigens in future vaccine studies ofL. donovani.
T-Cell Responses to theMycobacterium tuberculosis-Specific Antigen ESAT-6 in Brazilian Tuberculosis Patients
Authors: Cardoso Fernando L. L. , Antas Paulo R. Z. , Milagres Alexandre S. , Geluk Annemieke , Franken Kees L. M. C. , Oliveira Eliane B. , Teixeira Henrique C. , Nogueira Susie A. , Sarno Euzenir N. , Klatser Paul , Ottenhoff Tom H. M. , Sampaio Elizabeth P.
Published: November 2002

TheMycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-γ), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-γ secretion by peripheral blood mononuclear cells (PBMCs) in response toM. tuberculosisESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognizedM. tuberculosisESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-γ in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according toMycobacterium bovisBCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-γ in response to ESAT. The present data demonstrate the specificity of ESAT-6 ofM. tuberculosisand its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-γ production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis ofM. tuberculosisinfection in an area of TB endemicity.
Type 1 Immunity Provides Optimal Protection against Both Mucosal and SystemicTrypanosoma cruziChallenges
Authors: Hoft D. F. , Eickhoff C. S.
Published: November 2002

Chagas' disease results from infection withTrypanosoma cruzi, a protozoan parasite that establishes systemic intracellular infection after mucosal invasion. We hypothesized that ideal vaccines for mucosally invasive, intracellular pathogens likeT. cruzishould induce mucosal type 2 immunity for optimal induction of protective secretory immunoglobulin A (IgA) and systemic type 1 immunity protective against intracellular replication. However, differential mucosal and systemic immune memory could be difficult to induce because of reciprocal inhibitory actions between type 1 and type 2 responses. To test our hypotheses, we investigated the protective effects of type 1 and type 2 biased vaccines against mucosal and systemicT. cruzichallenges. Intranasal vaccinations were given with recombinant interleukin-12 (IL-12)- and IL-4-neutralizing antibody (Ab) for type 1 immune bias, or recombinant IL-4 and gamma interferon-neutralizing Ab for type 2 immune bias. Cytokine RNA and protein studies confirmed that highly polarized memory immune responses were induced by our vaccination protocols. Survival after virulent subcutaneousT. cruzichallenge was used to assess systemic protection. Mucosal protection was assessed by measuring the relative inhibition of parasite replication in mucosal tissues early after oralT. cruzichallenge, using both PCR and quantitative culture techniques. As expected, only type 1 responses protected against systemic challenges (P< 0.01). However, contrary to our original hypothesis, type 1 responses optimally protected against mucosal challenges as well (P< 0.05). Type 1 and type 2 biased vaccines induced similar secretory IgA responses. We conclude that future vaccines forT. cruziand possibly other mucosally invasive, intracellular pathogens should induce both mucosal and systemic type 1 immunity.
Chromosomal DNA Deletions Explain Phenotypic Characteristics of Two Antigenic Variants, Phase II and RSA 514 (Crazy), of theCoxiella burnetiiNine Mile Strain
Authors: Hoover T. A. , Culp D. W. , Vodkin M. H. , Williams J. C. , Thompson H. A.
Published: November 2002

After repeated passages through embyronated eggs, the Nine Mile strain ofCoxiella burnetiiexhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol.132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.
Up-Regulation of Th1-Type Responses in Mucosal Leishmaniasis Patients
Authors: Bacellar Olívia , Lessa Hélio , Schriefer Albert , Machado Paulo , Ribeiro de Jesus Amélia , Dutra Walderez O. , Gollob Kenneth J. , Carvalho Edgar M.
Published: November 2002

The cytokine profile produced by peripheral blood mononuclear cells (PBMC) in response to leishmania antigens and the ability of interleukin-10 (IL-10) and transforming growth factor β (TGF-β) to modulate the immune response were evaluated in 21 mucosal leishmaniasis patients. Patients with mucosal disease exhibited increased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion and decreased IL-10 secretion compared to patients with classical cutaneous leishmaniasis. CD4+Th1 cells were the main source of IFN-γ and TNF-α production in mucosal leishmaniasis patients. Evaluation of cytokine gene expression in PBMC of these patients showed that there was strong up-regulation of IFN-γ transcripts upon stimulation with leishmania antigen, in contrast to the baseline levels of IL-10 mRNA. IL-10 suppressed IFN-γ production by 48% in cell cultures from cutaneous leishmaniasis patients and by 86% in cell cultures from healthy subjects stimulated with purified protein derivative, whereas in similar conditions IL-10 suppressed IFN-γ production by 19% in cell cultures from mucosal leishmaniasis patients stimulated with leishmania antigen. TGF-β suppressed IFN-γ levels to a greater extent in healthy subjects than in mucosal leishmaniasis and cutaneous leishmaniasis patients. These data indicate that a poorly modulated T-cell response in mucosal leishmaniasis patients leads to production of high levels of proinflammatory cytokines, such as IFN-γ and TNF-α, as well as a decreased ability of IL-10 and TGF-β to modulate this response. These abnormalities may be the basis for the pathological findings observed in this disease.
Cloning and Porin Activity of the Major Outer Membrane Protein P1 from
Coxiella burnetii
Authors: Varghees Sunita , Kiss Kati , Frans Giovanni , Braha Orit , Samuel James E.
Published: November 2002

Coxiella burnetii
, the etiological agent of Q fever, is a gram-negative obligate intracellular bacterium. Two striking characteristics of this microorganism are its ability to thrive within a phagolysosome and its ability to persist in the environment outside a host cell. These abilities have been attributed to the existence of
C. burnetii
developmental cycle variants: large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC). Variants differ in protein profiles, including differential expression of a major outer membrane protein (MOMP) of
C. burnetii
, designated P1. The ∼29-kDa MOMP is highly expressed in LCV, down-regulated in SCV, and not apparent in SDC. We sought to characterize P1 through purification of native protein for N-terminal analysis, cloning, and functional studies. Highly purified P1, extracted from
C. burnetii
membranes by using the zwitterionic detergent Empigen, allowed the determination of N-terminal and internal peptide sequences. The entire P1 coding locus was cloned by PCR amplification based upon these peptide sequences, followed by inverse PCR. Comparison of the predicted P1 amino acid sequences among the
C. burnetii
isolates Nine Mile, Koka, Scurry, and Kerns indicated a high degree of conservation. Structural prediction suggests that the peptide has a predominantly β-sheet conformation, consistent with bacterial porins. Typical porin characteristics were observed for native P1, including detergent solubilization properties, heat modification of purified protein, and channel formation in a planar lipid bilayer. Characterization of differentially expressed P1 as a porin increases our understanding of the function of morphological variants and their role in pathogenesis.
Decreased Infectivity despite Unaltered C3 Binding by a ΔhbhAMutant ofMycobacterium tuberculosis
Authors: Mueller-Ortiz Stacey L. , Sepulveda Eliud , Olsen Margaret R. , Jagannath Chinnaswamy , Wanger Audrey R. , Norris Steven J.
Published: November 2002

HbhA ofMycobacterium tuberculosisis a multifunctional binding protein, binding to both sulfated sugars such as heparin and to human complement component C3. HbhA may therefore interact with host molecules and/or host cells duringM. tuberculosisinfection and play a role in the pathogenesis of this bacterium. The purpose of this study was to use allelic exchange to create anM. tuberculosisstrain deficient in expression of HbhA to determine whether this protein's C3-binding activity plays a role in the pathogenesis ofM. tuberculosis. An in-frame, 576-bp unmarked deletion in thehbhAgene was created usingsacBas a counterselectable marker. Southern blotting and PCR analyses confirmed deletion ofhbhAin the ΔhbhAmutant. The ΔhbhAmutant strain grew at a rate similar to that of the parent in broth culture and in J774.A1 murine macrophage-like cells but was deficient in growth compared to the parent strain in the lungs, liver, and spleen of infected mice. In addition, the ΔhbhAmutation did not reduce binding ofM. tuberculosisto human C3 or to J774.A1 cells in the presence or absence of serum, suggesting that in the absence of HbhA, other molecules serve as C3-binding molecules on theM. tuberculosissurface. Taken together, these data indicate that HbhA is important in the infectivity ofM. tuberculosis, but its ability to bind C3 is not required for mycobacterial adherence to macrophage-like cells. Using the ΔhbhAmutant strain, a secondM. tuberculosisC3-binding protein similar in size to HbhA was identified as HupB, but the role of HupB as a C3-binding protein in intact organisms remains to be determined.
Identification of a Novel Fimbrial Gene Cluster Related to Long Polar Fimbriae in Locus of Enterocyte Effacement-Negative Strains of Enterohemorrhagic
Escherichia coli
Authors: Doughty Stephen , Sloan Joan , Bennett-Wood Vicki , Robertson Marcus , Robins-Browne Roy M. , Hartland Elizabeth L.
Published: November 2002

Enterohemorrhagic
Escherichia coli
(EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an ∼90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames,
lpfA
to
lfpD
, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of
lpfA
O113
were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene,
lpfA
, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that
lpf
O113
may function as an adhesin in LEE-negative isolates of EHEC.
Identification and Genetic Characterization of PmrA-Regulated Genes and Genes Involved in Polymyxin B Resistance in
Salmonella enterica
Serovar Typhimurium
Authors: Tamayo Rita , Ryan Sara S. , McCoy Andrea J. , Gunn John S.
Published: November 2002

Salmonella enterica
serovar Typhimurium encounters antimicrobial peptides (AP) within the phagosomes of professional phagocytes and at intestinal mucosal surfaces.
Salmonella
serovar Typhimurium utilizes the two-component regulatory system PmrA-PmrB, which is activated in response to the environmental conditions encountered in vivo, to regulate resistance to several AP, including polymyxin B (PM). Random Mu
d
J transposon mutagenesis was used to identify PmrA-PmrB-regulated genes, as well as genetic loci necessary for PM resistance. Three different phenotypic classes of genes were identified: those necessary for PM resistance and regulated by PmrA, those necessary for PM resistance and not regulated by PmrA, and PmrA-regulated genes not required for PM resistance. Loci identified as necessary for PM resistance showed between 6- and 192-fold increased sensitivities to PM, and transposon insertion sites include
surA
,
tolB
, and
gnd
. PmrA-regulated loci identified included
dgoA
and
yibD
and demonstrated 500- and 2,500-fold activation by PmrA, respectively. The role of the identified loci in aminoarabinose modification of lipid A was determined by paper chromatography. The
gnd
mutant demonstrated a loss of aminoarabinose from lipid A, which was suggested to be due to a polar effect on the downstream gene
pmrE
. The remaining PM
s
mutants (
surA
and
tolB
), as well as the two PmrA-regulated gene (
yibD
and
dgoA
) mutants, retained aminoarabinose on lipid A.
yibD
,
dgoA
, and
gnd
(likely affecting
pmrE
) played no role in PmrA-regulated resistance to high iron concentrations, while
surA
and
tolB
mutations grew poorly on high iron media. All PM
s
mutants identified in this study demonstrated a defect in virulence compared to wild-type
Salmonella
serovar Typhimurium when administered orally to mice, while the PmrA-regulated gene (
yibD
and
dgoA
) mutants showed normal virulence in mice. These data broaden our understanding of in vivo gene regulation, lipopolysaccharide modification, and mechanisms of resistance to AP in enteric bacteria.
Enhanced Immunogenicity of a Genetic Chimeric Protein Consisting of Two Virulence Antigens ofStreptococcus mutansand Protection against Infection
Authors: Zhang Ping , Jespersgaard Christina , Lamberty-Mallory Leticia , Katz Jannet , Huang Yan , Hajishengallis George , Michalek Suzanne M.
Published: November 2002

The saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme ofStreptococcus mutanshave been implicated in the initial adherence ofS. mutansto saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation ofS. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect againstS. mutansinfection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P< 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P< 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P< 0.05) inS. mutanscolonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization ofS. mutanssuggests its potential as a vaccine antigen for dental caries.
Analysis ofSalmonella entericaSerotype-Host Specificity in Calves: Avirulence ofS. entericaSerotype Gallinarum Correlates with Bacterial Dissemination from Mesenteric Lymph Nodes and Persistence In Vivo
Authors: Paulin Susan M. , Watson Patricia R. , Benmore Annette R. , Stevens Mark P. , Jones Philip W. , Villarreal-Ramos Bernardo , Wallis Timothy S.
Published: November 2002

Host and bacterial factors that determine whetherSalmonellaserotypes remain restricted to the gastrointestinal tract or penetrate beyond the mucosa and cause systemic disease remain largely undefined. Here, factors influencingSalmonellahost specificity in calves were assessed by characterizing the pathogenesis of different serotypes.Salmonella entericaserotype Dublin was highly virulent intravenously, whereasS. entericaserotype Choleraesuis was moderately virulent. Both serotypes were virulent in calves infected orally. In contrast,S. entericaserotypes Gallinarum and Abortusovis were avirulent by either route. Serotypes Dublin, Gallinarum, and Abortusovis colonized the intestinal tract 24 h after oral inoculation, yet only serotype Dublin was consistently recovered from systemic tissues. Serotypes Dublin and Gallinarum invaded bovine intestines in greater numbers and induced greater enteropathogenic responses than serotypes Choleraesuis and Abortusovis. However, only serotype Dublin was able to persist within the intestinal mucosa, and use of a novel cannulation model demonstrated that serotype Dublin was able to pass through the mesenteric lymph nodes in greater numbers than serotype Gallinarum. Together, these results suggest that initial interactions with the intestinal mucosa do not correlate with host specificity, although persistence within tissues and translocation via efferent lymphatics appear to be crucial for the induction of bovine salmonellosis.
The Abundant Polyadenylated Transcript 2 DNA Sequence of the Pathogenic Protozoan Parasite
Entamoeba histolytica
Represents a Nonautonomous Non-Long-Terminal-Repeat Retrotransposon- Like Element Which Is Absent in the Closely Related Nonpathogenic Species
Entamoeba dispar
Authors: Willhoeft Ute , Buß Heidrun , Tannich Egbert
Published: November 2002

While comparing gene expression in the pathogenic organism
Entamoeba histolytica
and the closely related but nonpathogenic species
Entamoeba dispar,
we discovered that the
E. histolytica
abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in
E. dispar
. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites of variable length. Target site duplications were obviously generated during integration of ehapt2 into the
E. histolytica
genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous
E. dispar
genomic sequences. ehapt2 shares 3′ sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like element of
E. histolytica
, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous non-LTR retrotransposons. A comparison of various
E. histolytica
isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2 elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent and late transition of
E. histolytica
from a harmless gut commensal to an invasive pathogen.
Identification of a Fibronectin-Binding Protein from
Staphylococcus epidermidis
Authors: Williams Rachel J. , Henderson Brian , Sharp Lindsay J. , Nair Sean P.
Published: November 2002

Staphylococcus epidermidis
has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from
S. epidermidis
. A phage display library of
S. epidermidis
genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research
S. epidermidis
genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain
embp
. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of
S. epidermidis
, but not the binding of
Staphylococcus aureus
, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the
S. aureus
fibronectin-binding protein FnBPB, blocked binding of
S. aureus
to fibronectin but had a negligible effect on the binding of
S. epidermidis
.